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determinar a presena de anticorpos ige especficos para superantgenos estafiloccicos e o grau de sensibilizao mediada por esses , assim como se esses esto associados gravidade da asma em pacientes adultos .
estudo transversal incluindo asmticos adultos em acompanhamento ambulatorial em um hospital universitrio tercirio no rio de janeiro ( rj ) .
os pacientes foram alocados consecutivamente em dois grupos de gravidade da asma segundo critrios da global initiative for asthma : asma leve ( al ) , com asmticos leves intermitentes ou persistentes , e asma moderada ou grave ( amg ) .
foram determinados os nveis sricos de anticorpos ige antitoxinas estafiloccicas , e os resultados foram comparados por anlise estatstica .
foram includos 142 pacientes no estudo : 72 no grupo al ( mediana de idade = 46 anos ; 59 do sexo feminino ) e 70 do grupo amg ( mediana de idade = 56 anos ; 60 do sexo feminino ) .
na amostra geral , 62 pacientes ( 43,7% ) apresentaram resultados positivos para dosagens de anticorpos ige antitoxinas estafiloccicas : enterotoxina ( tx ) a , em 29 ( 20,4% ) ; txb , em 35 ( 24,6% ) ; txc , em 33 ( 23,2% ) ; e toxic shock syndrome toxin ( tsst ) , em 45 ( 31,7% ) . as mdias das dosagens sricas de anticorpos ige especficos anti - txa , txb , txc e tsst foram , respectivamente , de 0,96 u / l , 1,09 u / l , 1,21 u / l , e 1,18 u / l .
a presena de anticorpos ige sricos anti - txa , txb , txc e tsst , foi detectada em 43,7% nessa amostra de pacientes , mas no houve associao estatisticamente significativa entre seus resultados qualitativos ou quantitativos e gravidade clnica da asma .
staphylococcus aureus is a gram - positive bacterium that can colonize the human skin and respiratory tract .
the most important are toxic shock syndrome toxin ( tsst ) , staphylococcal enterotoxin a ( sea ) , seb , sec , sed , see , seg , seh , and sei , the activities of which include superantigen activity , pyrogenicity , and potentiation of lethality of other toxins .
the superantigen activity of staphylococcal toxins consists of direct stimulation of class ii mhc receptors and t cells , independently of antigen presentation by antigen - presenting cells , stimulating the proliferation and activity of cd4 and cd8 t lymphocytes .
this mechanism is related to the worsening of allergic diseases by the production of staphylococcal toxin - specific ige antibodies , as well as by a direct effect on tissue mast cells , leading to their degranulation .
in asthma patients
, staphylococcal toxins also act as superantigens , stimulating cd4 t lymphocyte proliferation and activity and leading to an increased production of staphylococcal toxin - specific ige antibodies , causing an allergic - type reaction by biding to mast cells in the respiratory tract .
this reaction results in the release of mediators such as histamine , kinins , platelet - activating factor , and arachidonic acid metabolites ( prostaglandins and leukotrienes ) , as well as of chemokines , eliciting immediate and late inflammatory responses ( by the recruitment and activation of neutrophils and eosinophils ) and culminating in asthma worsening .
staphylococcal superantigens have been shown to play roles in atopic dermatitis , rhinosinusitis , and asthma , being correlated with their severity .
with regard to asthma , kowalski et al . found ige antibodies to sea , sec , and tsst in 89.7% of 237 asthma patients ( mean levels of 1.096 3.25 ku / l ) ; although there was no significant difference between those with severe asthma and those with non - severe asthma in terms of the prevalence of staphylococcal toxin - specific ige antibodies ( 81.4% vs. 69.2% ) , mean levels were higher in the former than in the latter ( 1.65 3.25 ku / l vs. 0.54 0.72 ku / l ) .
in another study ( n = 210 ) , the same authors obtained similar results , the prevalence of staphylococcal toxin - specific ige antibodies being 76.1% in patients with severe asthma and 71.1% in those with non - severe asthma , mean levels being three times higher in the former than in the latter .
bachert et al . found a significant increase in staphylococcal toxin - specific ige antibodies in patients with severe asthma when compared with those with mild asthma and controls ( n = 70 ) .
in a more recent study ( n = 387 ) , the same group of authors found a significant increase in staphylococcal toxin - specific ige antibodies in patients with severe uncontrolled asthma ( 59.6% ) when compared with those with controlled asthma ( 40.8% ) and controls ( 13.0% ) .
high levels of staphylococcal toxin - specific ige antibodies have been found to be a risk factor for asthma ( or = 7.6 ) and severe asthma ( or = 11.09 ) .
in latin
america , there have been no studies correlating staphylococcal superantigens with the severity of asthma .
therefore , we investigated a population of asthma patients treated at a university hospital in the city of rio de janeiro , brazil , and having no risk factors for increased staphylococcal colonization or infection in order to correlate the clinical severity of asthma with the presence of staphylococcal toxin - specific ige antibodies and degree of ige - mediated sensitization .
this was a cross - sectional study including adult patients clinically and functionally diagnosed with asthma and receiving outpatient treatment at the clementino fraga filho university hospital , located in the city of rio de janeiro , brazil . between 2009 and 2013 ,
consecutive patients were divided into two groups according to the clinical severity of asthma based on the global initiative for asthma criteria
: the mild asthma ( ma ) group , comprising patients with mild intermittent or persistent asthma ; and the moderate or severe asthma ( msa ) group . according to the global initiative for asthma
,
asthma severity can be evaluated on the basis of the treatment required in order to control the disease .
patients with mild asthma are defined as those requiring only rescue medication , low - dose inhaled corticosteroids / leukotriene receptor antagonists , or a combination of the two .
patients with moderate asthma are defined as those using long - acting 2 agonists and inhaled corticosteroids at low or moderate doses .
patients with severe asthma are defined as those using long - acting 2 agonists and inhaled corticosteroids at high doses or other bronchodilators and anti - inflammatory drugs for asthma control .
the criteria for inclusion in the present study were as follows : being an adult patient clinically and functionally diagnosed with asthma ,
regardless of the presence of rhinitis and positive skin test results to aeroallergens .
the exclusion criteria were as follows : presence of copd , atopic dermatitis , or both ; asthma exacerbation in the last four weeks ; presence of respiratory infection or use of antimicrobial agents in the last six weeks ; use of systemic corticosteroid therapy for seven or more days in the last four weeks ; history of immunodeficiency , neoplasia , connective tissue disease , kidney failure , sinonasal polyposis , chronic sinus disease , cystic fibrosis , or bronchiectasis ; pregnancy ; smoking in the last twelve months ; and declining to participate in the study or give written informed consent .
the sample size calculation was based on a study by kowalski et al .
and was performed with a specific statistical calculation program ( openepi ) . for a paired relationship , with a 95% confidence interval and a power of 80% ,
the required sample size was calculated to be 140 ( 70 per group ) .
procedures included the following : clinical history taking ; physical examination ; routine tests ( including blood count , esr measurement , determination of total ige levels , parasitological stool examination , chest x - rays , and sinus x - rays ) ; pulmonary function tests ( spirometry and pef measurement ) ; skin prick tests to aeroallergens ; and determination of serum levels of ige antibodies ( to sea , seb , sec , and tsst ) .
spirometry was performed with a spirometer ( jaeger , wrzburg , germany ) , in accordance with the american thoracic society guidelines
and the reference values proposed by knudson et al .
a finding of obstruction and positive bronchodilator test results with reversal or significant improvement were consistent with asthma .
for pef measurement , a peak flow meter ( mini - wright afs ;
clement clarke international , essex , england ) was used , the reference values being those proposed by nunn and gregg .
skin tests to aeroallergens were performed with the use of the puncture technique and standard antigens .
for determination of serum levels of staphylococcal toxin - specific ige antibodies , an immunoassay system ( immunocap 100 ; phadia , uppsala , sweden ) was used .
, we used the student 's t - test or the mann - whitney test , as appropriate , through the analysis of the kolmogorov - smirnov and shapiro - wilk coefficients . in order to compare categorical variables
, we used the chi - square test or fisher 's exact test , as appropriate .
the sample size was calculated in order to provide a power of 80% , and values of p < 0.05 were considered statistically significant .
the present study was approved by the research ethics committee of the federal university of rio de janeiro clementino fraga filho university hospital .
all participating patients gave written informed consent , and the treatment provided to those who declined to participate in the study was in no way affected by their decision .
this was a cross - sectional study including adult patients clinically and functionally diagnosed with asthma and receiving outpatient treatment at the clementino fraga filho university hospital , located in the city of rio de janeiro , brazil . between 2009 and 2013 ,
consecutive patients were divided into two groups according to the clinical severity of asthma based on the global initiative for asthma criteria
: the mild asthma ( ma ) group , comprising patients with mild intermittent or persistent asthma ; and the moderate or severe asthma ( msa ) group . according to the global initiative for asthma
,
asthma severity can be evaluated on the basis of the treatment required in order to control the disease .
patients with mild asthma are defined as those requiring only rescue medication , low - dose inhaled corticosteroids / leukotriene receptor antagonists , or a combination of the two .
patients with moderate asthma are defined as those using long - acting 2 agonists and inhaled corticosteroids at low or moderate doses .
patients with severe asthma are defined as those using long - acting 2 agonists and inhaled corticosteroids at high doses or other bronchodilators and anti - inflammatory drugs for asthma control .
the criteria for inclusion in the present study were as follows : being an adult patient clinically and functionally diagnosed with asthma ,
regardless of the presence of rhinitis and positive skin test results to aeroallergens .
the exclusion criteria were as follows : presence of copd , atopic dermatitis , or both ; asthma exacerbation in the last four weeks ; presence of respiratory infection or use of antimicrobial agents in the last six weeks ; use of systemic corticosteroid therapy for seven or more days in the last four weeks ; history of immunodeficiency , neoplasia , connective tissue disease , kidney failure , sinonasal polyposis , chronic sinus disease , cystic fibrosis , or bronchiectasis ; pregnancy ; smoking in the last twelve months ; and declining to participate in the study or give written informed consent .
the sample size calculation was based on a study by kowalski et al .
and was performed with a specific statistical calculation program ( openepi ) . for a paired relationship , with a 95% confidence interval and a power of 80% ,
procedures included the following : clinical history taking ; physical examination ; routine tests ( including blood count , esr measurement , determination of total ige levels , parasitological stool examination , chest x - rays , and sinus x - rays ) ; pulmonary function tests ( spirometry and pef measurement ) ; skin prick tests to aeroallergens ; and determination of serum levels of ige antibodies ( to sea , seb , sec , and tsst ) . spirometry was performed with a spirometer ( jaeger , wrzburg , germany ) , in accordance with the american thoracic society guidelines
and the reference values proposed by knudson et al .
a finding of obstruction and positive bronchodilator test results with reversal or significant improvement were consistent with asthma .
for pef measurement , a peak flow meter ( mini - wright afs ;
clement clarke international , essex , england ) was used , the reference values being those proposed by nunn and gregg .
skin tests to aeroallergens were performed with the use of the puncture technique and standard antigens .
for determination of serum levels of staphylococcal toxin - specific ige antibodies , an immunoassay system ( immunocap 100 ; phadia , uppsala , sweden ) was used .
in order to compare numerical variables , we used the student 's t - test or the mann - whitney test , as appropriate , through the analysis of the kolmogorov - smirnov and shapiro - wilk coefficients . in order to compare categorical variables
, we used the chi - square test or fisher 's exact test , as appropriate .
the sample size was calculated in order to provide a power of 80% , and values of p < 0.05 were considered statistically significant .
the present study was approved by the research ethics committee of the federal university of rio de janeiro clementino fraga filho university hospital .
all participating patients gave written informed consent , and the treatment provided to those who declined to participate in the study was in no way affected by their decision .
a total of 142 patients were studied . of those , 72 ( 17 with mild intermittent asthma and 55 with mild persistent asthma )
were allocated to the ma group and 70 ( 53 with moderate asthma and 17 with severe asthma ) were allocated to the msa group .
the median age was 52.5 years ( 46 years in the ma group and 56 years in the msa group ) , females and white individuals having predominated . in the sample as a whole , the mean body mass index ( bmi ) was 27.09 kg / m ,
128 patients had rhinitis , 131 had positive skin test results to aeroallergens , and 99 had a family history of atopy . only 37 ( 26.1% ) had a history of smoking .
mean percent predicted pef was 72.59% , mean percent predicted pre - bronchodilator fev1 was 71.55% , and mean percent predicted post - bronchodilator fev1 was 81.48% .
mean eosinophil count was 4.4% , and mean total ige levels were 574.92 iu / ml .
table 1 shows the distribution of sociodemographic and clinical variables , and table 2 shows lung function parameters and laboratory findings in the ma and msa groups .
student 's t - test ( for age ) and chi - square test or fisher 's test ( for the remaining parameters ) .
* student 's t - test ( for age ) and chi - square test or fisher 's test ( for the remaining parameters ) .
table 2lung function parameters and laboratory findings in the study population , by asthma severity .
student 's t - test or mann - whitney test . of the sample as a whole ,
62 patients ( 43.7% ) tested positive for staphylococcal toxin - specific ige antibodies : sea , in 29 ( 20.4% ) ; seb , in 35 ( 24.6% ) ; sec , in 33 ( 23.2% ) ; and tsst , in 45 ( 31.7% ) .
the mean serum levels of ige antibodies to sea , seb , sec , and tsst were 0.96 u / l , 1.09 u / l , 1.21 u / l , and 1.18 u / l , respectively .
as can be seen in tables 3 and
, there were no statistically significant differences between the two groups regarding the frequency of ige - mediated sensitization and serum levels of staphylococcal toxin - specific ige antibodies .
table 3frequency of ige - mediated sensitization to staphylococcal toxins in the study population , by asthma severity .
sea : staphylococcal enterotoxin a ; seb : staphylococcal enterotoxin b ; sec : staphylococcal enterotoxin c ; and tsst : toxic shock syndrome toxin .
sea : staphylococcal enterotoxin a ; seb : staphylococcal enterotoxin b ; sec : staphylococcal enterotoxin c ; and tsst : toxic shock syndrome toxin .
table 4serum levels of staphylococcal toxin - specific ige antibodies in the study population , by asthma severity .
sea : staphylococcal enterotoxin a ; seb : staphylococcal enterotoxin b ; sec : staphylococcal enterotoxin c ; and tsst : toxic shock syndrome toxin . * chi - square test or mann - whitney test .
sea : staphylococcal enterotoxin a ; seb : staphylococcal enterotoxin b ; sec : staphylococcal enterotoxin c ; and tsst : toxic shock syndrome toxin . * chi - square test or mann - whitney test .
there were statistically significant differences between the two groups of patients in the present study regarding their clinical and sociodemographic characteristics ( including age , bmi , and prevalence of rhinitis ) .
the fact that the patients in the msa were significantly older than were those in the ma group might be due to the fact that asthma tends to be more severe in older individuals , especially those in whom the onset of asthma occurred at an older age .
our finding of a significantly higher bmi in the msa group is consistent with the literature , obesity having been reported to be a risk factor for and an aggravator of asthma .
recent studies have established a relationship between obesity - induced changes in the gastrointestinal and respiratory microbiome and the etiopathogenesis of obesity - related asthma .
our finding of a higher prevalence of rhinitis in the ma group suggests that atopy was more common in the ma group than in the msa group , supporting the concept that atopic manifestations tend to be less common in asthma patients with disease that is more severe .
with regard to the lung function parameters assessed in the present study , absolute and percent predicted pef
were significantly lower in the msa group , as were percent predicted pre - bronchodilator fev1 and percent predicted post - bronchodilator fev1 .
these findings were expected and are consistent with the literature , showing the usual correlation between clinical severity and lung function parameters .
of the 142 patients studied , 62 ( 43.7% ) tested positive for staphylococcal toxin - specific ige antibodies , ige antibodies to tsst being the most prevalent .
our findings are different from those of two studies in the literature and similar to those of two other studies . in one study , kowalski et al . found an 89.7% prevalence of positivity for staphylococcal toxin - specific ige antibodies in severe and non - severe asthma patients ; in another study , they found a 76.1% prevalence in patients with severe refractory asthma and a 71.1% prevalence in patients with non - severe asthma .
in one study , bachert et al . found a 38.1% prevalence of positivity for staphylococcal toxin - specific ige antibodies in patients with asthma ( independently of disease severity ) and a 62% prevalence in patients with severe asthma ; in another study , they found a 59.6% prevalence in patients with severe uncontrolled asthma and a 40.8% prevalence in patients with controlled asthma ,
the latter prevalence being closer to that found in the present study .
aureus , such as sinonasal polyposis , bronchiectasis , chronic bronchitis , and atopic dermatitis , were not included in the present study . by facilitating colonization or infection with s .
aureus , the aforementioned conditions can lead to increased quantities of staphylococcal toxins in the body , resulting in increased ige - mediated sensitization and , consequently , a heterogeneous population of asthma patients .
the studies conducted by kowalski et al .
and bachert et al .
had no such exclusion criteria and included patients with chronic sinus disease and sinonasal polyposis , as was the case with the most recent study by kowalski et al . ,
who nevertheless found no statistically significant differences between asthma patients with polyposis and those without regarding their levels of staphylococcal toxin - specific ige antibodies .
this might explain the differences between our results and those obtained by the two aforementioned groups of authors regarding the degree of ige - mediated sensitization .
aureus in the brazilian population ; therefore , it is currently impossible to determine whether or not it is lower than that in the european population .
it is also impossible to determine whether the allergic immune response to staphylococcal toxins is lower in asthma patients in brazil than in those in other countries . in the present study
, there were no statistically significant differences between the two groups regarding the frequency of staphylococcal toxin - specific ige antibodies .
our results are qualitatively similar to but quantitatively different from those obtained by kowalski et al . ,
who found significantly higher levels of staphylococcal toxin - specific ige antibodies in patients with severe asthma .
in addition , our results are qualitatively and quantitatively different from those obtained by bachert et al .
these differences are also due to the exclusion criteria used in the present study , which were not used in any of the aforementioned studies , and to specific characteristics of our study population , as previously mentioned . in the present study , ige antibodies to sea , seb , sec , and tsst
were found in 62 ( 43.7% ) of the 142 asthma patients analyzed , and neither the frequency of staphylococcal toxin - specific ige antibodies nor the serum levels of those antibodies were associated with the clinical severity of asthma .
these results are extremely relevant because this is the first study on this topic in latin america , the results of which differed from those of previous studies conducted in europe , indicating a " negative " association
. the limitations of the present study lie in the fact that it was a single - center study conducted at a tertiary care university hospital , in a single demographic area of brazil .
our primary objective was to evaluate the influence of serum levels of staphylococcal toxin - specific ige antibodies and their association with asthma severity , meaning that this was not a population prevalence study of ige sensitization in asthma patients and healthy individuals in our region . if that had been the case , a much larger sample size would have been required , and it would have been impossible to obtain that in a single - center study .
therefore , in the present study , each group served as the control group for the other , without the use of a third group , comprising healthy individuals .
multicenter studies in brazil and other latin american countries are needed in order to determine more accurately the role of ige - mediated sensitization to staphylococcal toxins as an aggravator of asthma , as well as to determine its prevalence in asthma patients and healthy individuals .
such studies should include asthma patients with and without diseases that can lead to increased colonization or infection with s .
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abstractobjective : to determine the presence of staphylococcal superantigen - specific ige antibodies and degree of ige - mediated sensitization , as well as whether or not those are associated with the severity of asthma in adult patients .
methods : this was a cross - sectional study involving outpatients with asthma under treatment at a tertiary care university hospital in the city of rio de janeiro , brazil .
consecutive patients were divided into two groups according to the severity of asthma based on the global initiative for asthma criteria : mild asthma ( ma ) , comprising patients with mild intermittent or persistent asthma ; and moderate or severe asthma ( msa ) .
we determined the serum levels of staphylococcal toxin - specific ige antibodies , comparing the results and performing a statistical analysis .
results : the study included 142 patients : 72 in the ma group ( median age = 46 years ; 59 females ) and 70 in the msa group ( median age = 56 years ; 60 females ) . in the sample as a whole , 62 patients ( 43.7% ) presented positive results for staphylococcal toxin - specific ige antibodies : staphylococcal enterotoxin a ( sea ) , in 29 ( 20.4% ) ; seb , in 35 ( 24.6% ) ; sec , in 33 ( 23.2% ) ; and toxic shock syndrome toxin ( tsst ) , in 45 ( 31.7% ) .
the mean serum levels of ige antibodies to sea , seb , sec , and tsst were 0.96 u / l , 1.09 u / l , 1.21 u / l , and 1.18 u / l , respectively .
there were no statistically significant differences between the two groups in terms of the qualitative or quantitative results .
conclusions : serum ige antibodies to sea , seb , sec , and tsst were detected in 43.7% of the patients in our sample .
however , neither the qualitative nor quantitative results showed a statistically significant association with the clinical severity of asthma .
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Objetivo:
Mtodos:
Resultados:
Concluses:
INTRODUCTION
METHODS
Patients
Procedures
Statistical analysis
Ethical aspects
RESULTS
DISCUSSION
|
sixty - four patients in nepal that met us department of defense enrollment criteria ( 9 ) for influenzalike illness were evaluated by using onsite rapid influenza tests ( optical immunoassay rapid diagnostic tests , thermo electron corp .
throat swab specimens were collected within the first 72 hours of onset of symptoms , routed through the armed forces research institute for medical sciences in bangkok , thailand , and shipped on dry ice to brooks city base in san antonio , texas , for clinical characterization and diagnosis using traditional culturing techniques and monoclonal antibody staining ( 10 ) .
antigenic analysis of select isolates was performed at the centers for disease control and prevention ( cdc ) in atlanta , georgia , by using the hemagglutination inhibition ( hi ) assay and postinfection ferret antisera ( 11 ) .
rna was extracted from 48-hour shell vial cultures ( 10 ) by using the magnapure lx ( roche molecular , mannheim , germany ) and rna isolation kit ii ( roche molecular ) according to the manufacturer 's protocols . for reverse transcription - polymerase chain reaction ( rt - pcr ) amplification ,
5 l rna was added to a 50-l master mixture containing 1 reaction buffer , 1.6 mmol / l mgso4 , 1 enzyme mixture , and 400 nmol / l primers ( h3-f7 , 5-act - atc - att - gct - ttg - agc-3 and h3r-1184 , 5-atg - gct - gct - tga - gtg - ctt-3 ) by using the superscript iii one - step rt - pcr system ( invitrogen , carlsbad , ca , usa ) .
pcr thermocycling consisted of an rt step at 50c for 30 min , hot start activation at 95c for 3 min , followed by 40 amplification cycles of 95c for 30 s , 52c for 15 s , and 68c for 1 min , with a final extension cycle at 68c for 7 min .
all pcr products were visualized after electrophoresis in 2% precast gels stained with ethidium bromide ( invitrogen ) under uv illumination .
the ha1 amplicon ( 1177 bp ) was sequenced by using the h3-f7 and h3r-1184 pcr primers ( described above ) and 2 additional internal oligonucleotides , h3r-466 ( 5-ggt - gca - acc - aat - tca - atc-3 ) and h3f-282 ( 5-cag - caa - ctg - tta - ccc-3 ) .
unincorporated fluorescent nucleotides were removed by using a dye ex 96-well plate kit ( qiagen ) according to the manufacturer 's recommendations .
nucleotide sequencing was performed by using the big dye terminator v3.1 kit and analyzed by using an abi 3100 genetic analyzer ( both from applied biosystems , foster city , ca , usa ) according to the manufacturer 's specifications .
multiple sequence alignments , protein translation , and phylogenetic analysis were performed with the dnastar ( dnastar inc . ,
three - dimensional ha protein structures were generated by using molmol ( 12 ) and the swiss - pdb viewer programs ( 13 ) .
ha nucleotide sequences for all 26 nepal isolates depicted in the phylogenetic analysis are available from genbank under accession nos .
sixty - four patients in nepal that met us department of defense enrollment criteria ( 9 ) for influenzalike illness were evaluated by using onsite rapid influenza tests ( optical immunoassay rapid diagnostic tests , thermo electron corp .
throat swab specimens were collected within the first 72 hours of onset of symptoms , routed through the armed forces research institute for medical sciences in bangkok , thailand , and shipped on dry ice to brooks city base in san antonio , texas , for clinical characterization and diagnosis using traditional culturing techniques and monoclonal antibody staining ( 10 ) .
antigenic analysis of select isolates was performed at the centers for disease control and prevention ( cdc ) in atlanta , georgia , by using the hemagglutination inhibition ( hi ) assay and postinfection ferret antisera ( 11 ) .
rna was extracted from 48-hour shell vial cultures ( 10 ) by using the magnapure lx ( roche molecular , mannheim , germany ) and rna isolation kit ii ( roche molecular ) according to the manufacturer 's protocols . for reverse transcription - polymerase chain reaction ( rt - pcr ) amplification ,
5 l rna was added to a 50-l master mixture containing 1 reaction buffer , 1.6 mmol / l mgso4 , 1 enzyme mixture , and 400 nmol / l primers ( h3-f7 , 5-act - atc - att - gct - ttg - agc-3 and h3r-1184 , 5-atg - gct - gct - tga - gtg - ctt-3 ) by using the superscript iii one - step rt - pcr system ( invitrogen , carlsbad , ca , usa ) .
pcr thermocycling consisted of an rt step at 50c for 30 min , hot start activation at 95c for 3 min , followed by 40 amplification cycles of 95c for 30 s , 52c for 15 s , and 68c for 1 min , with a final extension cycle at 68c for 7 min .
all pcr products were visualized after electrophoresis in 2% precast gels stained with ethidium bromide ( invitrogen ) under uv illumination .
the ha1 amplicon ( 1177 bp ) was sequenced by using the h3-f7 and h3r-1184 pcr primers ( described above ) and 2 additional internal oligonucleotides , h3r-466 ( 5-ggt - gca - acc - aat - tca - atc-3 ) and h3f-282 ( 5-cag - caa - ctg - tta - ccc-3 ) .
unincorporated fluorescent nucleotides were removed by using a dye ex 96-well plate kit ( qiagen ) according to the manufacturer 's recommendations .
nucleotide sequencing was performed by using the big dye terminator v3.1 kit and analyzed by using an abi 3100 genetic analyzer ( both from applied biosystems , foster city , ca , usa ) according to the manufacturer 's specifications .
multiple sequence alignments , protein translation , and phylogenetic analysis were performed with the dnastar ( dnastar inc . , madison , wi , usa ) software package .
three - dimensional ha protein structures were generated by using molmol ( 12 ) and the swiss - pdb viewer programs ( 13 ) .
ha nucleotide sequences for all 26 nepal isolates depicted in the phylogenetic analysis are available from genbank under accession nos .
clinical evaluations and throat specimens were obtained from 64 patients from 3 refugee camps in southeastern nepal ( figure 1 ) .
of the 64 patients , 61 were refugees from bhutan , 1 was a foreign aid worker from japan , and 2 were nepalese nationals .
most of the patients were < 10 years of age ; 36 were male and 28 were female .
none had previously been vaccinated against influenza and of the 64 specimens collected , 42 ( 66% ) tested positive for influenza a by culture .
the green circle shows the location of 3 bhutan refugee camps where the outbreak occurred in early july 2004 .
( map courtesy of http://www.maps.com ) hi was performed by using postinfection ferret antisera with reference antigens that included the 20042005 h3n2 vaccine seed strain ( a / wyoming/03/2003 ) and the 20052006 southern hemisphere h3n2 vaccine strain ( a / wellington/1/2004 ) . when compared with a / wyoming/03/2003 , 4 of 9 nepal isolates showed 4-fold lower titers ( 1:320 versus 1:1,280 hi units ) than that allowed for homologous titer of the reference antisera .
six of 9 nepal isolates were antigenically distinct when compared with the a / wellington/1/2004 strain and showed a 4-fold ( 1:160 versus 1:640 ) reduction in titer to ferret antisera ( table 1 ) .
* test antigens are considered antigenically different from the reference strain if hi titers show a 4-fold difference . a
rt - pcr - based molecular subtyping showed that all 42 specimens were the h3n2 influenza subtype .
twenty - six of the 42 influenza a positive samples were randomly selected for molecular characterization using direct nucleotide sequencing of the ha gene .
the 26 nepal isolates exhibited 99.8% nucleotide sequence identity and contained the fujian - like amino acid substitutions at positions 155 ( h155 t ) and 156 ( q156h ) in the ha protein ( table 2 ) .
alignment of the 329amino acid ha protein from 26 isolates obtained from this outbreak with the 2004/05 a / wyoming/3/03 vaccine strain and previous h3n2 vaccine strains indicated 4 evident amino acid changes present in most of the isolates ( table 2 ) .
all 4 amino acid changes observed within most of these outbreak isolates are present within a / california/7/04 , a variant strain selected as the h3n2 vaccine strain for the 20052006 influenza season . *
n , asparagine ; t , threonine ; h , histidine ; i , isoleucine ; p , proline ; k , lysine ; s , serine ; v , valine ; q , glutamine .
consensus sequence derived from a multiple sequence protein alignment of 26 ha1 hemagglutinin sequences from nepal .
of the 26 nepal strains examined , 24 exhibited a novel lysine - to - asparagine substitution at position 145 in the ha protein ( k145n ) .
this substitution is noteworthy because most strains characterized in 20032004 , including the fujian/411/2002 vaccine strain , contained a lysine ( k ) at this position .
prior to this outbreak , the us department of defense had only observed k145n substitutions in 6 strains obtained from ramstein , germany , ( data not shown ) in june 2004 .
additionally , all 26 nepal sequences exhibited a serine - to - asparagine substitution at position 189 ( s189n ) that had also been observed in the 6 isolates from germany , as well as in a few isolates from asia characterized at the end of the 20032004 influenza season .
two other substitution mutations in the ha1 hemagglutinin , i.e. , valine to isoleucine at position 226 ( v226i ) and serine to proline at position 227 ( s227p ) , were also observed in 24 ( 92% ) and 26 of 26 of the nepal isolates , respectively .
both substitutions differ from most influenza a h3n2 field isolates collected in 20032004 , including the fujian and wyoming vaccine strain for 20042005 ( table 2 ) .
the phylogeny of h3n2 ha proteins indicates a drifting of the nepal isolates from the a / fujian/411/03 and a / wyoming/03/03 vaccine strains and shows that these outbreak isolates have a higher genetic homology to a / wellington/1/04 , a prototype strain selected as the 20052006 southern hemisphere h3 vaccine strain ( figure 2 ) .
the a / wellington/1/04 strain contains 2 of the 4 amino acid changes ( s227p and s189n ) observed in the nepal isolates , but does not contain the k145n and v226i substitutions .
unrooted phylogenetic analysis of ha1 hemagglutinin nucleotide sequences from 26 nepal isolates and h3n2 vaccine and reference strains .
the nepal isolates have drifted from the 20042005 a / fujian/411/03 vaccine strain ( and a / wyoming/03/03 vaccine seed strain ) and are genetically equivalent to a / california/7/04 , the 20052006 northern hemisphere vaccine strain . a k145n substitution ( branch point indicated by the arrow )
was observed in 24 of 26 nepal isolates and represents a genetic marker for the dominant lineage of h3n2 viruses during the 20042005 season .
nucleotide and amino acid sequences for all nepal isolates are available from genbank under accession no .
the asterisk indicates isolates from table 2 that were antigenically distinct from a / wyoming/303 .
three - dimensional views of influenza ha proteins highlighting amino acid changes in a representative nepal isolate and the a / wyoming/3/03 vaccine strains are shown in figure 3a and b , respectively .
the mutation at position 145 ( shown in yellow ) , which is located adjacent to antibody - binding site a and within a known glycosylation site , introduces an asparagine - for - lysine substitution .
this substitution results in a more accessible receptor - binding cleft located directly above residue 145 ( comparing panels a and b ) . located above the receptor - binding pocket is a serine - to - asparagine change ( shown in green ) that possibly alters the regional surface topography at position 189 within antibody - binding site b. a serine - to - proline mutation at position 227 ( shown in magenta ) appears to marginally affect the ha surface features .
this substitution resides within antibody - binding site d , which corresponds to residues 225228 , which make up the left side of the receptor - binding pocket ( 14 ) .
interestingly , this proline residue is located within a barrel ( a protein motif consisting of an antiparallel sheet domain ) and does not appreciably alter the predicted protein structure , as shown by the absence of any substantial changes in the computer - modeled , 3-dimensional structure compared with the ha1 of a / wyoming/3/2003 .
three - dimensional top view of the ha1 hemagglutinin structures for a ) a representative a / nepal/1648/04 virus and b ) vaccine strain a / wyoming/3/03 .
most ( 24/26 ) of the nepal isolates contain a lysine to asparagine substitution ( shown in yellow ) at position 145 ( k145n ) .
magenta , residues 226 and 227 ; orange , residue 189 ; green , residues 155 and 156 ; yellow , residue 145 .
hemagglutinin molecules were generated by using the respective amino acid sequences with molmol ( 12 ) .
clinical evaluations and throat specimens were obtained from 64 patients from 3 refugee camps in southeastern nepal ( figure 1 ) .
of the 64 patients , 61 were refugees from bhutan , 1 was a foreign aid worker from japan , and 2 were nepalese nationals .
most of the patients were < 10 years of age ; 36 were male and 28 were female .
none had previously been vaccinated against influenza and of the 64 specimens collected , 42 ( 66% ) tested positive for influenza a by culture .
the green circle shows the location of 3 bhutan refugee camps where the outbreak occurred in early july 2004 .
hi was performed by using postinfection ferret antisera with reference antigens that included the 20042005 h3n2 vaccine seed strain ( a / wyoming/03/2003 ) and the 20052006 southern hemisphere h3n2 vaccine strain ( a / wellington/1/2004 ) . when compared with a / wyoming/03/2003 , 4 of 9 nepal isolates showed 4-fold lower titers ( 1:320 versus 1:1,280 hi units ) than that allowed for homologous titer of the reference antisera .
six of 9 nepal isolates were antigenically distinct when compared with the a / wellington/1/2004 strain and showed a 4-fold ( 1:160 versus 1:640 ) reduction in titer to ferret antisera ( table 1 ) .
* test antigens are considered antigenically different from the reference strain if hi titers show a 4-fold difference . a
rt - pcr - based molecular subtyping showed that all 42 specimens were the h3n2 influenza subtype .
twenty - six of the 42 influenza a positive samples were randomly selected for molecular characterization using direct nucleotide sequencing of the ha gene .
the 26 nepal isolates exhibited 99.8% nucleotide sequence identity and contained the fujian - like amino acid substitutions at positions 155 ( h155 t ) and 156 ( q156h ) in the ha protein ( table 2 ) .
alignment of the 329amino acid ha protein from 26 isolates obtained from this outbreak with the 2004/05 a / wyoming/3/03 vaccine strain and previous h3n2 vaccine strains indicated 4 evident amino acid changes present in most of the isolates ( table 2 ) .
all 4 amino acid changes observed within most of these outbreak isolates are present within a / california/7/04 , a variant strain selected as the h3n2 vaccine strain for the 20052006 influenza season . *
n , asparagine ; t , threonine ; h , histidine ; i , isoleucine ; p , proline ; k , lysine ; s , serine ; v , valine ; q , glutamine . consensus sequence derived from a multiple sequence protein alignment of 26 ha1 hemagglutinin sequences from nepal . of the 26 nepal strains examined ,
24 exhibited a novel lysine - to - asparagine substitution at position 145 in the ha protein ( k145n ) .
this substitution is noteworthy because most strains characterized in 20032004 , including the fujian/411/2002 vaccine strain , contained a lysine ( k ) at this position .
prior to this outbreak , the us department of defense had only observed k145n substitutions in 6 strains obtained from ramstein , germany , ( data not shown ) in june 2004 .
additionally , all 26 nepal sequences exhibited a serine - to - asparagine substitution at position 189 ( s189n ) that had also been observed in the 6 isolates from germany , as well as in a few isolates from asia characterized at the end of the 20032004 influenza season .
two other substitution mutations in the ha1 hemagglutinin , i.e. , valine to isoleucine at position 226 ( v226i ) and serine to proline at position 227 ( s227p ) , were also observed in 24 ( 92% ) and 26 of 26 of the nepal isolates , respectively .
both substitutions differ from most influenza a h3n2 field isolates collected in 20032004 , including the fujian and wyoming vaccine strain for 20042005 ( table 2 ) .
the phylogeny of h3n2 ha proteins indicates a drifting of the nepal isolates from the a / fujian/411/03 and a / wyoming/03/03 vaccine strains and shows that these outbreak isolates have a higher genetic homology to a / wellington/1/04 , a prototype strain selected as the 20052006 southern hemisphere h3 vaccine strain ( figure 2 ) .
the a / wellington/1/04 strain contains 2 of the 4 amino acid changes ( s227p and s189n ) observed in the nepal isolates , but does not contain the k145n and v226i substitutions .
unrooted phylogenetic analysis of ha1 hemagglutinin nucleotide sequences from 26 nepal isolates and h3n2 vaccine and reference strains .
the nepal isolates have drifted from the 20042005 a / fujian/411/03 vaccine strain ( and a / wyoming/03/03 vaccine seed strain ) and are genetically equivalent to a / california/7/04 , the 20052006 northern hemisphere vaccine strain . a k145n substitution ( branch point indicated by the arrow )
was observed in 24 of 26 nepal isolates and represents a genetic marker for the dominant lineage of h3n2 viruses during the 20042005 season .
nucleotide and amino acid sequences for all nepal isolates are available from genbank under accession no .
the asterisk indicates isolates from table 2 that were antigenically distinct from a / wyoming/303 .
three - dimensional views of influenza ha proteins highlighting amino acid changes in a representative nepal isolate and the a / wyoming/3/03 vaccine strains are shown in figure 3a and b , respectively .
the mutation at position 145 ( shown in yellow ) , which is located adjacent to antibody - binding site a and within a known glycosylation site , introduces an asparagine - for - lysine substitution .
this substitution results in a more accessible receptor - binding cleft located directly above residue 145 ( comparing panels a and b ) .
located above the receptor - binding pocket is a serine - to - asparagine change ( shown in green ) that possibly alters the regional surface topography at position 189 within antibody - binding site b. a serine - to - proline mutation at position 227 ( shown in magenta ) appears to marginally affect the ha surface features .
this substitution resides within antibody - binding site d , which corresponds to residues 225228 , which make up the left side of the receptor - binding pocket ( 14 ) .
interestingly , this proline residue is located within a barrel ( a protein motif consisting of an antiparallel sheet domain ) and does not appreciably alter the predicted protein structure , as shown by the absence of any substantial changes in the computer - modeled , 3-dimensional structure compared with the ha1 of a / wyoming/3/2003 .
three - dimensional top view of the ha1 hemagglutinin structures for a ) a representative a / nepal/1648/04 virus and b ) vaccine strain a / wyoming/3/03 .
most ( 24/26 ) of the nepal isolates contain a lysine to asparagine substitution ( shown in yellow ) at position 145 ( k145n ) .
magenta , residues 226 and 227 ; orange , residue 189 ; green , residues 155 and 156 ; yellow , residue 145 .
hemagglutinin molecules were generated by using the respective amino acid sequences with molmol ( 12 ) .
the 4 substitutions described represent a growing lineage of influenza a ( h3n2 ) viruses characterized since july 2004 .
three amino acid changes are confined within known antibody - binding sites , i.e. , the s189n change within antibody - binding site b ( 4,5 ) and the v226i and s227p changes residing in antibody - binding site d ( 4,5 ) . because of rotational restrictions , a proline substitution at position 227 ( s227p ) would typically give rise to considerable conformation change ; however , this particular substitution is located within a barrel motif and therefore has little effect on regional protein conformation . cumulatively , field isolates characterized subsequent to this outbreak continue to exhibit these 4 changes , and they appear to constitute a distinct branch in the phylogeny of ha sequences when compared with h3n2 isolates from the 20032004 season .
. this change may affect protein - protein interactions since it is immediately adjacent to antibody - binding site a , where neutralizing antibodies have been shown to bind ( 4,5 ) .
furthermore , since the k145n substitution is located within a glycosylation site , the charge alteration may affect glycosyl transferase activity , which results in altered glycosylation .
differences in glycosylation have been shown to contribute to antigenic variation by preventing antibody binding to antigenic sites ( 15 ) .
additionally , 3-dimensional analysis suggests this amino acid substitution may also promote enhanced receptor binding since the asparagine r group is shorter , which may make binding requirements less stringent and the receptor cleft more accessible .
the 3-dimensional depiction provides a unique regional residue perspective , demonstrating how the rapidly evolving ha surface antigens in the vaccine strain differ at the molecular level .
collectively , the clinical isolates obtained from this outbreak in nepal can not be considered antigenically distinct from the a / wyoming/3/03 vaccine strain because only 4 of 9 isolates evaluated exhibited 4-fold lower titers by hi ( table 1 ) .
furthermore , the varying reactivity noted in several isolates from this outbreak having identical ha1 sequences is suggestive that other viral antigens aside from the ha1 protein may have contributed to the antigenic variability observed in the hi panel . with the exception of a / nepal/1670/2004 and a / nepal/1672/2004 , all isolates evaluated by hi ( table 1 ) exhibited identical ha1 amino acid sequences and varying antigenicity profiles to a / wyoming/03/2003 reference antisera .
one explanation for this observation is that genetic differences in other influenza surface proteins contribute to the observed immunoreactivity .
alternative viral surface protein candidates include the neuraminidase , ha2 , and m2 glycoproteins , which have been shown to exhibit antigenic properties ( 1619 ) . in this report
, we describe the genetic analysis of the ha proteins from viruses obtained from an early season outbreak and compare them to current vaccine strains .
three amino acids changes ( s189n , i226v , and s227p ) were noted in known ( 4,5 ) antibody - binding sites ( table 2 ) . the fourth change ( k145n ) , which was located within a glycosylation site , may enhance viral binding since the smaller asparagine r group is located close to the ha receptor - binding cleft ( figure 3 ) .
phylogenetic analyses show that the nepal isolates make up a distinct branch in the evolution of h3n2 viruses when they are compared with vaccine and reference strains ( figure 2 ) . however , antigenic data appear more ambiguous , suggesting a multigenic effect that can not solely be attributed to properties of the influenza ha ( table 1 ) .
studies are in progress to characterize the neuraminidase , m2 , and ha2 proteins to determine the molecular basis responsible for antigenicity differences observed within isolates from this outbreak .
the k145n substitution change has become a marker for an increasingly large subset of the fujian - like viruses .
cdc and the us department of defense have recently characterized viruses with the k145n change in singapore , taiwan , china , australia , canada , and the united states . in february 2005 , who reported the emergence of a new influenza h3n2 strain in the united states .
the a / california/7/2004 strain , which was first identified in the united states in september 2004 , contains all 4 changes observed in isolates from this nepalese outbreak .
the a / california/7/2004 strain differs by only 1 amino acid in ha1 ( which is of no immunologic importance ) from most isolates from the outbreak in nepal .
all viruses characterized ( 150 globally isolated strains ) subsequent to the preparation of this report ( march 2005 ) by the us department of defense are genetically similar in amino acid sequence to these nepalese strains ( and the a / california strain ) .
most of the isolates ( 80% ) analyzed by cdc since october 2004 are antigenically related to a / california ( 20 ) , which indicates that this strain has emerged as the dominant influenza a h3n2 strain .
these data indicate that these viruses may persist as the dominant strain at the onset of the 20052006 influenza season . in february 2005 , who recommended inclusion of an a / california/7/2004-like strain in the 20052006 trivalent influenza vaccine to afford immunologic protection from this variant h3n2 virus .
our findings emphasize the importance of continued molecular surveillance for characterizing emerging influenza drift variants .
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worldwide emergence of variant viruses has prompted a change in the 20052006 h3n2 influenza a vaccine strain .
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Materials and Methods
Sample Collection and Antigenic Analysis
Molecular Analysis
Results
Epidemiologic and Laboratory Assessment
Antigenic Analysis
Molecular Analysis
Discussion
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rising levels of obesity are becoming a worldwide phenomenon and are increasingly identified as a health problem across the globe [ 14 ] .
higher weight has been associated with adverse health indicators and outcomes , including cardiovascular disease [ 512 ] , stroke [ 5 , 13 ] , cognitive and functional decline [ 1418 ] , metabolic syndrome [ 19 , 20 ] , inflammation [ 21 , 22 ] , and mortality [ 20 , 2325 ] .
obesity among aging populations is relatively recent and aging among people who have been obese for much of their lives is also a new phenomenon . from 1980 to 2004 , the prevalence of obesity in the us has continued to rise from about 17% to 25% for men aged 5059 . while obesity in england has also increased during this period , from approximately 9% in 1980 to 15% in 2004 for men aged 5564 , the level of obesity remains much lower in england .
additionally , the difference in obesity between the us and england is more pronounced for women .
the level of obesity in us women was about 24% in 1980 and rose to 37% in 2004 ( age 5059 ) ; in england , levels for women were 9% in 1980 and 14% in 2004 ( age 5564 ) .
the aim of this paper is to investigate differences in how obesity relates to indicators of physiological dysregulation in men and women of diverse populations .
this comparison will lead to an improved understanding of how obesity might be differentially related to health and mortality across cultures and lifestyles .
obesity was relatively rare in most populations until the second part of the last century , but it has now become common in many countries
. the us population is recognized as among the most obese in the world , although many other countries are now approaching the us level and most countries are experiencing increasing obesity .
this worldwide obesity epidemic began with the epidemiological revolution and the virtual elimination of infectious disease ; the decline in manual labor needed to provide sustenance with the industrial revolution ; and the increasing availability and decreasing cost of food [ 2729 ] .
obesity reflects some combination of calorie intake , diet content , and amount of physical activity . in some cultures ,
lack of physical activity can be a more important determinant of obesity ; in other cultures , overeating or food composition may be the more important determinant of obesity .
it is also true that within countries , individuals could differ in the causes of obesity .
for instance , changes in activity might be more characteristic of women or men resulting in different reasons for obesity by gender .
these differences may affect how obesity is related to other risk factors for poor health , and it may determine the overall health risk associated with obesity . if physical activity is maintained , the overall effect of obesity may be less than if the activity is not .
one indication of the cause of obesity may be the relationship between waist size and weight [ 30 , 31 ] . in societies where obese people are more physically active , waist size of the obese
waist circumference has also been linked to late life mortality , where high waist circumference has been associated with increased mortality among men and women in the netherlands , while high bmi was not associated with mortality .
this paper builds upon current obesity research by using both bmi and waist circumference to quantify obesity in order to determine how a combined indicator of weight and adiposity is related to physiological dysregulation in populations with different cultures , diets , behaviors , and epidemiological histories .
obesity has been related to many indicators of physiological dysregulation including cardiovascular risk factors such as hypertension and metabolic dysregulation in lipid levels or insulin resistance .
most studies that investigate the differences in biological risk associated with excess weight have examined western populations [ 33 , 36 ] .
comparative studies on the health risks associated with obesity that examined the us and england reported that obese americans had an increased risk of diabetes and a higher waist circumference [ 37 , 38 ] .
these studies suggested that differences in physical activity , diet , and social environments may explain these national differences .
while these differences have been observed between the us and england , these two western countries have roughly similar life expectancy , levels of living , history , and culture even while the us has poorer health by a number of indicators of disease prevalence and biological risk [ 36 , 39 ] .
comparative studies of the links between obesity and health outcomes and risk factors for obesity comparing western and non - western countries indicate important differences in the causes and consequences of obesity .
a comparison of the association of disease with overweight and obesity in japan and the us indicated that the associations were stronger in the us than in japanese women and that there was no association in japanese men .
links between social , demographic , and behavioral risk factors for obesity also differ markedly in japan , korea , and the us .
the availability of biomarker data from taiwan a middle - income country undergoing rapid economic growth , increasing obesity , and with life expectancy recently increasing to levels similar to that of the us and england allows for investigation of the biological risk associated with obesity in a population characterized by very different cultural , behavioral , socioeconomic , and dietary parameters .
we examine how elevated weight and obesity ( using an indicator that considers both bmi and waist circumference ) relate to having levels defined as clinical risk for cardiovascular , metabolic , and inflammatory markers in three aging societies that are now relatively similar in life expectancy but that differ in the timing of the epidemiological transition and obesity epidemic , history of economic development , socioeconomic levels , general lifestyle habits , health behaviors , and health care systems : the us , england , and taiwan .
finding differences in the relationship between obesity and indicators of physiological dysregulation in these three aging societies will clarify whether increases in weight gain are equally problematic across all countries .
we use data from three nationally representative samples : the us national health and nutrition examination survey ( nhanes , 20032006 ; n = 3855 ) , the english longitudinal study of ageing ( elsa , 2004 - 2005 ; n = 9139 ) , and the social environment and biomarkers of aging study ( sebas ) in taiwan ( 2000 ; n = 1023 ) .
these surveys collect information on demographics , as well as anthropometric , physical , and biological measures .
every year , approximately 5,000 individuals undergo detailed interviews and medical examinations , which include collection of several physiological measures .
nhanes utilizes a complex sampling design , and when weights are applied , the sample is representative of the noninstitutionalized american population .
we use the 20032006 data since nhanes data is released in two - year intervals , and this sample is centered on 2004 - 2005 , which matches the period in which elsa was collected .
for nhanes , we use individual - level data based on a sample of 1,513 fasting individuals aged 54 and older .
elsa includes participants drawn from households responding to the health survey for england ( hse ) in 1998 , 1999 , and 2001 and is representative of the english population aged 50 and older .
the core elsa sample ( wave 1 : 2002 - 2003 ) includes people living in an hse responding household who were born prior to march 1 , 1952 , and their partners who could be under age 50 . wave 4 of elsa ( 2008 - 2009 ) , which includes a nurse visit , includes wave 1 core members , if they are still alive and do not refuse further contact after the first interview at wave 1 .
it also includes a refresher sample to maintain the age structure of the sample ( in waves 3 and 4 ) , and their partners . for elsa , we use individual - level data based on a sample of 7,384 individuals aged 54 and older .
sebas is drawn from a follow - up survey of the survey of health and living status of the near elderly and elderly in taiwan ( also known as the taiwan longitudinal study of aging ( tlsa ) ) , a nationally representative survey of taiwanese adults ( including institutionalized individuals ) collected in 1989 , 1993 , 1996 , 1999 , and 2000 . in 2000 , a subsample of individuals was randomly selected for inclusion in sebas .
sebas consists of adults aged 54 and older in 2000 , with in - home interviews and medical exams taken in a hospital . for sebas ,
the sample averages 66.8 years of age in england , and the us and taiwan mean age is about the same ( table 1 ) .
there are more men in taiwan ( 56% ) and england ( 53% ) and fewer in the us ( 44% ) .
we examine the following indicators of physiological dysregulation often associated with obesity and also associated with increased risk for multiple adverse health outcomes and obesity [ 43 , 44 ] : ( 1 ) cardiovascular markers : high systolic ( sbp ) and diastolic blood pressure ( dbp ) ; ( 2 ) metabolic markers : high levels of blood lipids ( total and low - density lipoprotein ( ldl ) cholesterol , and fasting triglycerides ) , low levels of high - density lipoprotein ( hdl ) cholesterol , and high fasting glucose and glycated hemoglobin ; ( 3 ) high levels of inflammatory markers c - reactive protein ( crp ; available in nhanes and elsa ) and interleukin-6 ( il-6 ; available in sebas ) , as crp and il-6 have been positively associated with bmi . for each indicator
we use clinical cutpoints or widely used research - based cutpoints to indicate high levels of risk which are shown in table 1 [ 39 , 43 , 45 ] .
there has been debate as to the best indicator of obesity : body mass index ( bmi ) or waist circumference .
waist circumference is thought to be a better measure of abdominal adiposity than bmi and a better indicator of risk of poor health outcomes , including all - cause and cardiovascular mortality [ 46 , 47 ] . for this reason
we investigate the association between bmi and biomarkers across categories of bmi ( underweight < 18.5 kg / m , normal and overweight 18.529.9 kg / m , obese 3034.9 kg / m , and very obese 35 kg / m ) and waist circumference categorized as normal or high waist ( high waist : men 120 cm , women 88 cm ) . we create a composite measure of obesity and adiposity by categorizing individuals into five groups : ( 1 ) underweight and normal waist ( all underweight individuals had a normal waist circumference ) , ( 2 ) normal / overweight bmi ( termed normal bmi ) and normal waist circumference ( reference group ) , ( 3 ) normal / overweight bmi ( termed normal bmi ) and high waist circumference ( termed high waist ) , ( 4 ) obese and high waist ( all obese individuals had a high waist circumference ) , and ( 5 ) very obese .
we also evaluate an alternate definition for obesity in taiwan based on bmi 27 , as it has been suggested by some that obesity levels should be differentially defined for asians [ 48 , 49 ] .
a similar composite measure of obesity and adiposity was calculated using this alternate definition of bmi in taiwan . because these risk factors are all assumed to be associated with obesity and because dysregulation in multiple physiological systems has been shown to predict many of the poor health risk outcomes associated with aging , we also create two summary measures of risk based on the total number of at - risk levels of biomarkers , either 9 or 8 . because crp values for sebas are not available , this measure
is not included in the 9-item summary measure for taiwan , but a summary measure ( range 09 ) was calculated for taiwan using il-6 , another indicator of inflammation , instead of the crp values included for the us and england . a second alternate summary measure of biological risk , excluding the inflammatory marker ( range 08 ) ,
biological risk summary scores were computed for individuals who had missing values on no more than 3 biological markers .
we examine multiple covariates in our investigation of the relationship between obesity and biological risk .
self - reported use of antihypertensives was determined in all three countries , and use of lipid - lowering statins was only asked in the us sample .
dichotomous variables were created to indicate whether the respondent reported being a current smoker and participating in at least moderate physical activity for exercise ( e.g. , brisk walking , running , or swimming ) in the past 30 days ( for the us and england ) or generally exercising once a week ( for taiwan ) .
we use logistic regression models to determine the odds of having at - risk levels of a specific biomarker for obese men and women among the three populations . for all countries ,
the comparison group for bmi and waist circumference is the normal bmi and normal waist group .
the regressions included indicators of age , use of antihypertensives , current smoking status , and having exercised in the past 30 days .
ordinary least squares ( ols ) regression models were used to determine the relationship between the summary measures of biological risk and the composite measure of obesity and adiposity .
we begin by examining national differences in the high risk levels of individual biomarkers ( table 1 ) .
low levels or high risk levels of hdl cholesterol are also more common in taiwan .
high total and ldl cholesterol is more common among the english ; lower levels of plasma glucose , crp , and glycated hemoglobin are also characteristic of the english .
few adults in england have elevated levels of fasting glucose ( 2.2% ) , while this is observed in 17.3% and 13.2% of american and taiwanese adults .
most people in this age range are in the normal to overweight category ( 64.7% , 67.2% , and 89.4% in the us , england , and taiwan , respectively ) ( table 2(a ) ) .
americans are more likely to be obese ( 33.7% ) compared to the english ( 32.1% ) and taiwanese ( 7.2% ) . among the obese , americans are much more likely to be very obese : 13.4% of the total us sample , 10.1% in england , and about 1% in taiwan .
both among the obese and very obese , the average bmi is higher in the us and england compared to taiwan .
few are underweight in any country ( 1.7% , 0.8% , and 3.4% in the us , england , and taiwan , resp . ) .
when we examine waist circumference , the us has the highest average waist circumference , with 65.5% of americans , 55.9% of english , and 15.8% of taiwanese having a high waist size ( table 2(a ) ) .
this means that high waist characterizes a substantial number of those who would be categorized as normal weight in the us and england . among those in the normal and overweight group about half ( 49.5% ) of americans and a third ( 36.4% ) of the english
almost all obese individuals have a high waist in the us and england ( 98.3% in the us and 96.5% in england ) , but only 78.4% of the obese in taiwan also have a high waist . when we use the alternate obese cutpoint of 27 kg / m in taiwan , less than half of the obese individuals have a high waist ( not shown here ) .
americans exhibit the highest proportion of the older population taking antihypertensive medication ( 47.1% ) ( table 1 ) .
the percentage who reports taking antihypertensives is lower in england ( 32.0% ) and taiwan ( 28.6% ) .
americans are more likely to be current smokers ( 24.5% ) than persons in taiwan ( 22.5% ) and england ( 13.9% ) .
more than half of the population in all countries report having exercised in the past 30 days , with more english exercising ( 82.2% ) compared to taiwan ( 61.4% ) and the us ( 58.5% ) .
men with normal bmi and high waist have a greater likelihood than men with normal bmi and normal waist size of having high - risk levels of triglycerides in all three countries . in the us and england ,
men with high waist are more likely to have high levels of glycosylated hemoglobin and higher crp ; fasting glucose is also elevated among this group in the us ( table 3(a ) ) .
men who are obese in the us have fewer elevated risk factors than those with high waist who are not obese ; in the us , obese men are only more likely than normal weight men without high waist to have elevated glycated hemoglobin , fasting glucose and crp .
english men who are obese have more elevated risk : both blood pressure indicators , hdl cholesterol , triglycerides , glycated hemoglobin , and crp .
very obese men in england have the same elevated risk factors with the exception of dbp .
very obese men in the us are more likely to have elevated fasting glucose in addition to crp and glycated hemoglobin .
english and taiwanese women with normal weight and high waist are more likely to have elevated sbp , dbp , and glycosylated hemoglobin ; only british women with higher waist have significantly elevated triglycerides and only the taiwanese women had more hdl risk .
high risk crp is more common among both american and english women with normal bmi and high waist , and this risk of elevated crp is also higher in the obese and very obese ( table 3(b ) ) .
obese women in britain had elevations in the same markers as normal bmi and high waist english women , while obese women in the us only have elevated fasting glucose , glycated hemoglobin , and crp ; obese women in taiwan only had high dbp . with the exception of taiwan , levels of the inflammatory markers ( crp in the us and england ; il-6 in taiwan ) are more likely to be elevated among persons with a high waist and normal bmi , obese or very obese , compared to their normal bmi and normal waist counterparts . among men ,
an increase in the biological risk summary score ( range 09 ) is associated with having a high waist relative to being of normal bmi and normal waist in all three countries ( table 4(a ) ) .
being obese or very obese is also related to a higher biological risk summary score ( 09 ) for men in the us and england .
these equations explain 6 to 11 percent of the variance in the summary indicator of biological risk .
these relationships are similar for women ( table 4(b ) ) , with one exception : obese taiwanese women do not have a significantly increased biological risk compared to their normal weight and normal waist counterparts .
when we consider the alternate summary score that excludes our indicators of inflammation ( range 08 ) , being obese or very obese is no longer associated with a higher biological risk summary score in us men compared to men with a normal bmi and normal waist when controls for health behaviors and medication use are included ( table 5(a ) ) .
the size of the effects of the obesity categories is reduced on the 8-indicator summary measure in both england and the us , indicating the strong link between crp and obesity .
the r is also reduced in these equations for england and the us . for taiwan ,
women in england and taiwan , but not women in the us , with a higher waist but who are not obese have significantly higher physiological dysregulation compared to their normal bmi and normal waist counterparts .
obese women in all three countries have elevated risk and the very obese have even higher risk ( table 5(b ) ) . the alternate biological risk summary measure ( 08 )
yields different relationships between weight and physiological dysregulation in the us and taiwan . in the us ,
only very obese women have a higher alternate biological risk summary score ( 08 ) . in taiwan , obese and normal bmi and high waist women exhibit higher alternate biological risk summary scores compared to their normal bmi and normal waist counterparts , except when smoking status , physical activity , and use of hypertensives are included . figures 1(a ) and 1(b ) illustrate the predicted alternate biological risk score ( 08 ) for each weight category for men and women ( respectively ) aged 65 who are nonsmokers , do not engage in physical activity , and are currently taking antihypertensive medication .
this figure allows for country comparisons of individuals with these characteristics within each weight category and across weight categories .
the predicted values indicate that the us has the highest biological risk score within each respective weight category among men and women of the same age and lifestyle behaviors . with the exception of women in the normal bmi and high waist group ,
england has the second highest biological risk score within each weight category , followed by taiwan . among 65-year - old women with the noted lifestyle behaviors and with a normal bmi and high waist
when we consider the alternate bmi cutpoint for obesity in taiwan ( bmi 27 kg / m ) , our findings for the individual biomarkers and summary measures of biological risk are similar to using the bmi 30 kg / m cutoff for taiwan except that the category normal weight with high waist no longer differs from the omitted category ( results not shown ) .
this study observes three general findings about how biological risk is associated with obesity in three countries that differ in lifestyle and culture .
first , obesity is associated with physiological dysregulation in all countries with differences in the links between specific indicators of biological risk and obesity .
generally , obesity in england is associated with hypertension , dyslipidemia , and elevated glycated hemoglobin ; americans who are obese are not more likely to have hypertension . in taiwan ,
obese women are more likely to have elevated dbp and obese men have an increased risk of elevated triglycerides and glycated hemoglobin compared to their nonobese , normal waist counterparts .
our biological risk summary scores indicate that at all levels of weight physiological dysregulation was highest in the us , followed by england ( with one exception ) , with taiwanese exhibiting the lowest biological risk in all groups among the three countries .
second , these relationships remain after controlling for demographic factors , participation in physical activity , and other behavioral factors .
third , similar to obese older adults , high waist individuals with normal bmi also exhibit greater physiological dysregulation in all countries compared to their normal bmi and normal waist counterparts .
our finding of a higher physiological dysregulation , as shown by the alternate biological risk summary score , in taiwan compared to the us and england could be due to a couple of potential explanations .
the prevalence of obesity in the us and england is much higher than in taiwan , indicating an earlier initial rise in obesity relative to taiwan . from 1978 to 2002 , the proportion of obese americans and britons exhibited stark increases ( 1332% and 623% for men and women , resp . ) .
the estimates for obesity prevalence in taiwan indicate a recent increase for men but not women . from 19931996 to 2000 - 2001 , the age - adjusted prevalence of obesity rose from 10.5% to 15.9% for men and declined from 13.2% to 10.7% in women .
it may be that the lower levels of risk among older adults who have lived longer years with obesity could be a reflection of better pharmacologic control of physiological dysregulation ( e.g. , through statin use ) , which may in turn confer less biological risk in these populations compared to populations of currently obese taiwanese adults who may have more recently begun living with obesity .
a second reason for the observed country differences in obesity may be due to differences in dietary habits and lifestyle .
the us and england are two modern , western populations whose diets have been influenced by increased industrialization and have over time come to be characterized by high glycemic loads and high fatty acid composition .
taiwan , on the other hand , represents a country that has experienced the effects of the industrial and scientific revolutions later than that of the us and england but is currently rapidly undergoing economic development and demographic change .
the recent economic changes in taiwan may indicate that obese older adults in taiwan have more recently begun to consume high - fat diets , which could result in greater initial physiological dysregulation associated with access to western - influenced dietary habits . despite controlling for lifestyle behaviors thought to be linked with health , the country differences in obesity and physiological regulation remain .
moreover , the consideration of antihypertensives does not alter our substantive conclusions on these associations .
this suggests that despite the greater use of medications to treat hypertension in the us , obesity among americans is associated with greater overall biological risk than the other two countries .
this is supported by findings from the general population of americans relative to england , which report that the us is faced with greater health disadvantages than england in adulthood and across the life span .
we also note differences in biological profiles of obese individuals between the two westernized countries : the us and england . the excess risk of hypertension associated with obesity in england was not found in the us .
these differences may be due to the higher use of medications among americans compared to the english , with about 16% more men and 18% more women in the us aged 65 + taking antihypertensive medication compared to their british counterparts .
the greater use of hypertensive medications in the us is also noted when compared to japan and countries across europe .
two notable differences in country patterns of the relationship between obesity and physiological dysregulation by sex are found . among men in england and taiwan ,
the order of magnitude of physiological dysregulation increases with higher weight categories ; however , this is not observed for us men .
this difference may be due to our inability to consider statin use in england and taiwan , which may be particularly important in the relationship between obesity and physiological dysregulation for men .
conversely , the importance of considering statin use may be less vital to understanding the country differences in the association between obesity and physiological dysregulation among women , given that the relationship for women is more consistent across countries , namely in the us and england .
women , underweight corresponds with higher biological risk ( though nonsignificant ) compared to women with normal bmi and normal waist .
underweight among men in taiwan is significantly associated with much lower biological risk than their normal bmi and normal weight counterparts .
further studies will be required to explore possible explanations for these differences in physiological dysregulation .
the higher biological risk observed among normal bmi and high waist individuals relative to normal bmi and normal waist older adults builds upon previous studies that report on alternate indicators of body shape , which vary across countries .
the importance of waist circumference is underscored by our current study , as well as a growing body of literature on the predictive value of waist circumference on indicators of health .
higher rates of diabetes among older americans compared to britons have been accounted for by high waist circumference as opposed to bmi differences . additionally , increasing waist circumference is more predictive of greater risk of incident diabetes than bmi in middle - aged british men ( and the european prospective investigation into cancer and nutrition ( epic)-potsdam study ) .
waist circumference , as an indicator of central fat mass , is thought to be more strongly associated with disease risk , and in our case with physiological dysregulation , compared to bmi , which is considered a cruder index of adiposity .
banks and colleagues cite differences in physical activity , diet and greater psychosocial environmental challenges in america compared to england as potential mechanisms linking central adiposity and type 2 diabetes .
our study considers some of these possible mechanisms ( e.g. , physical activity and antihypertensive use ) but finds that they explain little of the relationship between biological risk and adiposity among the three countries .
together , these results highlight the importance of considering waist circumference in investigating the links between indicators of health and adiposity .
our finding of the biological risks associated with obesity among older taiwanese adults underscores the growing concern for risks associated with obesity in countries rapidly undergoing modernization . in comparing the biological risk of obese individuals among the three countries , we are able to use these international comparisons to our advantage to examine how differences in modernization influence the health of older adults in different populations
this may have potential health policy implications that underscore the importance of addressing and controlling the rising obesity epidemic that has become most widespread in countries , like the us and england , that have long experienced high economic growth and in countries currently undergoing rapid economic development .
the increasing use of biological information to inform our understanding of health represents an innovative method in biodemography that will further contribute to the testing of current comparative theory and the potential creation of new paradigms surrounding the influence of modernization on health .
first is the use of a broad range of biological markers across three large - scale population surveys . the inclusion of biological information as objective precursors of health allows , to some extent , a fairly comparable comparison of indicators of health across the different populations .
an exception to this uniform comparison of biomarkers across the three surveys is our use of inflammatory marker crp in the us and england and our inclusion of a different marker of inflammation ( il-6 ) in taiwan . of note , crp seems to be more strongly associated with obesity than il-6 .
a growing body of literature has made distinctions between bmi and waist circumference , namely , suggesting that waist circumference is a better indicator of abdominal obesity , which in turn has been associated with obesity - related health risks .
our findings generally report a similar association between increased biological risk and ( 1 ) normal bmi and high waist and ( 2 ) obese and high waist .
using the us nhanes , reported that when both waist circumference and bmi were included in their analyses , only waist circumference was a significant predictor of comorbidity . although this and other studies have suggested that waist circumference may be a better indicator of obesity and risk for adverse health outcomes , our study finds the two indicators to be similarly associated with biological risk across the three countries .
first , we examine population - based data from three countries at a single time point .
future studies of longitudinal data will allow for further investigations of the potential role of obesity on biological risk observed in the current associations .
second , we do not have measures of some lifestyle and medical behaviors for some of the datasets ( e.g. , statin use ) , which likely influence the relationship between obesity and biological risk .
as such , we are unable to include such factors in our analyses of all three countries .
it is possible that these lifestyle behaviors are key explanatory factors to the noted cross - country differences in obesity - related biological risk .
the cross - country differences in the relationship between increased biological risk for individuals who are obese and have a high waist underscore potential differences in health and lifestyle behaviors .
these behaviors may be a result of country differences in economic development that we are not able to observe in this study .
the country differences in the links between obesity and physiological dysregulation are particularly marked when comparing obesity among taiwanese older adults relative to westernized populations , such as the us and england .
further examination of these relationships over time and across other countries will contribute to our understanding of the potential factors responsible for these country - specific variations in biological risk , as obesity becomes increasingly more prevalent and older adults in various countries live more years with obesity and increased adiposity .
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excess weight has generally been associated with adverse health outcomes ; however , the link between overweight and health outcomes may vary with socioeconomic , cultural , and epidemiological conditions .
we examine associations of weight with indicators of biological risk in three nationally representative populations : the us national health and nutrition examination survey , the english longitudinal study of ageing , and the social environment and biomarkers of aging study in taiwan .
indicators of biological risk were compared for obese ( defined using body mass index ( bmi ) and waist circumference ) and normal weight individuals aged 54 + . generally , obesity in england
was associated with elevated risk for more markers examined ; obese americans also had elevated risks except that they did not have elevated blood pressure ( bp ) . including waist circumference in our consideration of bmi indicated different links between obesity and waist size across countries ; we found higher physiological dysregulation among those with high waist but normal bmi compared to those with normal waist and normal bmi .
americans had the highest levels of biological risk in all weight / waist groups .
cross - country variation in biological risk associated with obesity may reflect differences in health behaviors , lifestyle , medication use , and culture .
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1. Introduction
2. Methods
3. Results
4. Discussion
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medical tourism is illustrated as occurrence in which individuals travel abroad to receive healthcare services ( 1 ) .
it is a multi - billion dollar industry and countries like india , thailand , singapore , malaysia , belgium , costa rica , cuba , dubai , hungary , israel , jordan , south africa and many others are being benefited in their economy by this recent phenomenon ( 2 , 3 ) .
the prime driving factors in medical tourism are increased medical costs , increased insurance premiums , increasing number of uninsured or partially insured individuals in developed countries , long waiting lists for procedures in countries having public healthcare system , availability of high quality services at affordable price , and cheaper airfare . increased communication and internet access in developing countries
are other supporting factors , which help patients to develop awareness about international travel for medical care ( 47 ) .
estimations can vary but still some of reliable sources claim ; gross medical tourism revenue worldwide was more than us$ 40 billion in year 2004 and reached up to us$ 100 billion by year 2012 ( 8) . in year 2007
, it was estimated that around 750,000 us residents traveled abroad . accordingly , the base case estimated form 2007 to 2010 for the annual growth rate for outbound patients was 100 per cent in us ( 9 ) .
forbes business estimated around 1.25 million americans were expected to travel outside for medical treatment in year 2014 ( 10 ) .
after thorough review it was found that there is huge gap in literature available in medical tourism .
accounts detailing size and market of the industry are large in number yet there is scarcity of literature detailing about role and significance of different variables and linking them to form conceptual and theoretical framework which reflects decision making of international patients for taking part in medical tourism .
available literature explore various factors related to patients travel such as source of information , perceived risks , benefits of medical tourism and attributes of medical tourism destinations in sufficient amount but almost all of these related accounts are exploratory in nature and do not provide conceptual frameworks for testing .
many researchers ( 2 , 6 , 11 , 12 ) made calls to explore more about the decision making process based on conceptual models and test them empirically , so managers in medical tourism industry will be more acquainted about the needs and requirements of patients and design their future strategies accordingly and at the same time patients will also be benefited by receiving more quality services from the providers .
this article will draw a conceptual model based on patients source of information , perceived benefits , perceived risks , and medical tourism destination image with available literature that will be helpful to mangers to draw their future course of action for competitive advantage and at the same time fulfill the gap in available literature .
image formation agents are the factors which control the perception and evaluation of image ( 13 ) . researchers addressed amount and diversity of information sources that expose individuals including information related to destination acquired through visiting particular place .
various studies performed on destination selection behavior of tourists explored that with combination of other different factors , information sources explored by individuals determined certain destinations as possible alternatives ( 1317 ) .
consumer behavior studies have already established the effects of source of information on purchase behavior ( 18 ) .
amount and type of different information sources directly influence in development of cognitive image formation of destination ( 19 ) .
source of information is a vital antecedent of destination image formation and destination choice intention ( 20 ) .
there are four ( i ) professional advice ( tour operators , travel agents , airlines ) ( ii ) word of mouth ( friends , relatives , social media ) ( iii ) advertisement , and ( iv ) news / books / movies , different categories of type of information sources usually consider responsible for destination image formation ( 13 ) , this article use these four categories of source of information for medical tourism destinations image formation .
medical tourism facilitators are specialized in promotion of medical services abroad and offer supportive services such as assisting in selection of country and hospital , correspondence with doctors , travel arrangements , and arrangements of required paper work ( 21 , 22 ) .
judgment of agent related to travel of their clients has high influence on decisions of clients ( 23 ) .
these facilitators serve as motivators also due to providing much - needed assistant to those unenthusiastic potential international patients who do not want to make their trip arrangements by their own .
many hospitals and clinics linked themselves with airlines to promote their services and offer discounts ( 2 , 5 , 2426 ) .
literature on medical tourism also reveals the role of practitioner in promotion of medical tourism mainly in underdeveloped countries due to various reasons such as lack of resources , unavailability of equipment , unavailability of infra - structure , unavailability of specialized manpower , and unavailability of medication ( 2730 ) .
word of mouth and recommendation also has high influence in decision making of international patients in selection of destinations , hospitals , and doctors .
studies revealed large numbers of patients visit different countries for medical services were received recommendations from family members , friends , relatives , and colleagues ( 3134 ) .
websites and online forums created by experienced international patients to share their experiences with medical tourism are also considered as decisive source of information for those planning to take medical services abroad ( 35 , 36 ) .
printed materials are also used to promote the services by hospitals and clinics , for example air mauritius in - flight magazine provides details about procedures and services provided by the hair grafting clinics in mauritius ( 2 ) .
major medium of promotion of services by destination countries are trade fairs , travel markets / travel fairs , exhibitions , seminars and conferences to make potential patients informed about products and services offered by destinations .
some of these fairs and exhibitions are organized in collaboration with government agencies like tat , ministry of foreign affairs and department of export promotion but some providers organized these events by their own in cooperation with local institutes , medical schools and universities ( 37 ) .
print media play an important role in promotion and advertising medical tourism in major source countries , publish attractive and evidence based stories in their different segments related to health and travel .
los angeles time first examined about the growth of medical tourism and marked it as trend . later on new york times and los angeles times published stories of satisfied patients .
fox , cbs s 60 minutes and cnn aired their segments on patients traveling to medical services .
magazines like forbes and wall street journal analyzed the business aspect of medical tourism as brokers assertively marketed medical tourism to consumers , employers , and insurers ( 38 ) .
literature revealed that not every physical attribute of a destination has influence on image formation process .
there is substantial inequality between descriptive dimensions of image and the attributes which are considered important for decision making within individuals ( 39 ) .
it is generally presumed in marketing that products with similar characteristics will be equally preferred by the consumers , however , attributes , which make the product similar to other products , will not be necessarily same at the time of actual purchase . the importance of attributes will be change according to the need of consumer ( 40 ) . wish ( 1971 ) cited in ( 40 ) found that despite have many similarities individual like one country and dislike another one .
this is due to dimensions of liking may not be agree with the dimensions of similarity .
literature revealed that many researchers made distinction between physical and beneficial aspect of a product .
few researchers explained it as characteristic and benefits to the physical and beneficial aspect of product respectively ( 41 ) .
the typology of different attributes of product has been proven fruitful because by this a product s features can be segments into three groups as characteristic , beneficial , and imagery .
there are three components of vacation destination image formation ; ( i ) based on awareness : rely on the information sources , tourist believes about what a destination possesses , ( ii ) based on attitude : feelings and beliefs about destination , and ( iii ) based on expectations : expected benefits obtain from a tourist product ( 43 ) .
the above mention discussion clearly indicates that process of image formation is not just emphasis on physical attributes of destination but also depends on benefits or consumption values of product or service consumers hold in their minds .
benefits offered by a product or service are considered as consumption value of the same ( 44 ) .
sheth , newman and gross ( 1991 ) developed a theory known as theory of consumption values , which focuses on consumption values of products and services ( 45 ) .
conditional value. the decision making of consumer choice behaviour can be based on all five or any of the five consumption values .
consumption value model define the characteristics of functional value as price , credibility , and durability .
medical tourism destinations offer very low price for medical services to international patients in comparison to developed countries , and most of the time price of a procedure in india and other asian destination is equal to 1/10 of price in us or european countries ( 4 , 4648 ) .
quality of medical services is one of the aspects which drive medical tourism high towards success .
most of the hospitals in india provide medical care services to international patients are certified by jci which has reputation for hospital safety and regulatory management and recognized worldwide ( 49 ) .
high procedure success rate also forms image as quality medical tourism destination of various destinations at south asian , african and latin america ( 48 , 49 ) .
although availability of literature explain social value in medical tourism is less , little available literature clearly define the
social value aspect has been clearly observed in yemeni patients travel abroad for treatment ( 27 , 50 ) .
senior male members of the household consider it as opportunity to increase their reputation in society . availing expensive and prestigious medical care service in foreign countries and the associated sacrifices with it would make stories full of pride to tell to others ( 51 ) .
most of the citizens of yemen do not obtain resources to go for leisure travel at different parts of the world therefore traveling to foreign country for medical services provide chance to explore new territory , meet new people , and experience different social culture ( 27 , 50 ) .
however , the concept of social value and epistemic value may not be associated with patients from developed regions .
many researchers found in their studies that patients were highly motivated to travel to a particular destination due to emotional attachment with doctor , hospitals or destination ( 6 , 50 ) .
such as , many omani patients visit shiraz in iran for treatment due to religious and cultural familiarity ( 52 ) .
conditional value can be considered as major factor pushes patients to travel abroad because medical needs are crucial and considered as unavoidable so have great conditional value .
perceived risks is defined as perception of an individual about the probability that a particular action will lead them to a situation exposed with danger more than acceptable limit , and will lead to influence travel decision - making ( 53 ) . security and safety at the destination is major issue of concern by the potential travelers . in travel decision ,
making perception of risks has utmost importance due to its tendency to alter destination selection ( 54 ) .
researchers argued in favor of conducting study on perceived risks and destination image together ( 55 ) . in tourism literature , authors considered safety and security at destination as one of the pull factors caused destination image formation ( 13 , 14 , 56 ) .
however , in general cognitive image of destination does not engage with varied range of travel specific risks at destination and consider safety and risks as one of the many other attributes associated with destination ( 54 ) .
in addition , at the same time perceived risks are considered as potential inhibitors of travel ( 57 ) .
researchers argued that using cognitive image to understand the dimensions of travel risks will be a conceptual mistake ( 58 ) .
hence , perceived risks and cognitive image should consider individual variables for image related studies in travel .
credence goods as the quality of these good can not be assessed accurately even after consumption .
patients may vulnerable to many risks if consider to take medical services at medical tourism destinations .
researchers described six dimension of risks associated with health travelers visited israel as human - induced risks ,
financial risks , service quality risks , socio - psychological risk , natural disasters and car accident risk , and
after thorough literature review , authors categorized medical tourism destination perceived risks into three categories , as physical - health related risks , service related risks , and destination related risks. physical risks are considered as possibility of physical danger or injury detrimental to health where as health risks are considered as possibility to becoming sick while traveling or at destination ( 54 ) .
in medical tourism , international patients can be exposed with different diseases , so risk of contracting with a different kind of disease is even higher such as blood borne infection and infection due to improper screening and storage of blood , deep - vein thrombosis ( dvt ) to those patients returning home after surgery , language related risks and lose of money can also be a matter of concern while involved in medical tourism .
health risks for patients travel for organ transplant is even higher due to ignorance of standard protocol in donor selection ( 12 , 6062 ) .
literature revealed few american patients came in contact with non - tuberculous mycobecterial infection while taking treatment in hospitals abroad ( 63 , 64 ) .
researchers consider terrorism and kidnapping of rich patients and unstable economy of the destination as other risks associated with medical related travel .
apart from physical - health and service related risks , every destination have its own associated risks ; risks of terrorism , crime , and personal safety are associated with medical tourism destination due to falling in middle and lower middle - income countries in development status ( 63 ) . according to researchers , destination image is the total of ideas , beliefs , and impressions individuals possess about the attributes of destination and or activities available at a destination after processing information from various sources over a period of time ( 39 , 65 ) .
destination image as overall imagery picture individual obtains in his mind ( 66 ) . in the early studies on travel
, researchers used the concept of stereotypic image of a travel destination ( 67 , 68 ) . later on , to understand the destination image formation process researchers developed an alternative framework and argued destination image consist of two components i.e. attribute based and holistic ( 66 ) .
attribute based component refers to the perception of individual based on destination features and holistic component refers to imaginary mental image of destination .
later on , a bi - dimensional model was presented by researchers to represent destination image , consist of cognitive and affective image component ( 13 , 69 ) .
the cognitive component of destination image develops on knowledge and beliefs of destination based on tangible attributes , whereas affective component of image develops on emotions and feelings about the destination ( 70 , 71 ) .
researchers further studied affective image of destination and illustrate a four semantic differential scale to evaluate the affective component of destination image based on arousing
furthermore , cognitive component of destination image is an antecedent of affective component ( 72 ) .
thus , a distinctive image of a destination forms in tourists mind based on strength and weakness of attributes of cognitive and affective components . image of destination formed in consumer s mind is defined as aggregate of attributes and beliefs ( 73 ) .
it means if tourist has enough level of positive beliefs about attributes of destination it is expected that s / he has developed favorable attitude towards destination .
the process of destination image formation is described as mental construct of representation of destination based on information cues transmitted by image inducing agents chosen by individual ( 19 ) .
after thorough literature review authors identified four dimension of cognitive medical tourism destination image based of different attributes projected by different medical tourism destinations , as ; medical amenities , general infra - structure , tourism attractions , and
social environment. most of the hospitals websites are dominated with images stressing on sophistication , advanced technology , cleanliness and efficiency .
majority of these sites are in english highlight the variety of procedures , price , accreditation of hospitals , and affiliation of doctors , qualified and smart staff , lavishly furnished accommodations , and testimonials of past patients , and efficiency with different languages of staff .
command on technology is rarely missed in any form of marketing ( 12 , 46 , 74 , 75 ) .
based on mentioned qualities , medical tourism destinations project themselves as most suited destination for potential travelers ( 28 , 32 , 76 ) . to nullify the quality related concerns of international patients hospitals and medical tourism facilitators / brokers emphasis on the markers of quality services .
physicians trainings in world reputed institutes like national institute for health , johns hopkins university , university of birmingham , and other reputed universities are highlighted in the profiles of physicians .
prominent display of training and expertise of doctors achieve two goals ; established trustworthiness and mitigate concerns related to risk .
the accreditation awarded by jci is promoted by hospitals as mark of offering medical care of american standards ( 2 , 28 , 77 ) .
collaboration with prestigious hospitals in us and europe in also indicate seriousness towards providing quality services to patients by medical tourism hospitals at different destination ( 21 , 78 ) . distinguishing that major market drivers such as lack of health insurance and unaffordability are influencing patients to take procedures abroad , websites of brokerages predominantly display the comparative cost charts and price schedules .
most of the destinations claim providing significant cost services through different sources of promotion ( 5 , 79 , 80 ) .
most the promotions and advertisements show smiling , well - dressed and empathetic medical staff taking care of international patients .
many hospitals focus on multi - lingual staff providing services to patients speaking different major languages of the world .
language is also an influencing factor in decision making in selection of destination ( 11 , 37 ) .
facilities and quality of accommodation to international patients and their companions play important role in attracting the foreign patients .
essential supportive services like local transportation services , food , communication , and others are also important in attracting the patients to a destination .
thailand for example attracts more patients from developed countries because of already established quality tourism infrastructure offer excellent accommodation and hospitality services to international patients ( 81 ) . despite offering excellent medical care to international patients by hospitals in india most of the international patients visit india worry about accommodation quality , food hygiene , and personal safety ( 82 , 83 ) .
potential tourists prefer to receive medical services to the places where they are also interested for holidaying ( 75 , 84 ) . after focusing on cost and reliability
image formation agents are the factors which control the perception and evaluation of image ( 13 ) . researchers addressed amount and diversity of information sources that expose individuals including information related to destination acquired through visiting particular place .
various studies performed on destination selection behavior of tourists explored that with combination of other different factors , information sources explored by individuals determined certain destinations as possible alternatives ( 1317 ) .
consumer behavior studies have already established the effects of source of information on purchase behavior ( 18 ) .
amount and type of different information sources directly influence in development of cognitive image formation of destination ( 19 ) .
source of information is a vital antecedent of destination image formation and destination choice intention ( 20 ) .
there are four ( i ) professional advice ( tour operators , travel agents , airlines ) ( ii ) word of mouth ( friends , relatives , social media ) ( iii ) advertisement , and ( iv ) news / books / movies , different categories of type of information sources usually consider responsible for destination image formation ( 13 ) , this article use these four categories of source of information for medical tourism destinations image formation .
medical tourism facilitators are specialized in promotion of medical services abroad and offer supportive services such as assisting in selection of country and hospital , correspondence with doctors , travel arrangements , and arrangements of required paper work ( 21 , 22 ) .
judgment of agent related to travel of their clients has high influence on decisions of clients ( 23 ) .
these facilitators serve as motivators also due to providing much - needed assistant to those unenthusiastic potential international patients who do not want to make their trip arrangements by their own .
many hospitals and clinics linked themselves with airlines to promote their services and offer discounts ( 2 , 5 , 2426 ) .
literature on medical tourism also reveals the role of practitioner in promotion of medical tourism mainly in underdeveloped countries due to various reasons such as lack of resources , unavailability of equipment , unavailability of infra - structure , unavailability of specialized manpower , and unavailability of medication ( 2730 ) .
word of mouth and recommendation also has high influence in decision making of international patients in selection of destinations , hospitals , and doctors .
studies revealed large numbers of patients visit different countries for medical services were received recommendations from family members , friends , relatives , and colleagues ( 3134 ) .
websites and online forums created by experienced international patients to share their experiences with medical tourism are also considered as decisive source of information for those planning to take medical services abroad ( 35 , 36 ) .
printed materials are also used to promote the services by hospitals and clinics , for example air mauritius in - flight magazine provides details about procedures and services provided by the hair grafting clinics in mauritius ( 2 ) .
major medium of promotion of services by destination countries are trade fairs , travel markets / travel fairs , exhibitions , seminars and conferences to make potential patients informed about products and services offered by destinations .
some of these fairs and exhibitions are organized in collaboration with government agencies like tat , ministry of foreign affairs and department of export promotion but some providers organized these events by their own in cooperation with local institutes , medical schools and universities ( 37 ) .
print media play an important role in promotion and advertising medical tourism in major source countries , publish attractive and evidence based stories in their different segments related to health and travel .
los angeles time first examined about the growth of medical tourism and marked it as trend . later on new york times and los angeles times published stories of satisfied patients .
fox , cbs s 60 minutes and cnn aired their segments on patients traveling to medical services .
magazines like forbes and wall street journal analyzed the business aspect of medical tourism as brokers assertively marketed medical tourism to consumers , employers , and insurers ( 38 ) .
literature revealed that not every physical attribute of a destination has influence on image formation process .
there is substantial inequality between descriptive dimensions of image and the attributes which are considered important for decision making within individuals ( 39 ) .
it is generally presumed in marketing that products with similar characteristics will be equally preferred by the consumers , however , attributes , which make the product similar to other products , will not be necessarily same at the time of actual purchase . the importance of attributes will be change according to the need of consumer ( 40 ) . wish ( 1971 ) cited in ( 40 ) found that despite have many similarities individual like one country and dislike another one .
this is due to dimensions of liking may not be agree with the dimensions of similarity .
literature revealed that many researchers made distinction between physical and beneficial aspect of a product .
few researchers explained it as characteristic and benefits to the physical and beneficial aspect of product respectively ( 41 ) .
the typology of different attributes of product has been proven fruitful because by this a product s features can be segments into three groups as characteristic , beneficial , and imagery .
there are three components of vacation destination image formation ; ( i ) based on awareness : rely on the information sources , tourist believes about what a destination possesses , ( ii ) based on attitude : feelings and beliefs about destination , and ( iii ) based on expectations : expected benefits obtain from a tourist product ( 43 ) .
the above mention discussion clearly indicates that process of image formation is not just emphasis on physical attributes of destination but also depends on benefits or consumption values of product or service consumers hold in their minds .
benefits offered by a product or service are considered as consumption value of the same ( 44 ) .
sheth , newman and gross ( 1991 ) developed a theory known as theory of consumption values , which focuses on consumption values of products and services ( 45 ) .
conditional value. the decision making of consumer choice behaviour can be based on all five or any of the five consumption values .
consumption value model define the characteristics of functional value as price , credibility , and durability .
medical tourism destinations offer very low price for medical services to international patients in comparison to developed countries , and most of the time price of a procedure in india and other asian destination is equal to 1/10 of price in us or european countries ( 4 , 4648 ) .
quality of medical services is one of the aspects which drive medical tourism high towards success .
most of the hospitals in india provide medical care services to international patients are certified by jci which has reputation for hospital safety and regulatory management and recognized worldwide ( 49 ) .
high procedure success rate also forms image as quality medical tourism destination of various destinations at south asian , african and latin america ( 48 , 49 ) .
although availability of literature explain social value in medical tourism is less , little available literature clearly define the social value factor with - in the patient groups especially from less developed regions .
social value aspect has been clearly observed in yemeni patients travel abroad for treatment ( 27 , 50 ) .
senior male members of the household consider it as opportunity to increase their reputation in society . availing expensive and prestigious medical care service in foreign countries and the associated sacrifices with it would make stories full of pride to tell to others ( 51 ) .
most of the citizens of yemen do not obtain resources to go for leisure travel at different parts of the world therefore traveling to foreign country for medical services provide chance to explore new territory , meet new people , and experience different social culture ( 27 , 50 ) .
however , the concept of social value and epistemic value may not be associated with patients from developed regions .
many researchers found in their studies that patients were highly motivated to travel to a particular destination due to emotional attachment with doctor , hospitals or destination ( 6 , 50 ) . such as , many omani patients visit shiraz in iran for treatment due to religious and cultural familiarity ( 52 ) .
conditional value can be considered as major factor pushes patients to travel abroad because medical needs are crucial and considered as unavoidable so have great conditional value .
perceived risks is defined as perception of an individual about the probability that a particular action will lead them to a situation exposed with danger more than acceptable limit , and will lead to influence travel decision - making ( 53 ) . security and safety at the destination is major issue of concern by the potential travelers . in travel decision ,
making perception of risks has utmost importance due to its tendency to alter destination selection ( 54 ) .
researchers argued in favor of conducting study on perceived risks and destination image together ( 55 ) . in tourism literature , authors considered safety and security at destination as one of the pull factors caused destination image formation ( 13 , 14 , 56 ) .
however , in general cognitive image of destination does not engage with varied range of travel specific risks at destination and consider safety and risks as one of the many other attributes associated with destination ( 54 ) .
in addition , at the same time perceived risks are considered as potential inhibitors of travel ( 57 ) .
researchers argued that using cognitive image to understand the dimensions of travel risks will be a conceptual mistake ( 58 ) .
hence , perceived risks and cognitive image should consider individual variables for image related studies in travel .
credence goods as the quality of these good can not be assessed accurately even after consumption .
patients may vulnerable to many risks if consider to take medical services at medical tourism destinations .
researchers described six dimension of risks associated with health travelers visited israel as human - induced risks ,
financial risks , service quality risks , socio - psychological risk , natural disasters and car accident risk , and
after thorough literature review , authors categorized medical tourism destination perceived risks into three categories , as physical - health related risks , service related risks , and
destination related risks. physical risks are considered as possibility of physical danger or injury detrimental to health where as health risks are considered as possibility to becoming sick while traveling or at destination ( 54 ) . in medical tourism ,
international patients can be exposed with different diseases , so risk of contracting with a different kind of disease is even higher such as blood borne infection and infection due to improper screening and storage of blood , deep - vein thrombosis ( dvt ) to those patients returning home after surgery , language related risks and lose of money can also be a matter of concern while involved in medical tourism .
health risks for patients travel for organ transplant is even higher due to ignorance of standard protocol in donor selection ( 12 , 6062 ) .
literature revealed few american patients came in contact with non - tuberculous mycobecterial infection while taking treatment in hospitals abroad ( 63 , 64 ) .
researchers consider terrorism and kidnapping of rich patients and unstable economy of the destination as other risks associated with medical related travel .
apart from physical - health and service related risks , every destination have its own associated risks ; risks of terrorism , crime , and personal safety are associated with medical tourism destination due to falling in middle and lower middle - income countries in development status ( 63 ) .
according to researchers , destination image is the total of ideas , beliefs , and impressions individuals possess about the attributes of destination and or activities available at a destination after processing information from various sources over a period of time ( 39 , 65 ) .
destination image as overall imagery picture individual obtains in his mind ( 66 ) . in the early studies on travel
, researchers used the concept of stereotypic image of a travel destination ( 67 , 68 ) . later on , to understand the destination image formation process researchers developed an alternative framework and argued destination image consist of two components i.e. attribute based and holistic ( 66 ) .
attribute based component refers to the perception of individual based on destination features and holistic component refers to imaginary mental image of destination . later on ,
a bi - dimensional model was presented by researchers to represent destination image , consist of cognitive and affective image component ( 13 , 69 ) .
the cognitive component of destination image develops on knowledge and beliefs of destination based on tangible attributes , whereas affective component of image develops on emotions and feelings about the destination ( 70 , 71 ) .
researchers further studied affective image of destination and illustrate a four semantic differential scale to evaluate the affective component of destination image based on arousing
sleepy , pleasant unpleasant , exciting gloomy , and relaxing distressing ( 70 ) .
furthermore , cognitive component of destination image is an antecedent of affective component ( 72 ) .
thus , a distinctive image of a destination forms in tourists mind based on strength and weakness of attributes of cognitive and affective components . image of destination formed in consumer s mind is defined as aggregate of attributes and beliefs ( 73 ) .
it means if tourist has enough level of positive beliefs about attributes of destination it is expected that s / he has developed favorable attitude towards destination .
the process of destination image formation is described as mental construct of representation of destination based on information cues transmitted by image inducing agents chosen by individual ( 19 ) .
after thorough literature review authors identified four dimension of cognitive medical tourism destination image based of different attributes projected by different medical tourism destinations , as ; medical amenities , general infra - structure ,
social environment. most of the hospitals websites are dominated with images stressing on sophistication , advanced technology , cleanliness and efficiency .
majority of these sites are in english highlight the variety of procedures , price , accreditation of hospitals , and affiliation of doctors , qualified and smart staff , lavishly furnished accommodations , and testimonials of past patients , and efficiency with different languages of staff .
command on technology is rarely missed in any form of marketing ( 12 , 46 , 74 , 75 ) . based on mentioned qualities , medical tourism destinations project themselves as most suited destination for potential travelers ( 28 , 32 , 76 ) . to nullify the quality related concerns of international patients hospitals and medical tourism facilitators / brokers emphasis on the markers of quality services .
physicians trainings in world reputed institutes like national institute for health , johns hopkins university , university of birmingham , and other reputed universities are highlighted in the profiles of physicians .
prominent display of training and expertise of doctors achieve two goals ; established trustworthiness and mitigate concerns related to risk .
the accreditation awarded by jci is promoted by hospitals as mark of offering medical care of american standards ( 2 , 28 , 77 ) .
collaboration with prestigious hospitals in us and europe in also indicate seriousness towards providing quality services to patients by medical tourism hospitals at different destination ( 21 , 78 ) . distinguishing that major market drivers such as lack of health insurance and unaffordability are influencing patients to take procedures abroad , websites of brokerages predominantly display the comparative cost charts and price schedules .
most of the destinations claim providing significant cost services through different sources of promotion ( 5 , 79 , 80 ) .
most the promotions and advertisements show smiling , well - dressed and empathetic medical staff taking care of international patients .
many hospitals focus on multi - lingual staff providing services to patients speaking different major languages of the world .
language is also an influencing factor in decision making in selection of destination ( 11 , 37 ) .
facilities and quality of accommodation to international patients and their companions play important role in attracting the foreign patients .
essential supportive services like local transportation services , food , communication , and others are also important in attracting the patients to a destination .
thailand for example attracts more patients from developed countries because of already established quality tourism infrastructure offer excellent accommodation and hospitality services to international patients ( 81 ) . despite offering excellent medical care to international patients by hospitals in india most of the international patients visit india worry about accommodation quality , food hygiene , and personal safety ( 82 , 83 ) .
potential tourists prefer to receive medical services to the places where they are also interested for holidaying ( 75 , 84 ) . after focusing on cost and reliability
prominent databases ( 2013 , 2014 , 2015 ) have been searched to obtain desire literature related to medical tourism including science direct , utmj.org , nih.gov , nchu.edu.tw , palgrave - journals , medretreat , biomedcentral and so on and the keywords used are medical tourism , information sources , medical travel , health travel and so on .
google search was performed to get latest data related to medical tourism . to know in detail about the variables used in the framework scientific research databases
the key words used to find out research variables as well as relationship between researches variables were destination image , destination information sources , beneficial image , perceived travel risks , cognitive image , affective image , consumption values and so on . to obtain data about medical tourism various types of available literature were reviewed such as industry reports , market research reports , media reports and internet sources , however for identification and explanation of variables scientific literature published in reputed journals only were included .
the theory of planned ( tpb ) is an established and comprehensively tested model details the relationship between beliefs , attitude , intention , and actual behavior of consumers ( 85 ) . the model is perfectly applied in a variety of studies including leisure and tourism travel , and hospitality ( 36 , 8688 ) .
these research studies emphases on the factors such as motivations , information sources , attitudes , and visit intentions , thus authors argue that attitudinal theory provides a sound foundation to understand travel intentions of international patients and underpin the conceptual model discussed in this article .
studies explained that consumers access more information in case of high association of risks or benefits or both in a particular act ( 89 , 90 ) and perform rigorous information search in case of planning first time trip ( 91 ) because first time trip is associated with unknown risks as well as unknown leisure activities .
in general consumer behaviour practices risk - handling activity increases in case of high level of perceived risks .
importance of risk handling activity also determines by associated benefits gained after engaging with risk taking activity ( 92 ) .
the benefits of information search includes possibility of finding superior alternative to those already considered and the reduction in risk achieved from eliminating inferior , but a priori uncertain , alternatives ( 93 ) . with the discussion of above literature related to tourism
it could be assumed that medical tourism information sources will have positive influence on perceived travel benefits of international patients . whereas , information sources of medical tourism will negatively influence perceived travel risks of international patients .
destination image is considered as perceptions or impressions of destination held by tourists with respect to the expected benefit or consumption values .
the benefits sought by the travelers are highly associated with the image of destination and the affective image of destination is largely highly influenced by the motivations / benefits sought by individuals ( 13 , 44 ) .
the affective image of a destination is more positive in individual s mind when emotions evoked by the place coincide with the benefits sought ( 95 ) .
risks and constraints associated with travel have significant impact on destination image formation at the time of early decision - making process ( 96 ) . according to researchers ,
two dimensions significantly influence cognitive and affective image of destination ( 58 ) . on the basis of previous studies in tourism literature
it can be assumed that perceived travel benefits of international patients will positively influence the destination image , whereas perceived travel risks of international patients will negatively influence the destination image .
intention of visit related to travel is considered as tourists perceived likelihood to visit particular destination within a specific time period ( 97 ) .
thus , to be closely correlated to the travel behaviour intention of visit believes as an important outcome variable in tourism research ( 16 , 97 ) .
intention to visit a destination is highly influenced by cognitive / perceptual and affective evaluation ( 98 ) .
destination image is a direct antecedent of perceived quality , satisfaction and revisit intention and recommendation ( 99 ) . according to researchers
, there is significant theoretical and empirical link between cognitive destination image and behavioral intention ( 100 ) .
based on above discussion it could be assumed that image of medical tourism destination will positively influence visit intention of international patients . conceptual framework international patients travel decision making
the article is a sincere effort by authors to establish conceptual framework which explains decision - making process of international patients .
though , there has been advance in explaining different factors play important roles in international patients travel , however , there is scarcity of literature conceptualizing these factors into a framework and test it empirically .
literature reveals about associated benefits and risks related to travel abroad for medical purposes but do not conceptualize it in the process of decision - making .
sources of information in medical tourism play significant role in development of attitude as well as intention , has already been established in consumer behavior .
however , need is to test its significance in medical tourism decision - making process .
the proposed framework will encourage future researchers to test different variables empirically and establish the model of international patients travel behavior .
ethical issues ( including plagiarism , informed consent , misconduct , data fabrication and/or falsification , double publication and/or submission , redundancy , etc . ) have been completely observed by the authors .
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background : role of information source , perceived benefits and risks , and destination image has significantly been examined in travel and tourism literature ; however , in medical tourism it is yet to be examined thoroughly .
the concept discussed in this article is drawn form well established models in tourism literature.methods:the purpose of this research was to identify the source of information , travel benefits and perceived risks related to movement of international patients and develop a conceptual model based on well - established theory .
thorough database search ( science direct , utmj.org , nih.gov , nchu.edu.tw , palgrave - journals , medretreat , biomedcentral ) was performed to fulfill the objectives of the study.results:international patients always concern about benefits and risks related to travel .
these benefits and risks form images of destination in the minds of international patients .
different sources of information make international patients acquaint about the associated benefits and risks , which later leads to development of intention to visit .
this conceptual paper helps in establishing model for decision - making process of international patients in developing visit intention.conclusion:ample amount of literature is available detailing different factors involved in travel decision making of international patients ; however literature explaining relationship between these factors is scarce .
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Introduction
Literature Review
International Patients Source of Information
International Patients Perceived Travel Benefits
Consumption Value of Medical Tourism
International Patients Perceived Travel Risks
Medical Tourism Destination Image
Methods
Results
Conclusion
Ethical considerations
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squamous cell carcinoma of the head and neck ( hnscc ) is a heterogeneous disease that includes tumors arising from the mucosal epithelial surface of the oral cavity , oropharynx , hypopharynx , and larynx . although these tumors originate within different anatomic sites within the upper aerodigestive tract , they are histologically identical ( 95% of hnscc are squamous cell carcinomas ) , share common etiologic risk factors and overlapping metastatic target site profiles ( reviewed in [ 13 ] ) .
recent genetic analysis of human head and neck tumors has revealed common molecular alterations including p53 mutation , p14arf , and p16 methylation , as well as cyclin d and egfr amplification [ 36 ] . despite these similarities ,
the distinct anatomic subsites are associated with differing rates of regional metastasis for example , vocal cord lesions tend to metastasize less frequently than oropharyngeal or hypopharyngeal lesions . this variation may be attributed to differing densities of lymph draining vessels within each of the relevant subsites .
patients who exhibit metastases into the regional nodal basin exhibit a 50% decrease in survival irrespective of treatment [ 715 ] .
currently , it is the 5th leading cause of cancer by incidence and the 6th leading cause of cancer mortality in the world [ 16 , 17 ] .
recurrent and/or metastatic hnscc patients have a poor prognosis , with a median survival of less than 1 - 2 years [ 18 , 19 ] .
several lines of evidence indicate that cancer is a disease resulting from dynamic changes in the genome that promote the progressive transformation of normal human cells into highly malignant derivatives [ 20 , 21 ] . during this process ,
cancer cells acquire several unique capabilities including self - sufficiency in response to growth signals , insensitivity to antigrowth signals , evasion of programmed death ( apoptosis ) , limitless replicative potential , sustained angiogenesis as well as invasion and metastasis , reprogramming of energy metabolism , and avoiding immune destruction [ 21 , 22 ] .
detailed global genomic analyses of several human tumors has revealed that certain classes of signaling proteins appear to be targeted more frequently by oncogenic mutations .
receptor tyrosine kinases ( rtks ) are a good example . of the 59 transmembrane rtks identified to date ,
dysregulation of ~30 rtks are associated with neoplastic transformation and cancer progression [ 2325 ] .
interestingly , ninety percent of primary head and neck squamous cell cancers , irrespective of subsite , have alterations in members of the epidermal growth factor ( egf ) family of receptor tyrosine kinases ( erbbs ) , in particular erbb1/egfr .
ten to fifteen percent of tumors will also have an alteration in another egfr family member , the erbb2/her2/neu receptor [ 27 , 28 ] .
these findings suggest a strong etiologic role for rtk dysregulation in this type of tumors . given this association , patients with head and neck squamous cell cancers are well positioned to benefit from existing and future molecular targeted agents directed against oncogenic rtks such as egfr ( reviewed in ) .
rtks are a family of transmembrane proteins that mediate many important physiological processes in both normal and cancerous cells .
ligand binding to the extracellular domain of rtks induces receptor dimerization and activation of rtk activity .
subsequent autophosphorylation of the receptor at specific tyrosine residues within the cytoplasmic domain generates binding sites for proteins that relay downstream biological signals to regulate protein function , protein - protein interactions , and gene expression . under physiological conditions ,
rtk dysregulation can occur through several mechanisms including gene amplification or rtk overexpression , chromosomal translocation to produce constitutively active rtks , gain of function mutations or deletions that promote ligand - independent rtk activity , escape from negative regulatory mechanisms or local environmental changes , all of which lead to potent oncogenic signaling and hence neoplastic growth .
these complex signaling networks use multiple factors to drive the outcome of rtk signaling . although often depicted as linear pathways , they actually represent an integrated network with various modes of cross - talk , overlapping and distinct functions .
known signaling pathways involved in head and neck tumorigenesis include the phosphatidylinositol-3-kinase ( pi3k)-akt - mammalian target of rapamycin ( mtor ) , signal transducer and activator of transcription ( stats ) and raf kinase - mitogen - activated protein kinase kinase ( mek)-p42/p44 mitogen activated protein kinase ( mapk ) signaling pathways [ 1 , 30 ] .
this review highlights three rtk signaling pathways involved in head and neck squamous cell carcinoma ; egfr , the type 1 insulin - like growth factor receptor ( igf-1r ) and the hepatocyte growth factor ( hgf ) receptor ( met ) .
this short review will explore the relative contribution of each signaling axis to disease progression , potential modes of cross - talk , and targeted clinical approaches under investigation for disease management .
the egfr family of rtks is comprised of four different receptors known as erbb1 ( also referred to as egfr ) , erbb2 ( her2/neu in rodents ) , erbb3 ( her3 ) , and erbb4 ( her4 ) ( reviewed in [ 3133 ] ) . each receptor , with the exception of erbb3 , contain an intracellular tyrosine kinase domain that is activated by binding to extracellular egf - like ligands , which result in receptor dimerization and hence activation of downstream signaling cascades including mapk , pi3k / akt and stat signaling .
eleven egf - like ligands have been identified to date that can be categorized into four groups those that bind egfr only ( egf , transforming growth factor alpha ( tgf ) , and amphiregulin ) , those that bind to egfr and her4 ( heparin binding - egf , betacellulin and epiregulin ) , those binding directly to either her3 and her4 ( neuregulin 1 and neuregulin 2 ) and her4 binding only ( neuregulin 3 and neuregulin 4 ) ( reviewed in ) .
epigen , the most recently discovered member of the egf - like ligand family appears to be a low affinity and broad specificity ligand that effectively activates egfr .
erbb2 is considered a ligand - less coreceptor as it does not have any known ligands that bind directly with high affinity , despite its established role as a potent oncogene in several cancer types including breast , colorectal , nonsmall cell lung carcinoma ( nsclc ) and hnscc [ 36 , 37 ] .
aberrant egfr activity has been strongly linked to the etiology of 5890% of hnscc [ 26 , 38 ] .
these rates can vary due to the inclusion of cancers from different subsites within the head and neck , methods used to assess gene amplification and tumor scoring methods . in contrast to lung adenocarcinomas in which activating egfr mutations result in ligand - independent signaling [ 3943 ] , such activating egfr mutations are infrequent in hnscc [ 44 , 45 ] .
egfr gene amplification resulting in upwards of 12 copies per cell has been reported in hnscc patients compared to copy numbers detected in normal mucosa from noncancer patients .
this and other pathways of ligand - independent receptor activation that do not require egfr overexpression have been characterized as the likely drivers of egfr activity in hnscc .
egfr gene amplification remains a strong indicator for poor patient survival , radioresistance , and locoregional failure [ 4749 ] .
egfr overexpression is detected in healthy mucosa in cancer patients ( field cancerization ) that will increase in proportion to observed histological abnormalities such as hyperplasia , carcinoma in situ and invasive carcinoma , indicating that it is an early event in hnscc .
accordingly , significant effort has focused on egfr signaling as a therapeutic target for treating hnscc patients .
cetuximab , matuzumab and nimotuzumab represent humanized antiegfr antibodies , whereas gefitinib and erlotinib are small tyrosine kinase inhibitors ( tkis ) ( figure 1 ) .
cetuximab ( erbitux ) competitively inhibits endogenous ligand - binding to egfr and thereby inhibits subsequent receptor activation [ 5053 ] .
cetuximab is a valuable treatment option in head and neck patients as it synergizes with current treatment modalities .
cetuximab enhances the effects of many standard cytotoxic agents , including cisplatin ( the conventional platinum - fluorouracil chemotherapeutic ) , and in combination with chemotherapy it can elicit antitumor responses in tumors that previously failed to respond to that chemotherapy .
notably , cetuximab did not dramatically exacerbate the common toxic effects associated with radiotherapy of the head and neck , including mucositis , xerostomia , dysphagia , pain , weight loss , and performance status deterioration .
cetuximab has been approved for use in combination with radiation for treating patients with locally advanced hnscc and as monotherapy for patients with recurrent hnscc .
matuzumab ( formerly emd 72000 ) binds to egfr with high specificity and affinity to block receptor signaling , and also modulates antibody - dependent cellular cytotoxicity ( adcc ) when combined with cetuximab [ 5860 ] .
phase i clinical trials report excellent antitumor activity of matuzumab against several human tumor types including head and neck cancers .
a randomized phase iib , four - arm , open - label study recently assessed the safety and efficacy of nimotuzumab in combination with radiation therapy ( rt ) or chemoradiation therapy ( crt ) in patients with advanced ( stage iii or iva ) hnscc .
the addition of nimotuzumab to both the radiation and chemoradiation regimens was reported to improve the overall response rate , survival rate at 30 months , median progression - free survival and median overall survival .
a combined group analysis of the nimotuzumab arms versus the non - nimotuzumab arms demonstrated a significant difference in overall survival favoring nimotuzumab .
this study is compelling as patient response rates compare favorably with studies combining cetuximab with radiotherapy , but with fewer side effects .
gefitinib ( iressa ) is a small molecule tki - targeted to the intracellular active site for phosphorylation that has been tested in clinical trials involving hnscc patients , as a single agent or in combination with radiation treatment .
unfortunately , gefitinib has shown limited clinical efficacy with response rates of 1015% [ 63 , 64 ] .
erlotinib is a selective inhibitor of the egfr that also shows antitumor activity in hnscc comparable to standard combination chemotherapy .
another promising rtk under preclinical and clinical evaluation for head and neck cancers includes the igf-1r ( reviewed in [ 66 , 67 ] ) .
two ligands , insulin - like growth factor 1 ( igf1 ) and igf2 bind to igf-1r .
ligand binding to the igf-1r stimulates its intrinsic tyrosine kinase activity , activating downstream signaling networks including ras - raf , mapk and erk , and pi3k ( figure 1 ) to drive cellular functions such as cell growth , survival and differentiation .
it is widely accepted that the igf - axis activates antiapoptotic signaling , which in turn upregulates the pi3k - akt and mapk pathways in cancer cells .
additionally , igf - ir also regulates vascular endothelial growth factor ( vegf ) production , suggesting a role in tumor angiogenesis .
several studies indicate that igf-1r is overexpressed and functional in 94% of hnscc patient samples [ 70 , 71 ] .
consistent with this , igf - ir signaling significantly enhances the proliferation , motility and tumorigenicity of human head and neck cancer cell lines . igf-1r
down regulation in a hnscc cell line using antisense oligonucleotides resulted in a dose - dependent decrease in cellular proliferation , induction of apoptosis , caspase activation and reduced expression of proangiogenic cytokines such as vegf .
interest in targeting the igf-1r in hnscc was bolstered by the observation that treatment of head and neck cancer cells with either igf or egf resulted in igf - ir and egfr heterodimerization [ 71 , 72 ] .
however , only igf resulted in the phosphorylation of both receptors . using a mouse xenograft model for hnscc , treatment with antibodies against igf-1r , egfr or
it remains to be determined whether cellular cross - talk between igf-1r and egfr has an important role in determining the biological aggressiveness of hnscc or resistance to egfr - targeted therapies .
several monoclonal antibodies and tkis for igf-1r have been tested in preclinical studies and early phase clinical studies .
however , the efficacy of igf-1r - targeted therapy for treating patients with hnscc , particularly cross - talk with egfr , warrants further investigation . to date , the effect of blocking oncogenic igf-1r and egfr signaling have been studied more extensively in breast cancer cell lines [ 7375 ] .
treatment with gefitinib and ag1024 , a tki for igf-1r reduced cell proliferation when used as single agents and showed an additive effect when used in combination [ 76 , 77 ] . targeting igf-1r and egfr signaling is currently under evaluation in hormone - sensitive metastatic breast cancer using the igf-1r inhibitor osi-906 and the egfr tki erlotinib , although results are not yet available ( http://www.clinicaltrials.gov/ , identifier nct01205685 ) . similarly , an exploratory study to assess the modulation of biomarkers in hnscc patients treated preoperatively with cetuximab and/or imc - a12 , a humanized antiigf-1r monoclonal antibody is currently underway ( http://www.clinicaltrials.gov/ , identifier nct00617734 ) .
these studies will be critical for evaluating whether the use of anti - igf-1r and egfr - targeted treatments will be more effective than single - agent modalities for treating patients with hnscc .
the met receptor is a single pass transmembrane protein that upon binding its ligand hgf also known as scatter factor - promotes increased cell proliferation , survival and motility ( reviewed in [ 78 , 79 ] ) .
hgf is the only physiological ligand for met and is secreted as an inactive precursor polypeptide chain by mesenchymal cells .
hgf is proteolytically cleaved to form an active / heterodimer by a number of serine proteases including urokinase plasminogen activator ( upa ) , tissue - type plasminogen activator ( tpa ) , coagulation factors x. xi and xii .
met is a disulphide - linked / heterodimer derived from the proteolytic cleavage of a 170 kda precursor .
the chain and n - terminal region of the -chain form sema domain , a seven -propeller structure in which blades 2 and 3 bind to hgf .
the sema domain is flanked by a cysteine - rich region followed by four immunoglobulin repeats .
it is proposed that the cysteine - rich region and immunoglobulin repeat domains undergo a conformational change following hgf binding allowing for met dimerization [ 80 , 81 ] .
binding of hgf to met results in receptor autophosphorylation at key catalytic residues and subsequent recruitment of several cytosolic signaling molecules that are shared with the egfr and igf-1r signaling pathways , including the grb2/sos complex , the p85 regulatory subunit of pi3k , gab1 and jak / stat3 ( figure 1 ) .
subsequent activation of the mapk and jun - n - terminal kinase ( jnk ) pathways is responsible for the mitogenic and motogenic properties of met / hgf signaling resulting in invasive growth , depending on the physiological setting .
increased met signaling in human cancers can be the result of enhanced ligand - binding ( autocrine and paracrine ) , met overexpression or missense mutations that often induce constitutive kinase activity , failure of met down regulation and interactions with other cell surface receptors such as egfr ( reviewed in [ 8284 ] ) . met is overexpressed in 84% of hnscc patient samples .
interestingly , amplification of the met gene ( > 10 copies per cell ) is present only in 3 of 23 ( 13% ) tumor tissues .
hgf overexpression is detected in 45% of hnsccs , suggesting that hgf functions predominantly in a paracrine manner to drive met signaling in these cancers .
moreover , high levels of hgf are detected in hnscc patient plasma samples supporting the idea that ligand availability is not a limiting factor for met activation .
mutations in the met ligand - binding domain ( t230m / e168d ) , transmembrane or jm domain ( r988c , t1010i ) and the tyrosine kinase domain ( t1275i , v14333i ) have also been identified in hnscc tumor samples , although their relative contribution to hnscc progression remains to be determined .
two somatic met mutations have been detected in hnscc that result in constitutively active receptor signaling that confers an invasive phenotype when ectopically expressed in cell lines .
the y1230c mutation confers anchorage - independent growth and an invasive phenotype in transfected cells , whereas the y1235d met mutation stimulates epithelial cells to invade reconstituted basement membrane in the absence of hgf . in the case of the mety1235d mutation
, genomic analyses of hnscc patient samples detected the presence of this mutant allele in 50% of metastatic tumors versus 26% in primary tumors , raising the possibility that this could be a critical genetic lesion for the acquisition of a metastatic phenotype .
alternatively , increased met signaling could afford hnscc a selective advantage for growth and/or survival in metastatic sites , such as the lymph node and lung .
indeed several studies indicate that met overexpression correlates highly with lymph node metastasis , pathologic stage , and disease reoccurrence [ 8891 ] .
moreover , patient survival was significantly reduced in biopsy samples with positive met expression relative to negative met expression , suggesting the association of met with hnscc disease progression .
consistent with these findings , treatment with the tki pf-2341066 caused a significant reduction in tumor growth , a high level of apoptosis and cellular debris within the tumor using a xenograft animal model for hnscc .
selective inhibitors of met / hgf signaling include humanized monoclonal antibodies for hgf and met , and small - molecule tyrosine kinase inhibitors directed against met ( figure 1 ) .
although their efficacy for treating a variety of solid tumors is increasingly recognized , we await results of preclinical and clinical trials for head and neck cancer that are ongoing .
the humanized antibody amg 102 shows high potency towards the mature and processed form of hgf with no detected effects on proteolytic activation of prohgf .
amg 102 interferes with met signaling , by competing with hgf for binding to the chain of the met receptor . in phase
i clinical studies in patients with advanced solid tumors , 70% of patients had a best response in terms of achieving stable disease [ 93 , 94 ] .
importantly , no antiamg 102 antibodies were detected and circulating hgf levels were dose dependent .
another promising clinical therapeutic is the one - armed 5d5 humanized antibody ( oa5d5/metmab ) directed against met .
metmab binds met with high affinity , preventing hgf binding , met phosphorylation , receptor internalization and downstream signaling events and has been shown to inhibit tumor growth in animal models by more than 95% [ 95 , 96 ] .
metmab is currently in phase i / ii human clinical trials in comparison with erlotinib in patients with nsclc ( http://www.clinicaltrials.gov/ , identifier nct00854308 ) .
future clinical trials will be required to determine the suitability of amg102 and metmab as either single agents or combinatorial therapeutics for treating hnscc patients .
foretinib ( formerly xl880 ) is a tki whose primary targets include met and vegf , and to a lesser extent the platelet - derived growth factor ( pdgf ) receptor , ron , kit and tie2 rtks .
foretinib recently completed phase ii clinical trials in head and neck patients ( http://www.clinicaltrials.gov/ , identifier nct00725764 ) .
interim results suggest that after 12 months , 12 of 18 patients had stable disease .
a phase i dose - escalation study of the safety and pharmacokinetics of xl184 administered orally to patients with advanced malignancies ( showed that , on average , patients survived for more than 3 months with several up to 6 months while on treatment ) ( reviewed in ) . due to
encouraging data from this study , a randomized phase iii trial of xl184 in hnscc patients was initiated to investigate xl184 as a first - line treatment ( compared with placebo ) for survival benefit to patients with hnscc ( http://www.clinicaltrials.gov/ , identifier nct00704730 ) .
arq197 ( arqule ) is a nonatp - site competitive , selective small molecule inhibitor of the met intracellular region .
although the mechanism of arq197 is presently unknown , the results of phase i trials suggest potential antiinvasive activity for this compound .
overall , met , and hgf - targeted therapies have been well tolerated in clinical trials with negligible toxicities .
however , it remains to be determined whether met is a better therapeutic target than hgf .
clearly , in patients where met is activated by autocrine hgf secretion , both hgf and met targeted therapies may prove to be more efficacious treatment options .
acquired resistance is likely the result of several mechanisms including ( 1 ) egfr mutations initially present as well as those acquired during therapy , ( 2 ) receptor independent activation of downstream signaling cascades , ( 3 ) cross - talk with other rtks and converging signaling pathways and ( 4 ) environmental factors including inflammatory agents and viral infection .
resistance to cetuximab has been associated with the coexpression of the truncated egfr mutant , egfrviii with wild - type egfr .
egfrviii is the result of an in frame deletion of exons 27 spanning the extracellular ligand - binding domain .
the deletion results in a truncated egfr receptor that signals in a ligand - independent manner .
egfrviii expression has been detected in 42% of hnscc patient samples , and closely correlates with increased hnscc cell proliferation in vitro and increased tumor growth using in vivo xenograft models .
egfrviii preferentially activates the pi3k pathway instead of the ras / raf / mek pathway , which is activated by wild - type egfr . of particular interest to the therapeutic treatment of hnscc ,
egfrviii expression decreases the proliferative response of egfr expressing tumor cells to cetuximab treatment relative to vector control cells . in a recent study ,
egfrviii cells were shown to be resistant to the antiinvasive effects of cetuximab due to an increase in phosphorylation of stat3 rather than increased pi3k signaling .
egf - induced expression of the stat3 target gene hif1 was abolished by cetuximab in hnscc cells expressing wild - type egfr under hypoxic conditions , but not in egfrviii - expressing hnscc cells [ 102 , 103 ] .
these data suggest a role for egfrviii in mediating hnscc resistance to cetuximab . despite egfrs critical role in the development of hnscc
, clinical data indicate modest clinical benefits for locoregional control and survival of head and neck cancer patients treated with egfr - targeted therapies .
hnscc patients resistant to cetuximab , often succumb to local tumor recurrence as well as regional and distant metastasis .
the addition of cetuximab to radiation therapy was reported to show improved locoregional disease control , progression - free survival , and overall survival in patients with locally advanced hnscc .
however the data revealed a disproportionate benefit of cetuximab with radiotherapy to oropharyngeal cancer patients when compared to patients treated with hyperfractionated radiotherapy .
accumulating evidence suggests that human papilloma virus ( hpv ) 16 status ( hpv+ ) is an important prognostic factor associated with a favorable outcome in a subset of head and neck cancers , including oropharyngeal and tonsilar cancers .
hpv+ tumors tend to have unique genetic aberrations including decreased egfr expression , whereas increased igf-1r levels characteristic of hnscc appear to be independent of hpv status .
clinically , hpv+ tumors are characterized by more favorable patient prognosis regarding disease - free survival as well as overall survival [ 104 , 105 ] , possibly as a result of increased genomic stability associated with global gene hypermethylation in hpv+ tumors .
thus it will be interesting to determine whether hpv+ status explains some of the benefits derived from the addition of cetuximab to radiotherapy in this subset of hnscc patients . at present
, there are few clinical indicators of which hnscc patients will most likely respond to egfr - targeted therapies .
accordingly , strategies to optimize egfr - targeted therapy remain an active area of research .
additional mechanisms that result in egfr activation include activating mutations in downstream signaling components or cross - talk between different rtk pathways . activating mutations in the pi3ka oncogene
occurs in 10% of hnscc tumors whereas elevated levels of phosphorylated stat3 correlates with lymph node metastasis and poor patient prognosis [ 108110 ] .
conversely , h - ras mutations are infrequent in hnscc cases ( less than 5% ) , although a higher incidence has been detected in asian populations and correlates with areca nut chewing [ 111 , 112 ] .
met signaling has been shown to contribute to resistance in cell lines derived from multiple tumor types including breast , gastric and lung .
in one key study , nsclc with activating mutations in the egfr acquire resistance to the tki gefitinib and erlotinib , by amplification of the met gene to maintain akt and her3 signaling .
these studies underscore the role of cross - talk between rtks to preferentially signal through the pi3k - akt survival pathway as a mechanism for acquired drug resistance .
the relevance of met as a mechanism for escape from egfr - targeted therapy in head and neck cancers remains to be determined .
hypoxia results in the transcriptional upregulation of met gene expression via hif1 in a number of tumors including head and neck , often downstream of egfr signaling . in normoxia
, hydroxylation of 2 prolines in hif1 enables its binding to the von hippel - lindau tumor suppressor protein ( pvhl ) linking hif1 to a ubiquitin ligase complex . during hypoxia , minimal or no hydroxylation occurs enabling hif1 to avoid proteasomal degradation and
dimerize to other hif family members such as hif1 and coactivators , to form an active transcriptional hif complex on the hypoxia response element ( hre ) of target genes such as met .
the ubiquitin ligase catalyzes polyubiquitination of hif1 targeting it for proteasomal degradation . under hypoxic conditions ,
increased met signaling directs the invasive growth program , enabling cells to invade more oxygenated tissues .
since met has been reported to promote invasive and angiogenic effects in the tumor microenvironment , the use of hgf / met inhibitors may afford a means of impairing tissue colonization as well as tumor vascularization in head and neck cancer patients .
studies on other solid tumor types , most notably glioblastoma , indicate a role for igf-1r upregulation in resistance to egfr - targeted therapies .
igf-1r mediates resistance to anti - egfr therapy in primary glioblastoma through the continued activation of the pi3k / akt survival pathway .
the apparent cooperation between igf-1r and egfr in promoting hnscc pathogenesis as well as resistance to egfr - targeted therapy , suggests an advantage to cotargeting these signaling axes for the treatment of head and neck cancers . to date , the effect of blocking oncogenic igf-1r and egfr signaling have been studied more extensively in breast cancer lines .
treatment with gefitinib and ag1024 , a tki for igf-1r reduced cell proliferation when used as single agents and showed an additive effect when used in combination [ 76 , 77 ] .
targeting igf-1r and egfr signaling is currently under evaluation in hormone - sensitive metastatic breast cancer using the igf-1r inhibitor osi-906 and the egfr tki erlotinib , although results are not yet available ( http://www.clinicaltrials.gov/ , identifier nct01205685 ) .
similarly , an exploratory study to assess the modulation of biomarkers in hnscc patients treated preoperatively with cetuximab and/or imc - a12 , a humanized antiigf-1r monoclonal antibody is currently underway ( http://www.clinicaltrials.gov/ , identifier nct00617734 ) .
these studies will be critical for evaluating whether the use of antiigf-1r and egfr - targeted treatments will be more effective than single - agent modalities for treating patients with hnscc .
targeted therapies that block egfr , met , and igf-1r signaling in head and neck cancers continue to show promising results in preclinical studies and clinical trials .
however , it is difficult to predict which patients are most likely to benefit from these therapeutics and potential side effects during long - term in vivo use . given the interplay between these rtk signaling pathways and the mediocre results obtained with monotherapy regimens thus far , clinical trials will be required to determine how egfr- , met- , and igf-1r - targeted therapies can be used in combination in order to definitively abrogate their common downstream oncogenic signaling networks .
although gaps in our knowledge concerning the role of met and igf-1r in head and neck tumorigenesis , as well as acquired resistance to antiegfr therapies remain to be addressed , efforts to translate current information towards clinical applications continue to be impressive .
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molecular therapeutics for treating epidermal growth factor receptor-(egfr- ) expressing cancers are a specific method for treating cancers compared to general cell loss with standard cytotoxic therapeutics .
however , the finding that resistance to such therapy is common in clinical trials now dampens the initial enthusiasm over this targeted treatment .
yet an improved molecular understanding of other receptor tyrosine kinases known to be active in cancer has revealed a rich network of cross - talk between receptor pathways with a key finding of common downstream signaling pathways .
such cross talk may represent a key mechanism for resistance to egfr - directed therapy .
here we review the interplay between egfr and met and the type 1 insulin - like growth factor receptor ( igf-1r ) tyrosine kinases , as well as their contribution to anti - egfr therapeutic resistance in the context of squamous cell cancer of the head and neck , a tumor known to be primarily driven by egfr - related oncogenic signals .
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1. Introduction
2. EGFR Amplification in Head and Neck Cancers
3. Targeting IGF-1R Signaling in Head and Neck Cancers
4. A Role for Met/HGF Signaling in Head and Neck Cancers
5. Understanding Resistance to EGFR-Targeted Therapies in HNSCC
6. Conclusions
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this in vitro study was conducted in the chemistry and biochemistry institute , university of veterinary medicine , vienna , austria between june and august 2013 in accordance with the guidelines of the european union council ( 86/609/eu ) for the use of laboratory animals .
the work does not require approval from the ethics committee as it used mouse embryos under the fifteenth day of gestation .
primary mesencephalic cell cultures were prepared from c57/b16 embryos according to radad et al.10 to summarize , embryonic mouse mesencephala were dissected on the fourteenth day of gestation and cut into small pieces in a drop of dulbecco s phosphate - buffered saline ( dpbs ) ( invitrogen , darmstadt , germany ) , 2 ml of 0.2% trypsin solution ( invitrogen , darmstadt , germany ) and 2 ml of 0.02% dnase i solution ( roche , berlin , germany ) were added and the tissue was subsequently incubated in a water bath at 37c for 7 minutes ( min ) .
then , 2 ml of trypsin inhibitor ( 0.125mg / ml ) ( invitrogen , darmstadt , germany ) were added , the tissue was centrifuged at 100 g for 4 min and the supernatant was aspirated .
the tissue pellet was triturated 2 - 3 times with a fire - polished pasteur pipette , each time 0.02% dnase i ( invitrogen , darmstadt , germany ) was included in the medium .
dissociated cells were plated at a density of 257,000 cells / cm in dulbecco s modified eagle s medium ( dmem ) ( sigma aldrich , hamburg , germany ) supplemented with 4 mm glutamine , 10 mm 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid ( hepes ) buffer , 30 mm glucose , 100 iu / ml penicillin , 0.1 mg / ml streptomycin , and 10% heat - inactivated fetal calf serum ( sigma aldrich , hamburg , germany ) .
the medium was exchanged on the first day in vitro ( div ) and on the third div . on the fifth div ,
half of the medium was replaced by serum - free dmem containing 0.02 ml b-27/ml ( invitrogen , darmstadt , germany ) dmem .
serum - free supplemented dmem was used for feeding from the sixth div , and subsequently replaced every second day . a stock solution of tq ( sigma aldrich , hamburg , germany ) ( 10 mm )
four sets of cultures were treated as follows : the first set of cultures was treated with dmso and kept as untreated controls .
the second set of cultures was treated with tq ( 0.01 , 0.1 , 1 , and 10 m ) on the eighth div for 4 consecutive days to investigate the effect of tq on the survival of dopaminergic neurons .
the third set of cultures was treated with 10 m of mpp on the tenth div for 48 hours ( h ) .
the fourth set of cultures was concomitantly treated with tq ( 0.01 , 0.1 , 1 , and 10 ) , and 10 m of mpp on the tenth div for 48 h. dopaminergic neurons were identified immunocytochemically by staining tyrosine hydroxylase .
cultures were rinsed carefully with phosphate buffered saline ( pbs , ph 7.2 ) at the end of each treatment and fixed in 4% paraformaldehyde for 45 min at 4c . after washing with pbs , cells were permeabilized with 0.4% triton x-100 for 30 min at room temperature .
cultures were washed 3 times with pbs and incubated with 5% horse serum ( vectastain abc elite kit , biozol diagnostica vertrieb gmbh , eching , germany ) for 90 min to block nonspecific binding sites . to determine the number of thir in cultures
, cells were sequentially incubated with anti - th primary antibody overnight at 4c , biotinylated secondary antibody ( vectastain ) , and avidin - biotin - horseradish peroxidase complex ( vectastain ) for 90 min at room temperature and washed with pbs between stages .
the reaction product was developed in a solution of diaminobenzidine ( 1.4 mm ) in pbs containing 3.3 mm hydrogen peroxide and stained cells were counted with a nikon inverted microscope in 10 randomly selected fields per well at 10x magnification .
cellular injury was quantitatively assessed by measuring the activity of lactate dehydrogenase ( ldh ) released from damaged cells into the culture medium .
the reaction was initiated by mixing 0.2 ml of cell - free supernatant ( diluted 1:1 with aqua dest . ) with potassium phosphate buffer containing - nicotinamide adenine dinucleotide ( nadh ) and sodium pyruvate ( 0.18 and 0.62 mm in potassium phosphate buffer ) in a final volume of 0.5 ml in 1 ml cuvettes .
the decrease of nadh was spectrophotometrically ( novaspec ii , ge healthcare europe gmbh , freiburg , germany ) monitored .
the ldh activity was calculated from the slope of the decrease in optical density at 334 nm over a 3 min - time period .
the ldh release is proportional to the number of damaged or destroyed cells.11,12 lysotracker deep red ( life technologies , invitrogen , grand island , ny , usa ) is a red fluorescence dye used for labeling acidic organelles in live cells including autophagolysosomes .
cultures were treated with 1 m of tq ( a concentration that significantly protected dopaminergic neurons in mpp - treated cultures ) on the eighth div and co - administered with mpp ( 10 m ) on the tenth div for 2 days . on the twelfth div ,
culture medium was aspirated and cultured cells were incubated with a new medium containing 100 nm lysotracker deep red fluorescence dye ( life technologies , invitrogen , grand island , ny , usa ) for 15 - 30 min at 37c . after washing with dpbs
, cultured cells were photographed on a nikon inverted microscope equipped with epifluorescence attachment using a rhodamine filter set ( 580/590 , g-2a ) and a coolpix 990 digital camera ( nikon , otawara , japan ) .
5,5,6,6-tetrachloro-1,1,3,3-tetraethylbenzimidazolyl - carbocyanine ( jc-1 ) is a lipophilic cationic dye that selectively enters into mitochondria . in healthy cells with high mitochondrial membrane potential ( m )
the dye remains in the monomeric from with green fluorescence in case of apoptotic or damaged cells .
the jc-1 red : green ratio is used to estimate changes in m.13 the jc-1 was dissolved in dmso and further diluted in dmem ( 10 g / ml final concentration ) .
after removal of the culture medium , cells were loaded with jc-1 for 15 min at 37c , rinsed twice with pbs , and photographed on a nikon inverted microscope equipped with epifluorescence attachment using a rhodamine filter set ( 520 dm/520 ba , b-2a ) and a coolpix 990 digital camera ( nikon , otawara , japan ) .
fluorescence intensity of the red : green ratio was determined semi quantitively by using adobe photoshop software .
cells were fixed with 4% paraformaldehyde for 45 min at 4c . after washing with pbs ( ph 7.2 ) ,
the dapi solution ( 2 m final concentration ) was added to the cultures at room temperature for 5 min in the dark .
after washing with dpbs , 3 photos were taken randomly from each well with a coolpix 990 digital camera connected to an inverted microscope with epifluorescence attachment using an ultraviolet filter ( nikon , otawara , japan ) .
nuclei with condensed and fragmented chromatin were counted when the photos were analyzed with adobe photoshop software .
data was obtained from 12 wells ( from 2 repeats ) for each treatment condition .
comparisons were made using anova and post - hoc duncan s test using the statistical analysis system program 1998 ( sas institute inc . ,
this in vitro study was conducted in the chemistry and biochemistry institute , university of veterinary medicine , vienna , austria between june and august 2013 in accordance with the guidelines of the european union council ( 86/609/eu ) for the use of laboratory animals .
the work does not require approval from the ethics committee as it used mouse embryos under the fifteenth day of gestation .
primary mesencephalic cell cultures were prepared from c57/b16 embryos according to radad et al.10 to summarize , embryonic mouse mesencephala were dissected on the fourteenth day of gestation and cut into small pieces in a drop of dulbecco s phosphate - buffered saline ( dpbs ) ( invitrogen , darmstadt , germany ) , 2 ml of 0.2% trypsin solution ( invitrogen , darmstadt , germany ) and 2 ml of 0.02% dnase i solution ( roche , berlin , germany ) were added and the tissue was subsequently incubated in a water bath at 37c for 7 minutes ( min ) .
then , 2 ml of trypsin inhibitor ( 0.125mg / ml ) ( invitrogen , darmstadt , germany ) were added , the tissue was centrifuged at 100 g for 4 min and the supernatant was aspirated .
the tissue pellet was triturated 2 - 3 times with a fire - polished pasteur pipette , each time 0.02% dnase i ( invitrogen , darmstadt , germany ) was included in the medium .
dissociated cells were plated at a density of 257,000 cells / cm in dulbecco s modified eagle s medium ( dmem ) ( sigma aldrich , hamburg , germany ) supplemented with 4 mm glutamine , 10 mm 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid ( hepes ) buffer , 30 mm glucose , 100 iu / ml penicillin , 0.1 mg / ml streptomycin , and 10% heat - inactivated fetal calf serum ( sigma aldrich , hamburg , germany ) .
the medium was exchanged on the first day in vitro ( div ) and on the third div . on the fifth div ,
half of the medium was replaced by serum - free dmem containing 0.02 ml b-27/ml ( invitrogen , darmstadt , germany ) dmem .
serum - free supplemented dmem was used for feeding from the sixth div , and subsequently replaced every second day .
a stock solution of tq ( sigma aldrich , hamburg , germany ) ( 10 mm ) was prepared in dimethyl sulfoxide ( dmso ) .
four sets of cultures were treated as follows : the first set of cultures was treated with dmso and kept as untreated controls .
the second set of cultures was treated with tq ( 0.01 , 0.1 , 1 , and 10 m ) on the eighth div for 4 consecutive days to investigate the effect of tq on the survival of dopaminergic neurons .
the third set of cultures was treated with 10 m of mpp on the tenth div for 48 hours ( h ) .
the fourth set of cultures was concomitantly treated with tq ( 0.01 , 0.1 , 1 , and 10 ) , and 10 m of mpp on the tenth div for 48 h.
cultures were rinsed carefully with phosphate buffered saline ( pbs , ph 7.2 ) at the end of each treatment and fixed in 4% paraformaldehyde for 45 min at 4c . after washing with pbs , cells were permeabilized with 0.4% triton x-100 for 30 min at room temperature .
cultures were washed 3 times with pbs and incubated with 5% horse serum ( vectastain abc elite kit , biozol diagnostica vertrieb gmbh , eching , germany ) for 90 min to block nonspecific binding sites . to determine the number of thir in cultures
, cells were sequentially incubated with anti - th primary antibody overnight at 4c , biotinylated secondary antibody ( vectastain ) , and avidin - biotin - horseradish peroxidase complex ( vectastain ) for 90 min at room temperature and washed with pbs between stages .
the reaction product was developed in a solution of diaminobenzidine ( 1.4 mm ) in pbs containing 3.3 mm hydrogen peroxide and stained cells were counted with a nikon inverted microscope in 10 randomly selected fields per well at 10x magnification .
cellular injury was quantitatively assessed by measuring the activity of lactate dehydrogenase ( ldh ) released from damaged cells into the culture medium .
the reaction was initiated by mixing 0.2 ml of cell - free supernatant ( diluted 1:1 with aqua dest . ) with potassium phosphate buffer containing - nicotinamide adenine dinucleotide ( nadh ) and sodium pyruvate ( 0.18 and 0.62 mm in potassium phosphate buffer ) in a final volume of 0.5 ml in 1 ml cuvettes .
the decrease of nadh was spectrophotometrically ( novaspec ii , ge healthcare europe gmbh , freiburg , germany ) monitored .
the ldh activity was calculated from the slope of the decrease in optical density at 334 nm over a 3 min - time period .
lysotracker deep red ( life technologies , invitrogen , grand island , ny , usa ) is a red fluorescence dye used for labeling acidic organelles in live cells including autophagolysosomes .
cultures were treated with 1 m of tq ( a concentration that significantly protected dopaminergic neurons in mpp - treated cultures ) on the eighth div and co - administered with mpp ( 10 m ) on the tenth div for 2 days . on the twelfth div ,
culture medium was aspirated and cultured cells were incubated with a new medium containing 100 nm lysotracker deep red fluorescence dye ( life technologies , invitrogen , grand island , ny , usa ) for 15 - 30 min at 37c . after washing with dpbs ,
cultured cells were photographed on a nikon inverted microscope equipped with epifluorescence attachment using a rhodamine filter set ( 580/590 , g-2a ) and a coolpix 990 digital camera ( nikon , otawara , japan ) .
5,5,6,6-tetrachloro-1,1,3,3-tetraethylbenzimidazolyl - carbocyanine ( jc-1 ) is a lipophilic cationic dye that selectively enters into mitochondria . in healthy cells with high mitochondrial membrane potential ( m )
the dye remains in the monomeric from with green fluorescence in case of apoptotic or damaged cells .
the jc-1 red : green ratio is used to estimate changes in m.13 the jc-1 was dissolved in dmso and further diluted in dmem ( 10 g / ml final concentration ) .
after removal of the culture medium , cells were loaded with jc-1 for 15 min at 37c , rinsed twice with pbs , and photographed on a nikon inverted microscope equipped with epifluorescence attachment using a rhodamine filter set ( 520 dm/520 ba , b-2a ) and a coolpix 990 digital camera ( nikon , otawara , japan ) .
fluorescence intensity of the red : green ratio was determined semi quantitively by using adobe photoshop software .
cells were fixed with 4% paraformaldehyde for 45 min at 4c . after washing with pbs ( ph 7.2 ) ,
the dapi solution ( 2 m final concentration ) was added to the cultures at room temperature for 5 min in the dark . after washing with dpbs
, 3 photos were taken randomly from each well with a coolpix 990 digital camera connected to an inverted microscope with epifluorescence attachment using an ultraviolet filter ( nikon , otawara , japan ) .
nuclei with condensed and fragmented chromatin were counted when the photos were analyzed with adobe photoshop software .
data was obtained from 12 wells ( from 2 repeats ) for each treatment condition .
comparisons were made using anova and post - hoc duncan s test using the statistical analysis system program 1998 ( sas institute inc . ,
treatment of cultures with tq ( 0.01 , 0.1 , 1 , and 10 m ) on the eighth div for 4 consecutive days produced no significant effects on either the survival rate or the morphology of thir neurons ( data not shown ) .
treatment of cultures with mpp ( 10 m on the eighth div for 48 h ) decreased the number of dopaminergic neurons by around 40% compared with untreated control cultures ( figure 1a ) .
surviving neurons after mpp treatment showed fewer , shortened , and thickened neurites ( figure 1b ) .
co - treatment of cultures with tq ( on the eighth div for 4 days ) and mpp ( 10 m on the tenth div for 48 h ) prevented dopaminergic cell loss by around 25% at 0.1 and 1 m ( figure 1a ) , and improved the morphology of surviving neurons compared to mpp - treated cultures ( figure 1b ) .
anti - th immunohistochemical staining of cultured cells showing : a ) survival of dopaminergic neurons in primary mesencephalic cell cultures .
100% corresponds to the total number of thir neurons after 12 div in untreated controls .
values represent the meansem of 3 independent experiments with 4 wells in each treatment . in each well , 10 randomly selected fields were counted for th immunocytochemistry ( # p=0.001 , * p=0.008 , + p=0.009 ) .
the mpp - treated cultures showed thir neurons with few , shortened and thickened neuritis ( arrows ) .
treatment with tq improves the morphology of thir neurons compared to mpp - treated cultures .
th - tryosine hydrolase , thir - tyrosine hydroxylase immunoreactive , div - day in vitro , sem - standard error of mean , mpp - 1-methyl-4-phenylpyridinium , tq - thymoquinone the tq attenuated mpp - induced ldh increase in primary mesencephalic cell culture .
the mpp ( 10 m from the tenth to twelfth div ) increased ldh release in the culture medium by 145% compared with untreated cultures ( figure 2 ) .
the tq significantly decreased ldh release in the culture medium by around 70% at 0.1 and 0.1 m concentrations compared with mpp - treated cultures ( figure 2 ) .
div - day in vitro , sem - standard error of mean , mpp - 1-methyl-4-phenylpyridinium , tq - thymoquinone the tq increased lysotracker deep red fluorescence and the red : green fluorescence ratio of jc-1 , and decreased mpp - induced apoptotic cell death in primary mesencephalic cell culture .
lysotracker deep red fluorescent intensity increased 3 folds ( 682% ) in the cultures co - treated with tq and mpp compared with the cultures treated with mpp alone ( 222% ) ( figure 3a ) . in parallel , cultures co - treated with mpp and tq showed higher red fluorescence than the cultures treated with mpp alone ( figure 3b ) .
lysotracker deep red fluorescence staining of cultured cells showing : a ) lysotracker deep red fluorescence intensity in primary mesencephalic cell cultures .
100% corresponds to the density of lysotracker deep red in primary mesencephalic cell cultures after 12 div .
fluorescence intensity was determined densitometrically from 12 randomly selected micrographs in each experiment ( 3 photos from each well ) .
( # p=0.05 , * p=0.0001 ) b ) representative micrographs showing that treatment of cultures with tq increased lysotracker deep red fluorescence intensity compared with mpp - treated cultures .
div - day in vitro , sem - standard error of mean , tq - thymoquinone , mpp - 1-methyl-4-phenylpyridinium treatment of cultures with mpp ( 10 m on the tenth div for 48 h ) caused dissipation of m .
cultures treated with mpp showed a significant decrease in red : green fluorescence ratio of jc-1 by around 17% compared with untreated controls ( figure 4a ) . however , co - treatment of mpp - treated cultures with 1 m tq from the eighth - twelfth div significantly increased m as it increased the red : green fluorescence ratio of jc-1 by around 24% compared with mpp - treated cultures ( figure 4a ) .
as shown in figure 4b , mpp - treated cultures co - administered with tq displayed much higher red fluorescence than the cultures treated with mpp alone .
5,5,6,6-tetrachloro-1,1,3,3-tetraethylbenzimidazolyl - carbocyanine ( jc-1 ) fluorescence staining of cultured cells showing : a ) red : green fluorescence ratio of jc-1 in primary mesencephalic cell cultures .
100% corresponds to the red : green fluorescence ratio of jc-1 in primary mesencephalic cell cultures after 12 div .
red : green fluorescence ratio of jc-1 was determined densitometrically from 12 randomly selected micrographs in each experiment ( 3 photos from each well ) .
( # p=0.0001 , * p=0.0001 ) b ) representative micrographs showing that treatment of cultures with tq increased red fluorescence compared to mpp+-treated cultures which exhibits marked green fluorescence .
tq - thymoquinone , mpp - 1-methyl-4-phenylpyridinium staining of cultured cells with the nuclear fluorescence dye , dapi revealed that mpp ( 10 m on the tenth div for 48 h ) increased the number of nuclei showing apoptotic features by 139% compared with untreated cultures ( figure 5a ) .
against mpp , tq was shown to decrease the number of apoptotic nuclei by around 100% compared with mpp - treated cultures ( figure 5a ) .
4,6-diamidino-2-phenylindole fluorescence staining of cultured cells showing : a ) number of nuclei showing apoptotic features with condensed and fragmented chromatin in primary mesencephalic cell cultures .
100% corresponds to the number of apoptotic nuclei in untreated control cultures after 12 div .
( # p=0.0001 , * p=0.0001 ) b ) representative micrographs showing that treatment of cultures with tq decreased the number of apoptotic nuclei compared to mpp - treated cultures .
tq - thymoquinone , mpp - 1-methyl-4-phenylpyridinium summary of the neuroprotective effect of tq against mpp treatment in primary mesencephalic cell culture .
treatment of cultures with tq ( 0.01 , 0.1 , 1 , and 10 m ) on the eighth div for 4 consecutive days produced no significant effects on either the survival rate or the morphology of thir neurons ( data not shown ) .
treatment of cultures with mpp ( 10 m on the eighth div for 48 h ) decreased the number of dopaminergic neurons by around 40% compared with untreated control cultures ( figure 1a ) .
surviving neurons after mpp treatment showed fewer , shortened , and thickened neurites ( figure 1b ) .
co - treatment of cultures with tq ( on the eighth div for 4 days ) and mpp ( 10 m on the tenth div for 48 h ) prevented dopaminergic cell loss by around 25% at 0.1 and 1 m ( figure 1a ) , and improved the morphology of surviving neurons compared to mpp - treated cultures ( figure 1b ) .
anti - th immunohistochemical staining of cultured cells showing : a ) survival of dopaminergic neurons in primary mesencephalic cell cultures .
100% corresponds to the total number of thir neurons after 12 div in untreated controls .
values represent the meansem of 3 independent experiments with 4 wells in each treatment . in each well , 10 randomly selected fields were counted for th immunocytochemistry ( # p=0.001 , * p=0.008 , + p=0.009 ) .
the mpp - treated cultures showed thir neurons with few , shortened and thickened neuritis ( arrows ) .
treatment with tq improves the morphology of thir neurons compared to mpp - treated cultures .
th - tryosine hydrolase , thir - tyrosine hydroxylase immunoreactive , div - day in vitro , sem - standard error of mean , mpp - 1-methyl-4-phenylpyridinium , tq - thymoquinone the tq attenuated mpp - induced ldh increase in primary mesencephalic cell culture .
the mpp ( 10 m from the tenth to twelfth div ) increased ldh release in the culture medium by 145% compared with untreated cultures ( figure 2 ) .
the tq significantly decreased ldh release in the culture medium by around 70% at 0.1 and 0.1 m concentrations compared with mpp - treated cultures ( figure 2 ) .
div - day in vitro , sem - standard error of mean , mpp - 1-methyl-4-phenylpyridinium , tq - thymoquinone the tq increased lysotracker deep red fluorescence and the red : green fluorescence ratio of jc-1 , and decreased mpp - induced apoptotic cell death in primary mesencephalic cell culture .
lysotracker deep red fluorescent intensity increased 3 folds ( 682% ) in the cultures co - treated with tq and mpp compared with the cultures treated with mpp alone ( 222% ) ( figure 3a ) . in parallel , cultures co - treated with mpp and tq showed higher red fluorescence than the cultures treated with mpp alone ( figure 3b ) .
lysotracker deep red fluorescence staining of cultured cells showing : a ) lysotracker deep red fluorescence intensity in primary mesencephalic cell cultures .
100% corresponds to the density of lysotracker deep red in primary mesencephalic cell cultures after 12 div .
fluorescence intensity was determined densitometrically from 12 randomly selected micrographs in each experiment ( 3 photos from each well ) .
( # p=0.05 , * p=0.0001 ) b ) representative micrographs showing that treatment of cultures with tq increased lysotracker deep red fluorescence intensity compared with mpp - treated cultures .
div - day in vitro , sem - standard error of mean , tq - thymoquinone , mpp - 1-methyl-4-phenylpyridinium treatment of cultures with mpp ( 10 m on the tenth div for 48 h ) caused dissipation of m .
cultures treated with mpp showed a significant decrease in red : green fluorescence ratio of jc-1 by around 17% compared with untreated controls ( figure 4a ) . however , co - treatment of mpp - treated cultures with 1 m tq from the eighth - twelfth div significantly increased m as it increased the red : green fluorescence ratio of jc-1 by around 24% compared with mpp - treated cultures ( figure 4a ) .
as shown in figure 4b , mpp - treated cultures co - administered with tq displayed much higher red fluorescence than the cultures treated with mpp alone .
5,5,6,6-tetrachloro-1,1,3,3-tetraethylbenzimidazolyl - carbocyanine ( jc-1 ) fluorescence staining of cultured cells showing : a ) red : green fluorescence ratio of jc-1 in primary mesencephalic cell cultures .
100% corresponds to the red : green fluorescence ratio of jc-1 in primary mesencephalic cell cultures after 12 div .
red : green fluorescence ratio of jc-1 was determined densitometrically from 12 randomly selected micrographs in each experiment ( 3 photos from each well ) .
( # p=0.0001 , * p=0.0001 ) b ) representative micrographs showing that treatment of cultures with tq increased red fluorescence compared to mpp+-treated cultures which exhibits marked green fluorescence .
tq - thymoquinone , mpp - 1-methyl-4-phenylpyridinium staining of cultured cells with the nuclear fluorescence dye , dapi revealed that mpp ( 10 m on the tenth div for 48 h ) increased the number of nuclei showing apoptotic features by 139% compared with untreated cultures ( figure 5a ) .
against mpp , tq was shown to decrease the number of apoptotic nuclei by around 100% compared with mpp - treated cultures ( figure 5a ) .
4,6-diamidino-2-phenylindole fluorescence staining of cultured cells showing : a ) number of nuclei showing apoptotic features with condensed and fragmented chromatin in primary mesencephalic cell cultures .
100% corresponds to the number of apoptotic nuclei in untreated control cultures after 12 div .
( # p=0.0001 , * p=0.0001 ) b ) representative micrographs showing that treatment of cultures with tq decreased the number of apoptotic nuclei compared to mpp - treated cultures .
tq - thymoquinone , mpp - 1-methyl-4-phenylpyridinium summary of the neuroprotective effect of tq against mpp treatment in primary mesencephalic cell culture .
in the present study , tq was investigated to ascertain whether it protected mesencephalic dopaminergic neurons against mpp - induced cell death through activation of enzymatic degradation , preservation of mitochondrial function , and inhibition of apoptotic cell death .
clearly , mpp was found to significantly decrease the survival of dopaminergic neurons and increase the release of ldh into the culture medium .
the mpp toxicity involves its selective uptake by dopaminergic neurons through the dopamine transporter and inhibition of mitochondrial complex i activity with subsequent mitochondrial depolarization.14 in parallel , the use of jc-1 fluorescence dye in our current study showed that mpp significantly decreased the m of cultured cells as indicated by the decreasing red : green fluorescence ratio of jc-1 .
similar mpp - induced reduction of m was reported in other in vitro disease models.15,16 mitochondrial damage has long been implicated in the death of nigrostriatal dopaminergic neurons in both pd patients and experimental models.17,18 staining of primary dopaminergic cultures with blue - fluorescent dapi nucleic acid stain showed that a significant number of the cells displayed features of apoptosis , most notably chromatin condensation , and fragmentation .
previously , tang et al19 and xu et al16 demonstrated that mpp caused apoptotic cell death in pc12 and sh - sy5y cells .
the mpp - induced apoptosis was reported to occur as the result of disruption of mitochondrial transmembrane potential and opening of the permeability transition pore.20 similar to our previous report,9 co - treatment of primary mesencephalic cell cultures with tq and mpp was found to protect dopaminergic neurons and decreased the release of ldh into the culture medium . since that time , no evidence in the literature has shown how tq protected dopaminergic neurons in the primary mesencephalic cell culture .
staining of cultures with lysotracker deep red showed that tq significantly increased the red fluorescence of the dye compared with mpp - treated cultures , indicating enhancement of the formation of many autophagolysosomes , the sites of lysosomal degradation , by tq .
this is supported by the findings of he and klionsky21 who correlated the fluorescent signals of lysotracker deep red to the upregulation of autophagy in zebrafish . increased red fluorescence of lysotracker deep red
is attributed to the formation of many autophagosomes and autophagolysosomes that retain much dye as the result of increasing their acidification . using jc-1 fluorescent dye showed that tq significantly enhanced m as it increased the red : green fluorescence ratio of jc-1 compared with mpp - treated cultures .
the tq was similarly found to protect rat cortical neurons against ethanol- and a1 - 42-induced neurotoxicity through inhibition of mitochondrial membrane depolarization.22,23 counting of apoptotic nuclei using blue - fluorescent dapi nucleic acid stain indicated that tq decreased mpp - induced apoptotic cell death in primary mesencephalic cell cultures . in accordance ,
ullah et al22 reported that tq inhibited apoptotic cell death in ethanol - treated rat cortical neurons and attributed this effect of tq to the preservation of mitochondrial integrity .
zhang et al24 reported that mitochondrial clearance protected cultured cortical neurons against ischemia - reperfusion - induced cell damage . in conclusion , correlating such results would therefore suggest that tq might activate a lysosomal degradative process in dopaminergic neurons , where clearance of damaged mitochondria results in reduced mitochondria - mediated apoptotic cell death .
this might raise the possibility of using tq as a potentially therapeutic intervention in pd patients .
whenever a manuscript contains material ( tables , figures , etc . ) which is protected by copyright ( previously published ) , it is the obligation of the author to obtain written permission from the holder of the copyright ( usually the publisher ) to reproduce the material in neurosciences .
please submit copies of the material from the source in which it was first published .
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objectives : to investigate potential mechanisms mediating the neuroprotective effect of thymoquinone ( tq ) on dopaminergic neurons.methods:this study was conducted in the chemistry and biochemistry institute , university of veterinary medicine , vienna , austria between june and august 2013 .
primary cultures were prepared from embryonic mouse mesencephala ( ofi / spf ) at gestation day 14 .
four sets of cultures were kept untreated , treated with tq on the eighth day in vitro ( div ) for 4 days , treated with 1-methyl-4-phenylpyridinium ( mpp+ ) on the tenth div for 48 hours and co - treated with thymoquinone and mpp+ . on the twelfth div
, cultures were subjected to immunohistochemistry against tyrosine hydroxylase and fluorescent staining using lysotracker deep red , 5,5,6,6-tetrachloro-1,1,3,3-tetraethyl benzimidazolylcarbocyanine ( jc-1 ) and 4,6-diamidino-2-phenylindole stains.results:the mpp+ decreased the number of dopaminergic neurons by 40% , and increased the release of lactate dehydrogenase ( ldh ) into the culture medium .
the tq significantly rescued dopaminergic neurons and decreased the release of ldh at the concentrations of 0.1 and 1 m . the tq significantly shifted the red fluorescent intensity of the lysotracker deep red , increased the mitochondrial membrane potential as it increased the red : green florescent ratio of jc-1 , and decreased mpp+-induced apoptotic cell death.conclusion:the tq protects dopaminergic neurons in primary mesencephalic culture by enhancing lysosomal degradation that clears damaged mitochondria and inhibits mitochondria - mediated apoptotic cell death .
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Methods
Preparation of primary mesencephalic cell culture
Study design and treatment of primary mesencephalic cell cultures
Identification of tyrosine hydroxylase immunoreactive (THir) neurons
Measurement of lactate dehydrogenase activity
Fluorescence staining
Results
Effect of TQ on the survival of dopaminergic neurons
Discussion
None
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it is generally accepted that microleakage between the filling materials and root canal
walls might adversely affect the outcome of root canal treatment .
therefore , it is critical the complete
sealing of the root canal system after cleaning and shaping in order to avoid the
bacterial penetration and re - infection of the root and periapical tissues .
the association of gutta - percha cones
and root canal sealer has been traditionally used for this purpose . however , in the last
decade , the dentin adhesive technology has been incorporated into the root canal filling
techniques to reduce apical and coronal leakage by bonding to root canal walls .
etch - and - rinse adhesives have been
tested with resin cements and the
combination of a dentin - bonding agent and an epoxy resin - based root canal sealer
significantly reduced apical leakage . in restorative dentistry , the self - etch adhesive systems have shown less technique
sensitivity , with reliable long - term performance of a two - step mild self - etch
adhesive . following this trend ,
pentron clinical technologies
( wallingford , ct , usa ) has developed the epiphany system , which contains a self - etch
primer , a dual - cured composite resin sealer and a polyester - based thermoplastic
root - filling material ( resilon ; resilon research llc , madison , ct , usa ) .
according to shipper , et al .
( 2004 )
this material has been shown to be more resistant to bacterial leakage than epoxy
resin - based sealers for filling root canals . in endodontics ,
the controversy about the performance of adhesive systems inside the
root canal remains . despite that ,
other possibility is combining epiphany primer with ah plus in an attempt to add the
hybridization capacity to the gold standard endodontic sealer .
the reason for this is
that the removal of the smear layer with ethylenediamine tetraacetic acid ( edta ) does
not provide the same etching pattern usually associated with the hybrid layer , which is
considered an important factor for dentin bonding .
the majority of studies have evaluated apical or coronal microleakage , and few have
focused gap formation at the dentin / sealer interface .
so far , no correlation between microleakage and gap
formation has been established in the literature .
thus , it is reasonable to believe that
the association of both methods would provide a more precise evaluation of the adhesive
interface .
the aim of this study was to compare the apical sealing and gap formation of ah
plus / gutta - percha with epiphany system .
in addition , the opportunity was taken to assess
the effect of the association of epiphany primer and ah plus / gutta - percha on apical
sealing and gap formation .
the null hypotheses tested were as follows : ( 1 ) there is no
difference in apical microleakage or ( 2 ) apical gap formation among the experimental
groups and ( 3 ) there is no correspondence between apical microleakage and the presence
of gaps .
thirty - nine lower single - rooted human premolars with straight root canals and fully
developed apices ( local ethics committee approval 177/05 ) were cleaned and submitted
to 18.5 kgy gamacell radiation ( nuclear energy research institute , so paulo ,
sp , brazil ) , and stored in saline solution at 4c .
after endodontic access ,
the real working length ( rwl ) was established 1 mm short of the apical foramen .
a
crown - down technique was used ( up to k - file # 50 ) under constant irrigation with 0.5%
naocl .
the smear layer was removed with 17% edta ( 5 ml ) and 0.5% naocl ( 5
ml ) followed by saline
solution ( 15 ml ) .
three coats of nail polish were applied to
external root surfaces except for the apical 2 mm .
the teeth were randomly ( http://www.random.org ) divided into
3 experimental groups ( n=11 ) .
the endodontic sealers were
prepared according to the manufacturer 's instructions and the cold lateral
condensation filling technique ( lc ) was used according with the following
description : an iso # 50 master gutta - percha cone was lightly coated with ah plus sealer ( ah
plus ; dentsply detrey , konstanz , germany ) and placed into the
canal to rwl .
a size b finger spreader ( dentsply maillefer , ballaigues , switzerland )
was then inserted into the canal to a level approximately 1 mm short of rwl .
lc with
accessory gutta - percha cones was performed until the entirely filled root canal .
the
excess gutta - percha was removed with a heated plugger and then compacted
vertically .
adhesive - modified technique was used for bonding ah plus to intraradicular dentin . a
paper point soaked with epiphany primer
was used to etch dentin ( 30 s ) and the excess
was removed with paper points .
the coronal surface of the root filling was
light - cured for 40 s ( 600 mw / cm ) .
the positive controls ( n=3 ) were left unfilled and coated as described earlier . the
negative controls ( n=3 ) were filled and totally coated , including the apical
foramina .
the openings were sealed ( cavit w , 3 m espe , st paul , mn , usa ) , and
stored in a chamber held at 100% humidity and 37c for 7 days .
next , all teeth
were immersed in a 50-wt% aqueous silver nitrate solution ( agno3 , ph7.0 )
in the darkness that was buffered using naoh 0.1 n for 24 h at 37c .
the silver - impregnated teeth
were rinsed and placed in photodeveloper ( 8 h ) in fluorescence light to reduce the
silver ions into metallic silver .
lake bluff ,
il , usa ) , longitudinally sectioned in an isomet 1000 precision saw ( buehler ) at low
speed ( 200 rpm ) with a water - cooled diamond blade .
the interfaces were etched with a
35% phosphoric acid solution ( 5 s ) , rinsed with distilled water ( 30 s ) and gently
air - dried .
then , specimen preparation followed the protocol 1 for apical microleakage
analysis using scanning electron microscopy / energy dispersive spectroscopy ( sem / eds ) ;
or the protocol 2 for gaps analysis using sem according to the following
description : specimens were fixed , dehydrated in ascending grades of ethanol and final chemical
drying in hmds ( hexamethyldisilazane , sigma - aldrich inc . ,
louis , mo , usa ) for 10
min , and covered with
carbon ( sputter coater scd 050 , bal - tec ag , balzers , liechtenstein ) .
the apical 5 mm
of the root canal filling were divided into 5 regions of 1 mm to evaluate
microleakage by sem leo stereoscan 440 ( leo electron microscopy ltd . ,
cambridge ,
england ) using back scattered electrons ( bse ) mode ( figure 1a ) .
the eds ( inca software , oxford , uk , england ) was performed in
lower magnification within a pre - determined area ( 300 m ) ( figure 1b ) , and in a higher magnification , the
identification of silver was made punctually to determine its exact location ( figure 1c ) .
each 1 mm region was classified
according to the following scores : 0 ( absence of leakage in both interfaces ) and 1
( presence of leakage in at least one of the interfaces ) . in figures 1d and 1e
scanning electron microscopy ( sem ) micrographs using back scattered electrons
( bse ) mode and corresponding energy dispersive spectroscopy ( eds ) spectrum of
agno3 leakage : ( a ) - the apical 5 mm of the root canal filling
divided into 1 mm - regions .
pointer at the detected metallic silver ; ( b ) area
( 300 m ) scanned for the existence of silver and
respective eds spectrum ( arrow ) and ( c ) - in detail , punctual eds confirmation
of its exact location .
gp = gutta - percha ; d = dentin impressions of polyvinyl siloxane ( aquasil , ulv , dentsply detrey ) were
made of the interfaces and the surfaces replicated with epoxy resin
( epon - thin , buhler ) .
the replicas were covered with carbon to
investigate the presence of gaps at the dentin / sealer and sealer / cone interfaces , as
described previously , using secondary electrons ( se ) mode .
as the gaps were not
continuous , for each 1 mm - region , two interfaces were analyzed ( figure 2a ) and classified as : type 0 : both interfaces were
gap - free , type 1 : gap at dentin / sealer interface ( figure 2b ) , type 2 : gap at sealer / cone interface ( figure 2c ) , and type 3 : both types of gaps present .
therefore ,
for each experimental group , 11 replicas were prepared ; 55 regions were evaluated and
accordingly classified .
scanning electron microscopy ( sem ) micrographs using secondary electrons ( se )
and back scattered electrons ( bse ) mode of the replica and the section of the
same specimen , respectively .
( a ) - the apical 5 mm of the root canal filling
divided into 1 mmregions .
classification of the types of gaps : ( b ) type 1 :
gap between dentin / sealer ( pointer ) ; air voids were present within the sealer
( open arrow ) ; ( b ' ) - silver penetration was evident along the dentin / sealer
interface , into dentinal tubules in a reticular form and granular aspect on the
dentin surface ( arrowhead ) ; ( c ) type 2 : gap between sealer / cone ( pointer ) -
cohesive fracture of the sealer ( open arrow ) ; ( c ' ) - the area marked in figure
c at higher magnification : silver deposition into the dentinal tubules in a
reticular form and granular aspect within the sealer layer ( arrowhead ) could be
seen ; ( the sealer thickness was indicated between black / white arrowheads .
gp = gutta - percha ; d = dentin ; r = resilon ) the data obtained for apical microleakage and types of gaps were statistically
analyzed ( =0.05 ) using the ordinal logistic regression model and correspondence analysis in the minitab statistical software
program ( minitab inc .
the counting of regions analyzed for gaps was also computed and adjusted in a
generalized linear model with poisson distribution and logarithmic link function .
the
sealers groups ( ah plus , ah primer and epiphany ) and the type of gap were considered
as explanatory variables and ah plus and the type 0 as references .
thirty - nine lower single - rooted human premolars with straight root canals and fully
developed apices ( local ethics committee approval 177/05 ) were cleaned and submitted
to 18.5 kgy gamacell radiation ( nuclear energy research institute , so paulo ,
sp , brazil ) , and stored in saline solution at 4c .
after endodontic access ,
the real working length ( rwl ) was established 1 mm short of the apical foramen .
a
crown - down technique was used ( up to k - file # 50 ) under constant irrigation with 0.5%
naocl .
the smear layer was removed with 17% edta ( 5 ml ) and 0.5% naocl ( 5
ml ) followed by saline
solution ( 15 ml ) .
three coats of nail polish were applied to
external root surfaces except for the apical 2 mm .
the teeth were randomly ( http://www.random.org ) divided into
3 experimental groups ( n=11 ) .
the endodontic sealers were
prepared according to the manufacturer 's instructions and the cold lateral
condensation filling technique ( lc ) was used according with the following
description :
an iso # 50 master gutta - percha cone was lightly coated with ah plus sealer ( ah
plus ; dentsply detrey , konstanz , germany ) and placed into the
canal to rwl .
a size b finger spreader ( dentsply maillefer , ballaigues , switzerland )
was then inserted into the canal to a level approximately 1 mm short of rwl .
lc with
accessory gutta - percha cones was performed until the entirely filled root canal .
the
excess gutta - percha was removed with a heated plugger and then compacted
vertically .
adhesive - modified technique was used for bonding ah plus to intraradicular dentin . a
paper point soaked with epiphany primer
was used to etch dentin ( 30 s ) and the excess
was removed with paper points .
the coronal surface of the root filling was
light - cured for 40 s ( 600 mw / cm ) .
the positive controls ( n=3 ) were left unfilled and coated as described earlier . the
negative controls ( n=3 ) were filled and totally coated , including the apical
foramina .
the openings were sealed ( cavit w , 3 m espe , st paul , mn , usa ) , and
stored in a chamber held at 100% humidity and 37c for 7 days .
next , all teeth
were immersed in a 50-wt% aqueous silver nitrate solution ( agno3 , ph7.0 )
in the darkness that was buffered using naoh 0.1 n for 24 h at 37c .
the silver - impregnated teeth
were rinsed and placed in photodeveloper ( 8 h ) in fluorescence light to reduce the
silver ions into metallic silver .
lake bluff ,
il , usa ) , longitudinally sectioned in an isomet 1000 precision saw ( buehler ) at low
speed ( 200 rpm ) with a water - cooled diamond blade .
the interfaces were etched with a
35% phosphoric acid solution ( 5 s ) , rinsed with distilled water ( 30 s ) and gently
air - dried .
then , specimen preparation followed the protocol 1 for apical microleakage
analysis using scanning electron microscopy / energy dispersive spectroscopy ( sem / eds ) ;
or the protocol 2 for gaps analysis using sem according to the following
description :
specimens were fixed , dehydrated in ascending grades of ethanol and final chemical
drying in hmds ( hexamethyldisilazane , sigma - aldrich inc . , st .
louis , mo , usa ) for 10
min , and covered with
carbon ( sputter coater scd 050 , bal - tec ag , balzers , liechtenstein ) .
the apical 5 mm
of the root canal filling were divided into 5 regions of 1 mm to evaluate
microleakage by sem leo stereoscan 440 ( leo electron microscopy ltd . ,
cambridge ,
england ) using back scattered electrons ( bse ) mode ( figure 1a ) .
the eds ( inca software , oxford , uk , england ) was performed in
lower magnification within a pre - determined area ( 300 m ) ( figure 1b ) , and in a higher magnification , the
identification of silver was made punctually to determine its exact location ( figure 1c ) .
each 1 mm region was classified
according to the following scores : 0 ( absence of leakage in both interfaces ) and 1
( presence of leakage in at least one of the interfaces ) . in figures 1d and 1e
scanning electron microscopy ( sem ) micrographs using back scattered electrons
( bse ) mode and corresponding energy dispersive spectroscopy ( eds ) spectrum of
agno3 leakage : ( a ) - the apical 5 mm of the root canal filling
divided into 1 mm - regions .
pointer at the detected metallic silver ; ( b ) area
( 300 m ) scanned for the existence of silver and
respective eds spectrum ( arrow ) and ( c ) - in detail , punctual eds confirmation
of its exact location .
impressions of polyvinyl siloxane ( aquasil , ulv , dentsply detrey ) were
made of the interfaces and the surfaces replicated with epoxy resin
( epon - thin , buhler ) .
the replicas were covered with carbon to
investigate the presence of gaps at the dentin / sealer and sealer / cone interfaces , as
described previously , using secondary electrons ( se ) mode .
as the gaps were not
continuous , for each 1 mm - region , two interfaces were analyzed ( figure 2a ) and classified as : type 0 : both interfaces were
gap - free , type 1 : gap at dentin / sealer interface ( figure 2b ) , type 2 : gap at sealer / cone interface ( figure 2c ) , and type 3 : both types of gaps present .
therefore ,
for each experimental group , 11 replicas were prepared ; 55 regions were evaluated and
accordingly classified .
scanning electron microscopy ( sem ) micrographs using secondary electrons ( se )
and back scattered electrons ( bse ) mode of the replica and the section of the
same specimen , respectively .
( a ) - the apical 5 mm of the root canal filling
divided into 1 mmregions .
classification of the types of gaps : ( b ) type 1 :
gap between dentin / sealer ( pointer ) ; air voids were present within the sealer
( open arrow ) ; ( b ' ) - silver penetration was evident along the dentin / sealer
interface , into dentinal tubules in a reticular form and granular aspect on the
dentin surface ( arrowhead ) ; ( c ) type 2 : gap between sealer / cone ( pointer ) -
cohesive fracture of the sealer ( open arrow ) ; ( c ' ) - the area marked in figure
c at higher magnification : silver deposition into the dentinal tubules in a
reticular form and granular aspect within the sealer layer ( arrowhead ) could be
seen ; ( the sealer thickness was indicated between black / white arrowheads .
gp = gutta - percha ; d = dentin ; r = resilon ) the data obtained for apical microleakage and types of gaps were statistically
analyzed ( =0.05 ) using the ordinal logistic regression model and correspondence analysis in the minitab statistical software
program ( minitab inc .
the counting of regions analyzed for gaps was also computed and adjusted in a
generalized linear model with poisson distribution and logarithmic link function .
the
sealers groups ( ah plus , ah primer and epiphany ) and the type of gap were considered
as explanatory variables and ah plus and the type 0 as references .
the ordinal logistic regression analysis demonstrated that there was no statistically
significant difference ( p>0.05 ) among the groups tested and types of gaps over
the logit of microleakage .
the model was adjusted to identify the
effect of the type of gap on microleakage .
the type 3 data were not considered isolated
in the analysis ( very few regions ) but joined with type 2 data ( figure 2c ; table 1 ) .
the
adjustment tests of the model presented high p - values for pearson 's test ( p=0.900 ) and
deviance ( p=0.852 ) .
the adjusted model demonstrated that the effect of the type of gap
on the logit of microleakage is significant when compared to the
presence of types 1 and 2 ( figure 2b and 2c , respectively ) of gap with the absence of gap
( p=0.047 ) .
the correspondence analysis also showed the association between microleakage
and both types of apical gaps ( figure 3 ) .
correspondence analysis of microleakage vs. type of gap : ( inf.0 ) - absence of
leakage ; ( inf.1 ) - leakage up to the 1 millimeter ; ( inf.2 )
leakage
up to the 2 mm ; ( inf.3 ) leakage up to the 3
millimeter ; ( inf.5 ) leakage up to the 5 millimeter ; ( f.0 ) absence
of gaps ; ( f.1 ) gaps type 1 ; ( f.2 ) - gaps types 1 and 2 .
the absence of leakage
is associated with the absence of gap ; leakage up to the 1 millimeter
is associated with type 1 ; and more extensive leakage is associated with types 1
and 2 frequency and type of gaps in the apical 5 mm of root canal fillings percentage of regions with gaps .
type 0 : absence of gaps ; type 1 : gaps between
dentin and sealer ; type 2 gaps between sealer and cone the generalized linear model with poisson distribution ( table 1 ) found no difference between ah plus and ah primer ( p=0.497 ) in
distribution of each type of gap , but they presented more type 0 regions
( p<0.001 ) .
there was a significant interaction between epiphany and the type of
gap ( p<0.003 ) and for this group , there were more type 1 regions .
based on the findings of this study , the first null hypothesis could not be rejected .
recently some studies have shown similar results between ah plus and epiphany on apical
sealing ability using the fluid filtration method .
consistent with these
studies , our results indicate that the epiphany system is as effective as ah
plus / gutta - percha in preventing microleakage based on the chemical tracer penetration
analysis by sem and the use of epiphany primer does not reduce the microleakage of ah
plus . nevertheless , other researches pointed out that the epiphany system provided the
greatest resistance to the movement of fluids or dye leakage
test , when compared to epoxy
resin - based root canal sealers , but others have just indicated the opposite .
these apparent discrepancies can perhaps be explained by the
methodology used and their variables .
these results agree with those of previous studies in which , microleakage markers were
used to evaluate apical sealing of the same endodontic sealers .
the tracer
selected was agno3 , which may penetrate into dentinal tubules due to their
physical and chemical characteristics including : concentration , smaller molecular size ,
its neutral ph , diffusion coefficient and service life up to 168 h post - preparation time of
solution .
another reason for
this choice is that metallic silver deposits may be observed by sem using back scattered
electrons ( bse ) mode . on the other hand ,
the results of the current study disagree with the findings of
shipper , et al .
an
explanation for this would be that their studies have evaluated coronal and not apical
bacterial leakage . indeed , coronal seal might be favorably influenced by photoactivation
of the material which is unlikely to occur in the apical third .
furthermore , it is speculated that in in
vivo studies ( dogs ) , the high release of calcium hydroxide ( 41.46 mg / l ) that
would occur during the process of epiphany sealer solubilization , would make the medium
alkaline , resulting in acceleration of the periapical tissue repair process .
this study has shown that the presence of apical gaps at the sealer / dentin or
sealer / cone interfaces had an effect on apical microleakage .
the epiphany group
presented more type 1 gaps than the other groups ( table
1 ) , rejecting the second null hypothesis .
these results corroborate with
previous observation of gaps in the apical 4 mm of epiphany or ah plus - filled root
canals .
stress generated
during the polymerization shrinkage of epiphany sealer has probably influenced the
integrity loss at the sealer / dentin interface .
in addition , the cavity configuration factor ( c - factor ) is
highly unfavorable for adhesion inside root canals .
moreover , it is known that analysis of gap formation of
vertically sectioned root filled teeth hides the risk of artifacts during sectioning .
therefore , in this study , the sections were made at low speed under water cooling .
the
resin - epoxy replicas at the dentin / sealer and sealer / cone interfaces were made before
the specimens had been prepared for sem examination in order to differentiate genuine
gaps from artifactual gaps created after vacuum desiccation in conventional scanning
electron microscopes .
few studies have focused on apical sealing from two separate perspectives , such as
microleakage and apical gap formation .
sem was used to evaluate these perspectives , but it was restricted to a descriptive
approach .
a systematization of the observations , complemented by a statistical analysis , such as
the one performed in this study , could provide greater methodological accuracy and
impartiality for comparing the experimental groups .
the correspondence of analysis
between the types of gaps and linear extent of agno3 leakage ( fig .
3 ) confirms the effect of gaps on apical
microleakage , expressed by the silver deposition into gaps that extended deeply inside
dentinal tubules ( fig .
regarding to statistical analysis , the ordinal logistic regression analysis model was
used due to microleakage was considered categorized ordinal response variable ( ranging
from 0 - no leakage to 5 - leakage up to the fifth millimeter ) .
the correspondence
analysis is a method that leads to visualize the association between two categorical
variables , in this case microleakage and the type of gap .
the generalized linear model
with poisson which is an extension of usual regression model was performed to quantify
the distribution of types of gaps .
this result could be explained by two hypotheses : ( 1 )
the low degree of conversion of the epiphany sealer and ( 2 ) the formation of hydrogel at the bond interface resulting
from the incomplete evaporation of the epiphany primer solvent .
the second hypothesis could also explain the inefficacy
of epiphany primer in improving the sealing capacity of ah plus .
moreover , there is a
chemical incompatibility between these two materials , since the epoxy resin sealers do
not copolymerize with methacrylate resin - based adhesives .
other studies should be carried out to clarify the
sealing ability of the root - filled materials studied .
it may be concluded that none of the tested materials completely sealed the apical 5 mm .
the presence
of gaps had an effect on apical microleakage for all materials . comparing the materials ,
epiphany system presented more regions containing gaps between the dentin and the sealer
( type 1 ) . in view of these findings ,
clinically , it can be suggested that ah plus would
provide a better apical seal .
this study was partially supported by capes ( coordenao de
aperfeioamento de pessoal de nvel superior)/institutional
qualification program ( pqi n : 0090/03 - 4 ) .
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objectivethe aim of this study was to compare the correspondence between gap formation and
apical microleakage in root canals filled with epoxy resin - based ( ah plus )
combined or not with resinous primer or with a dimethacrylate - based root canal
sealer ( epiphany ) .
material and methodsthirty - nine lower single - rooted human premolars were filled by the lateral
condensation technique ( lc ) and immersed in a 50-wt% aqueous silver nitrate
solution at 37c ( 24 h ) .
after longitudinal sectioning , epoxy resin
replicas were made from the tooth specimens .
both the replicas and the specimens
were prepared for scanning electron microscopy ( sem ) .
the gaps were observed in
the replicas .
apical microleakage was detected in the specimens by sem / energy
dispersive spectroscopy ( sem / eds ) .
the data were analyzed statistically using an
ordinal logistic regression model and analysis of correspondence ( =0.05 ) .
resultsepiphany presented more regions containing gaps between dentin and sealer
( p<0.05 ) .
there was correspondence between the presence of gaps and
microleakage ( p<0.05 ) .
microleakage was similar among the root - filling
materials ( p>0.05 ) .
conclusionsthe resinous primer did not improve the sealing ability of ah plus sealer and the
presence of gaps had an effect on apical microleakage for all materials .
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INTRODUCTION
MATERIAL AND METHODS
Specimen preparation
Group AH Plus
Group AH Primer
Group epiphany
Positive and negative controls
Protocol 1
Protocol 2
RESULTS
DISCUSSION
CONCLUSIONS
ACKNOWLEDGEMENTS
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according to the u.s . national institutes of health , up to 80% of human bacterial infections involve biofilm - associated microorganisms 1 . among these , implant - related infections
do still have a tremendous impact in orthopaedics and trauma 2 , with high social and economic costs 3 , 4 , posing challenging diagnostic and therapeutic dilemmas 5 .
in fact , peri - prosthetic joint infection ( pji ) remains one of the most feared complications in orthopaedic surgery and among the first reasons for implant failure 6 .
moreover , given the increasing number of hip and knee arthroplasties performed , the prevalence of this complication is rising , with increasing costs for national health systems and increasing biological costs for the patients , such as loss or reduced joint function and deterioration in their physical and psychological health 7 . according to the widely accepted model of the ' race for the surface ' for pji development ,
host and bacterial cells compete for surface colonization , with a low probability of bacterial attachment if host cells adhere to implant first , and vice versa . in the event of bacterial adhesion to an implant ,
in addition , the matrix protects the biofilm cells from various microbicidal agents and stresses , including dehydration , toxins , ultraviolet light , chemical disinfectants , temperature and osmotic shock , and lead them to increased resistance against antimicrobials 9 , 10 . to address the limited efficacy of existing antibiotics in the treatment of established bacterial biofilms , novel approaches are required to prevent bacterial adhesion and biofilm formation 11 .
adhesion is a necessary first step in microbial colonization and pathogenesis and provides a good theoretical target for new preventive and treatment strategies 12 .
bacterial adhesion to surfaces can be divided into a first , reversible phase and a second , irreversible phase .
once an implant is inserted into the body , it is covered by a conditional protein layer composed of host proteins , such as albumin and complement , that act as a reservoir of several receptors for bacterial adhesive ligands , mediating adhesion of free - floating bacteria to the surface of the biomaterials 14 , 15 ; these first adhesions are , however mechanically and biologically unstable .
few minutes after this first , reversible phase , bacterial clusters attached to the surface starts to express biofilm related genes , produce glycocalyx and form mature biofilm , thus transforming the adhesion from reversible to irreversible 16 .
full - formed biofilm can be found few hours after the first bacterial adhesion 17 .
antimicrobial surface coatings can be based on an anti - adhesive principle that prevents bacteria to adhere and form biofilms 18 .
in fact , some polymer coatings , like the hydrophilic polymethacrylic acid , polyethylene oxide or protein - resistant polyethylene glycol can be applied to the surface of titanium implants and result in significant inhibition of bacterial adhesion 19 - 22 .
hydrophobic and superhydrophobic surface treatment technologies have also shown a great repellent antibacterial effect in preclinical studies 23 - 25 .
however , clinical application of completely novel coating technologies and compounds , not otherwise previously tested in humans , appears particularly challenging 26 .
bacterial colonization can also be blocked by an inhibitor interfering with ligand - receptor interaction for bacterial attachment .
one of these inhibitors could be hyaluronic acid ( ha ) , a glycosaminoglycan made up of glucuronic acid and n - acetylglucosamine disaccharide units .
it is a uniform , linear and unbranched molecule , with highly variable length and molecular weight ( up to 106 da ) .
it is abundant in skin ( up to 56% ) and in connective tissues , with a turnover ranging from several hours to a few days depending on tissues .
hyaluronic acid constitutes one of the main components of extracellular matrices . because of its biological properties , ha has several clinical applications ( aesthetic surgery , dermatology , dentistry , orthopedics and opthalmology ) 27 .
extensive studies on the chemical and physicochemical properties of ha and its physiological role in humans , together with its versatile properties , such as its biocompatibility , non - immunogenicity , biodegradability , and viscoelasticity , have proved that it is an ideal biomaterial for medical and pharmaceutical applications 28 , 29 . among its various properties ,
several studies have recently shown the ability of ha to protect against various infectious agents 30 , depending on ha concentration and molecular weight 31 , 32 , while more recently ha interference on bacterial adhesion and biofilm formation has been extensively investigated 33 .
given its high biocompatibility and well known safety profile and the anti - adhesive capabilities , ha and its composites represent an attractive , non - antibiotic , option to mitigate the impact of biofilm - related infections in various clinical settings including implant - related infections . aim of this review is to provide an update of the current evidence concerning ha ability to reduce / prevent bacterial adhesion and biofilm formation .
nearly two decades ago , pavesio et al . 34 were probably the first to describe the ability of ha to resist bacterial adhesion , with particular reference to staphylococcus epidermidis , and its non - fouling properties 35 , proposing coated polymeric medical devices ( e.g. , intraocular lenses , stents and catheters ) to reduce implant - related infections .
in particular , a hydrophilic ha overlayer , linked to the surface of polymethylmethacrylate intraocular lenses ( iols ) , was shown to be able to prevent fibroblasts adhesion and to greatly reduce staphyloccous epidermidis adhesion to the implant surface 36 .
the impact of slime dispersants and anti - adhesives on in vitro biofilm formation on iols was further investigated by kadry and co - workers 37 , using a staphyloccous epidermidis wild strain , isolated from a patient with endophtalmitis ; the authors reported the ability of hyaluronan to reduce bacterial adhesion to iols to 30% , compared with untreated control cells .
the authors suggested the use of adjuvant therapy such as dispersants or anti - adhesives , in addition to the antibiotics in irrigating solutions for bacterial ocular infections .
more recently , the in vitro antiadhesive and antibiofilm activity of hyaluronic acid towards bacterial species commonly isolated from respiratory infections was investigated by drago et al .
33 . in this study , the interference exerted on bacterial adhesion was evaluated by using hep-2 cells , while the antibiofilm activity was assessed by means of spectrophotometry after incubation of biofilm with hyaluronic acid and staining with crystal violet .
the experimental findings clearly demonstrated how hyaluronic acid is able to interfere with bacterial adhesion to a cellular substrate in a concentration - dependent manner .
moreover , staphylococcus aureus biofilm was found to be more sensitive to the action of ha , compared to that produced by haemophilus influenzae and moraxella catarrhalis . concerning more specifically the antimicrobial activity , ha
has also been shown to exert varied bacteriostatic , but not bactericidal , dose - dependent effects on different microorganisms in the planktonic phase 31 , 38 . in this
regard , radaeva et al . reported the inhibiting activity of ha with respect to some pseudomonas species 39 , while ardizzoni and co - workers 30 investigated the effects of ha on 15 atcc bacterial strains , representative of clinically relevant bacterial and fungal species .
their results showed that different microbial species and , sometimes , different strains belonging to the same species , are differently affected by ha . in particular , staphylococci , enterococci , streptococcus mutans , two escherichia coli strains , pseudomonas aeruginosa , candida glabrata and c. parapsilosis displayed a ha dose - dependent growth inhibition , while no ha effects were detected in e. coli atcc 13768 and c. albicans and s. sanguinis was favoured by the highest ha dose . comparing the potential bacteriostatic effect of some of the most commonly used biomatrix materials ( collagen type i , hyaluronic acid , hydroxyapatite , polylactic acid and polyglycolic acid ) on the growth over the first 12h of exposure of some of the most common orthopaedic bacterial pathogens ( staphylococcus aureus , staphylococcus epidermidis , -hemolytic streptococcus , pseudomonas aeruginosa ) , carlson and co - workers 38 found that ha had the most significant bacteriostatic properties on the studied organisms .
31 investigated the potential bacteriostatic effect of hyaluronic acid in different concentrations and molecular weight on oral and non - oral microorganisms ( staphylococcus aureus , propionibacterium acnes , actinobacillus actinomycetemcomitans , pavotella oris and porphyromonas gingivalis ) with potential application in dentistry surgery ; the results showed that different hyaluronan solutions exerted varied bacteriostatic effects on all the bacterial strains .
the authors concluded that the clinical application of hyaluronan in form of membranes , gels , or sponges during surgical therapy may reduce bacterial contamination of the surgical wound , thereby lessening the risk of postsurgical infection and promoting more predictable regeneration .
concerning possible orthopaedic applications , in 2004 harris and richards 40 showed the visualization and quantification of s. aureus adhering to a variety of different treated / coated titanium surfaces . in their study ,
coating titanium with sodium hyaluronate significantly decreased the density of s. aureus adhering to the surfaces and its potential use in osteosynthesis , orthopaedics or dental applications was suggested out . in a very recent review on polysaccharide - based coatings , that have been proposed over the last ten years to impede biofilm formation on material surfaces exposed to bacterial contamination ,
hyaluronic acid was discussed as one of the most studied , with demonstrated non - fouling properties on glass surfaces 41 ; displaying hydrophilic characteristics ( contact angle of 22 ) , this coating was in fact reported to reduce adhesion of s. epidermidis and e. coli by several orders of magnitude compared to the unmodified glass slide .
similarly , adhesion of s. aureus on ti foils functionalized with hyaluronic acid - catechol was lower than on pristine substrates .
based on ha antiadhesive properties , a novel ha - based hydrogel has been recently proposed , in order to protect implanted biomaterials in orthopaedics , trauma and dental surgery from bacterial colonization 42 ; this fast - resorbable hydrogel coating , composed of covalently linked hyaluronan and poly - d , l - lactide ( defensive antibacterial coating , dac , novagenit srl , mezzolombardo , italy ) , has been found to have a synergistic antibiofilm activity with various antibacterials and able to be effectively manually spread onto the surface of various biomaterials commonly used in orthopaedics , trauma and dental surgery 43 ( fig .
1 ) . the ability to completely cover even sand - blasted titanium surface and resist scraping has in fact been confirmed by scanning electron microscopy ( sem ) analysis ( cf .
this is an important requirement in order to reduce the exposed surface of a biomaterial , thus creating a uniform coating of the surface and leaving no pores or cracks that could eventually be colonized by planktonic bacteria . in unpublished experiments ( novagenit srl , data on file ) , in order to evaluate dac ability to prevent bacterial adhesion , 200 mg of hydrogel were homogenously spread on the surface of sterile titanium discs .
hydrogel - coated substrates and uncoated substrates ( controls ) were then placed into sterile 6-wells polystyrene plates and overlaid with a standardized inoculum ( 10 cfu / ml ) of bacterial cells for 15 , 30 , 60 and 120 minutes .
the remaining adhered cells were detached by adding a solution of 0.1% w / v dithiothreitol ( dtt ) ( sigma - aldrich , milan , italy ) to each well and stirring for 15 minutes at room temperature .
then , 100 l of each sample were plated onto tryptic soy agar ( tsa ; merck , darmstadt , germany ) and incubated at 37c for 24 hours for cfu counts .
the results showed that the adhesion density of s. aureus on titanium discs pre - treated with dac , was significantly lower than adhesion on untreated controls at each time point ( fig .
2 ) . in particular , reductions of adhered bacteria equal to 86.8% , 80.4% ,
74.6% and 66.7% vs untreated discs were observed after 15 , 30 , 60 and 120 minutes of incubation , respectively , while an increase of adhesion density during time was observed for both control and pre - treated discs ( fig .
further analyses were conducted to show the ability to dislodge previously adhered bacteria ; to this aim , titanium discs were placed into sterile 6-wells polystyrene plates and overlaid with a standardized inoculum ( 10 cfu / ml ) of bacterial cells in order to allow the adhesion of bacterial cells .
afterwards , 200 mg of hydrogel were spread on the surface of contaminated titanium discs in order to remove previously adhered bacteria .
non - adherent bacteria were removed by rinsing with sterile saline , while the remaining adhered cells were detached by adding 0.1% dtt as previously described .
then , 100 l of each sample were plated onto tsa and incubated at 37c for 24 hours for cfu counts .
the results showed that dac hydrogel treatment of discs reduced the amount of adhered bacteria in respect to control discs after 15 , 30 , 60 and 120 minutes of 84.0% , 72.8% , 72.3% and 64.3% , respectively ( fig.4 ) .
once again , an increase of adhesion density during time was observed for both control and treated discs ( fig .
dac hydrogel showed similar or superior in vitro activity , compared to various antibacterials and a synergistic activity when used in combination ( fig .
were grown on chrome - cobalt devices in 6-wells polystyrene plates containing tsb for 24 hours at 37c .
then , growth medium was removed together with non - adherent bacteria and new broth added .
the plates were incubated at 37c in ambient air , until a visible biofilm was obtained .
gentamycin and vancomycin were tested at a final concentration of 20 mg / ml . similarly , when mixed with the hydrogel , 60 mg of gel powder were reconstituted with 1 ml of water for injections containing gentamicin or vancomycin at 20 mg / ml concentration .
amount of biofilm at each time was determined before hydrogel and antibiotic agents addition and after 0.5 , 1 , 2 , 4 , 6 , 24 and 48 hours of incubation by a spectrophotometric assay .
in particular , at each time , broth was removed and biofilm stained with crystal violet .
after elution of the stain from implants with absolute ethanol , the amount of biofilm was quantified by reading optical density ( o.d . ) at a wavelength of 595 nm against blank ( consisting of ethanol ) .
amount of biofilm at each time was compared with that formed on the same type of implant before treatment .
each assay was performed in duplicate and repeated for three times . at each time point , both for gentamycin and vancomycin showed only a partial inhibition of biofilm formation ( ca .
40 - 50% for vancomycin ) , with minor difference between the two studied microorganisms . on the other side ,
50% in comparison to the untreated controls , while a combination of the hydrogel with either antibacterial resulted in a larger reduction of biofilm formation ( approximately 75 to 80% in comparison with untreated controls ) .
both these experimental studies show the ability of the dac hydrogel to significantly reduce bacterial adhesion and biofilm formation of common bacterial pathogens , thus potentially providing an effective protection of the implant ; however , these data also point out how , in the clinical setting , in the absence of an adequate immune response from the host and/or of sufficient local levels of antibiotics , a passive antiadhesive coating 18 like ha can be overcome by the remaining bacteria in a time - dependent manner .
for this reason , any passive antiadhesive coating of implants 44 should probably better be seen as a tool to reduce and delay bacterial adhesion and biofilm formation to a variable degree , also depending on the local environment , the contaminating bacterial species and initial bacterial load ; this may still provide an additional advantage to the host 's cells to first colonize the implanted biomaterial and win the competition with the microorganisms that may eventually be present , thus contributing to reduce the occurrence of implant - related infections .
several clinical local applications of ha to reduce the impact of biofilm - related infections have been reported , in different clinical settings , with favourable results and no adverse events .
45 recently described topical administration of hyaluronic acid in children with recurrent or chronic middle ear inflammations and chronic adenoiditis . in this prospective , single - blind , randomised controlled study , otoscopy , tympanometry and pure - tone audiometry in children which received the daily topical administration of normal 0.9% sodium chloride saline solution ( control group ) or 9 mg of sodium hyaluronate in 3 ml of a 0.9% sodium saline solution was performed .
the final analysis was based on 116 children ( 49.1% boys ; mean age , 62.9 17.9 months ) : 58 in the control group and 58 in the study group . at the end of follow - up ,
the prevalence of patients with impaired otoscopy was significantly lower in the study group ( p value = 0.024 ) compared to baseline but not in the control group . in comparison with baseline ,
the prevalence of patients with impaired tympanometry at the end of the follow - up period was significantly lower in the study group ( p value = 0.047 ) but not in the control group .
the reduction in the prevalence of patients with conductive hearing loss ( chl ) ( p value = 0.008 ) and those with moderate chl ( p value = 0.048 ) was significant in the study group , but not in the control group .
the mean auditory threshold had also significantly improved by the end of treatment in the study group ( p value = 0.004 ) but not in the control group .
several studies have also reported the beneficial effect of topical ha in chronic urinary tract infections ( uti ) .
in contrast to traditional antibiotic therapy , which aims at eradicating pathogens , treatment with ha targets bacterial adherence to the bladder mucosa with the presumption that a damaged glycosaminoglycan mucous layer facilitates bacterial adherence and therefore recurrent uti 46 . among others 47 , 48 ,
lipovac and colleagues evaluated the efficacy of nine ha bladder instillations over 6 months in 20 women with a history of recurrent uti .
their status was assessed prospectively but compared with a retrospective review of patients ' charts .
the number of infections per year per patient was significantly reduced ( from 4.990.92 to 0.560.82 , p>0.001 ) and the mean time to recurrence ( from 76.724.6 to 178.325.5 days , p>0.001 ) was prolonged significantly .
nevertheless 65% of treated patients were free of recurrences until the end of study ( 47.6 weeks ) 49 .
were able to provide a higher level of evidence by reporting a prospective , randomized , double - blind , placebo - controlled study , in which a significant reduction of 77% ( p<0.0002 ) in the uti rate per patient per year versus placebo was observed at the end of the study .
moreover , mean time to uti recurrence was significantly prolonged ( 185.278.7 versus 52.733.4 days , p<0.001 ) after treatment compared with placebo .
overall urinary symptoms and quality of life measured by questionnaires significantly improved compared with placebo 50 .
very recently a multicentre european study confirmed the efficacy of intravesical administration of combined hyaluronic acid and chondroitin sulphate ( cs ) for the treatment of female recurrent urinary tract infections 51 .
a total of 276 adult women received intravesical administration of ha+cs or standard of care ( antimicrobial/ immunoactive prophylaxis/ probiotics / cranberry ) . at follow - up ,
181 patients treated with ha+cs and 95 patients treated with standard of care from 7 centres were available .
the crude and adjusted ors ( 95% ci ) for bacteriologically confirmed recurrence within 12 months were 0.77 ( 0.46 to 1.28 ) and 0.51 ( 0.27 to 0.96 ) , respectively .
studies were also undertaken to determine the effect on clinical variables , sub - gingival bacteria and local immune response brought about by application of hyaluronan - containing gels in early wound healing after scaling and root planing ( srp ) in dentistry 52 , 53 . in the study reported from eick et al .
the exclusion criteria were : antibiotics intake in the 6 months before the study , periodontal treatment received during the previous year , pregnancy , nursing , smoking , chronic diseases such as diabetes mellitus or rheumatoid arthritis , and allergy to ingredients in the drug . in the test group ( n=17 ) , a 0.8% hyaluronan - containing gels ( ha ) was introduced into all periodontal pockets during srp and a 0.2% ha gel was applied by the patients onto the gingival margin twice daily during the following 2 weeks while the control group ( n=17 ) was treated with srp only ; no placebo was used . probing depth ( pd ) and clinical attachment level ( cal )
were recorded at baseline and after 3 and 6 months , and subgingival plaque and sulcus fluid samples were taken for microbiologic and biochemical analysis . in both groups , pd and cal
the changes in pd and the reduction of the number of pockets with pd5 mm were significantly higher in the test group after 3 ( p=0.014 and 0.021 ) and 6 ( p=0.046 and 0.045 ) months .
six months after srp , the counts of treponema denticola were significantly reduced in both groups ( both p=0.043 ) , as were those of campylobacter rectus in the test group only ( p=0.028 ) .
although to date no surface modification has been reported to be able to fully prevent bacterial adhesion and biofilm formation 55 , available data show that hyaluronic acid has a proven in vitro antiadhesive / antibiofilm effect against some of the most common pathogens and it has been used safely , alone or in combination with other polymers , with satisfactory results in different conditions associated with biofilm - related chronic infections .
clinical data in various applications , including dentistry , urology , wound management , dermatology and orthopedics , allow to consider the potential use of ha as a protective coating barrier of implants particularly safe and feasible on a large scale basis .
while antibacterial coatings to mitigate the occurrence of implant- and biofilm - related infections are regarded as one of the most needed technology , currently only few and insufficient options are available for clinical use in orthopedics and trauma surgery 18 .
considering the pathogenesis of implant - related infections , any protection offered by a fully biocompatible antiadhesive barrier , like ha and some of its derivatives , could be extremely useful to reduce the tremendous burden of implant - related infections .
on the other hand , it should be noted that hyaluronic acid as a passive protective barrier has some limits . among others ,
the antiadhesive / antibiofilm effect is limited and may vary , depending on the type of the microorganism , the bacterial load , the local environment , etc .
; moreover , ha protection may be neutralized by the possible ability of some bacteria to produce hyaluronidase , an enzyme that catalyzes the degradation of hyaluronic acid 56 , while collagen and hyaluronan may even become possible ligands for microbial attachment in particular situations 57 , 58 .
to overcome at least some of these limits , possible loading of hyaluronic - based hydrogels with antibiotics is technically feasible and has been proposed by different authors 59 - 62 , being a possible option for future developments and large scale clinical applications , provided that regulatory requirements can be met .
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living in biofilms is probably the most common condition for bacteria and fungi and biofilm - related infections account for the majority of bacterial infectious diseases worldwide.among others biofilm - related infections , those associated with implanted biomaterials have an enormous and still largely underestimated impact in orthopaedics and trauma , cardio - surgery and several other surgical disciplines.given the limited efficacy of existing antibiotics in the prevention and treatment of bacterial biofilms , new strategies are needed to protect implants and host tissues , overcoming the striking ability of the microorganisms to adhere on different surfaces and to immediately protect themselves by forming the biofilm matrix.adhesion is a necessary first step in microbial colonization and pathogenesis and provides a potential target for new preventive and treatment approach.among various polymers , tested as antibacterial coatings , hyaluronic acid and some of its composites do offer a well - established long - term safety profile and a proven ability to reduce bacterial adhesion and biofilm formation.aim of the present review is to summarize the available evidence concerning the antiadhesion / antibiofilm activity of hyaluronic acid and some of its derivatives to reduce / prevent bacterial adhesion and biofilm formation in various experimental and clinical settings .
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Introduction
Anti-adhesive and anti-biofilm properties of hyaluronic acid and its composites
Clinical applications of hyaluronic acid to prevent bacterial adhesion
Conclusion
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health - related quality of life ( hrqol ) is a multidimensional concept that defines a person 's health based on specific aspects , such as physical , emotional , and social functioning and general welfare ( 1 ) .
the assessment of hrqol consists of an evaluation of the degree to which these aspects are decreased by symptoms , incapacities , and limitations caused by disease ( 2 ) .
the assessment of hrqol has been used as a measure complementary to bone mineral density to evaluate and monitor the burden of osteoporosis on a patient 's daily life ( 3 ) .
there are several instruments that can be used to assess the quality of life of individuals with osteoporosis , including the osteoporosis assessment questionnaire , the quality of life questionnaire for osteoporosis ( optqol ) , the osteoporosis quality of life questionnaire , and the questionnaire of the european foundation for osteoporosis ( qualeffo ) ( 4 - 7 ) .
the qualeffo , a specific tool used to evaluate subjects with vertebral fractures and that includes questions on pain , physical functioning , social functioning , general health perception and mental functioning , has been shown to be repeatable and consistent ( 7 - 8 ) .
vertebral fractures are the most frequent osteoporotic fractures , occurring in at least 30% of the elderly population ( 9 ) , and have important clinical implications ( 10 - 14 ) .
these fractures are associated with increased risks of new osteoporotic fractures and mortality , especially in older women ( 10 - 11 ) .
only one - third of vertebral fractures are symptomatic ; therefore , patients may be unaware of their presence .
indeed , in studies based on the radiographic screening of populations , the incidence of all vertebral fractures has been estimated to be three times higher than the incidence of hip fractures , and only 30% of people with vertebral fractures were found to have received medical attention ( 12 ) . women with vertebral fractures can also experience decreased hrqol due to physical limitations and psychosocial disabilities ( 13 - 14 ) .
some studies have assessed the impact of vertebral fractures on hrqol in older women in many countries ( 15,16 ) , but few such epidemiological studies have been conducted in brazil .
moreover , most of the studies conducted in this country have been performed in ambulatory or institutionalized individuals ( 17,18 ) .
therefore , this study evaluated the impact of vertebral fractures on the quality of life of healthy , community - dwelling women aged 65 years or older using the qualeffo .
this study was performed using the framework of the so paulo ageing & health study ( spah ) , which was a population - based , cross - sectional study ( 9 ) .
the inclusion and exclusion criteria were the same as those of the core study ( 9 ) .
all of the individuals were apparently healthy and showed no evidence of malabsorption , chronic diarrhea , hepatic disease , severe chronic diseases , or cancer .
current or previous bisphosphonate use was also an exclusion criterion ( 9 ) . quality of life was assessed through individual interviews using the validated qualeffo with 41 questions covering five domains : pain ( 5 questions ) , physical functioning ( 17 questions ) , social functioning ( sevn questions ) , general health perception ( three questions ) , and mental functioning ( nine questions ) ( 7 ) .
the total score for each domain was obtained by summing the scores of all questions for that domain and submitting this sum to a linear transformation to a scale ranging from 0 to 100 , where 0 corresponds to the worst hrqol and 100 to the best hrqol .
radiographs of the lumbar and thoracic spine centered on l2 and t7 , respectively , were obtained for all participants , with 40 " between the tube and the film .
the identification of vertebral fractures was performed by two individuals with experience in the field of analyzing vertebral fractures .
they were blinded to each other 's assessments , and when the results conflicted , a consensus between the two individuals was reached .
the agreement between the assessments of the two individuals was 96% , and the kappa coefficient was 0.82 .
each identified fractured vertebra was assigned a grade based on the genant sq scale , where mild ( grade 1 ) corresponds to a 20 - 25% reduction in the anterior , middle , and/or posterior height ; moderate ( grade 2 ) corresponds to a 26 - 40% reduction in any height ; and severe ( grade 3 ) corresponds to a reduction of over 40% in any height .
the height ( without shoes ) of each participant was measured to the nearest 0.1 cm with a wall - mounted stadiometer .
the weight of each participant ( without shoes and wearing only light clothing ) was measured to the nearest 0.25 kg using a double - beam balance scale .
the body mass index ( bmi ) was calculated by dividing the participant 's weight ( kilograms ) by her height squared ( square meters ) , and the subjects were categorized using the following cutoff points proposed by the world health organization ( who ) : normal weight = bmi<25 ; overweight = 25bmi<30 ; and obese = bmi30 ( 20 ) .
information regarding health , lifestyle and risk factors for osteoporosis / fractures was obtained through individual interviews .
women who had had two or more falls in the last 12 months were defined as chronic fallers ( 21 ) .
physical activity was classified as ( a ) low , does not even perform housework ; ( b ) moderate , performs regular housework , walks irregularly , and gardens ; and ( c ) high , performs regular physical activity aside from her daily routine at least twice a week for 30 min ( 22 ) .
regarding concomitant diseases , those mentioned at the time of the interview were noted , as well as those diagnosed during the physical examination . systemic arterial hypertension ( sah ) was defined as a history of hypertension with the use of antihypertensive drugs or a systolic blood pressure>140 mmhg and/or diastolic blood pressure>90 mmhg , which was measured with a standard sphygmomanometer with the subject seated for at least 5 minutes prior to the measurement ( 23 ) .
participants taking oral hypoglycemic agents or insulin or those with fasting blood glucose levels126 mg / dl were considered to be diabetic ( 24 ) .
the bmd was measured by dual x - ray absorptiometry ( dxa ) using hologic densitometry equipment ( hologic inc .
bedford , ma , usa , discovery model ) in the following regions : lumbar spine , femoral neck , and total femur .
anatomically abnormal vertebrae were excluded from the analysis of the lumbar spine only if they were clearly abnormal and were not assessable within the resolution of the system or if there was a difference in the t - score of more than 1.0 between the vertebra in question and adjacent vertebrae , as recommended by the international society for clinical densitometry ( iscd ) ( 25 ) .
the precision of the bmd measurements was determined based on standard iscd protocols ( 26 ) .
we calculated the least significant change with 95% confidence to be 0.033 g / cm for the spine , 0.047 g / cm for the femoral neck , and 0.039 g / cm for the total femur . according to the classification criteria of the iscd ( international society of clinical densitometry ) , the lowest t - score among the three sites ( lumbar spine , femoral neck , and total femur ) was used to classify each participant as having osteoporosis , osteopenia , or normal bone density .
thus , the individuals with t - scores that were 2.5 standard deviations or more below the scores for healthy controls for the peak bone mass were diagnosed with osteoporosis , individuals with t - scores between 2.5 and 1.0 standard deviations below the scores for healthy controls were diagnosed with osteopenia , and individuals with t - scores greater than 1.0 standard deviation below the scores for healthy controls were classified as normal ( 26 ) .
the sample size of 180 was based on a standard deviation of 15% ( 27 ) for the total qualeffo score and a two - sided 5% significance level .
the study had 95% power to detect a difference of 10 points in the total qualeffo score .
the results for the quantitative variables are expressed as the mean ( standard deviation ) , and results for the qualitative variables are described by the absolute and relative ( % ) frequencies .
demographic characteristics and the qualeffo results were compared between women with and without fractures using the mann - whitney - wilcoxon test for quantitative variables and the chi - square test for qualitative variables .
the associations between the qualeffo scores and potential determinants of quality of life were assessed using the wilcoxon rank - sum test or the kruskal - wallis test .
correlations between continuous variables and the qualeffo questionnaire data were tested using the spearman correlation coefficient ( rs ) .
generalized linear models with gamma distributions and logarithmic link functions were performed to determine the influence of the vertebral fractures on the qualeffo score .
variables with a statistical significance better than 0.1 ( p<0.1 ) in the bivariate tests were included in these models , and statistically significant variables ( p<0.05 ) were retained in the final model .
this study was conducted in compliance with the ethical principles of the helsinki declaration ( 2008 ) and local applicable laws and regulations .
this study was approved by the local ethics and research committee ( research protocol 1110/07 ) .
this study was performed using the framework of the so paulo ageing & health study ( spah ) , which was a population - based , cross - sectional study ( 9 ) .
the inclusion and exclusion criteria were the same as those of the core study ( 9 ) .
all of the individuals were apparently healthy and showed no evidence of malabsorption , chronic diarrhea , hepatic disease , severe chronic diseases , or cancer .
quality of life was assessed through individual interviews using the validated qualeffo with 41 questions covering five domains : pain ( 5 questions ) , physical functioning ( 17 questions ) , social functioning ( sevn questions ) , general health perception ( three questions ) , and mental functioning ( nine questions ) ( 7 ) .
the total score for each domain was obtained by summing the scores of all questions for that domain and submitting this sum to a linear transformation to a scale ranging from 0 to 100 , where 0 corresponds to the worst hrqol and 100 to the best hrqol .
radiographs of the lumbar and thoracic spine centered on l2 and t7 , respectively , were obtained for all participants , with 40 " between the tube and the film .
the identification of vertebral fractures was performed by two individuals with experience in the field of analyzing vertebral fractures .
they were blinded to each other 's assessments , and when the results conflicted , a consensus between the two individuals was reached .
the agreement between the assessments of the two individuals was 96% , and the kappa coefficient was 0.82 .
each identified fractured vertebra was assigned a grade based on the genant sq scale , where mild ( grade 1 ) corresponds to a 20 - 25% reduction in the anterior , middle , and/or posterior height ; moderate ( grade 2 ) corresponds to a 26 - 40% reduction in any height ; and severe ( grade 3 ) corresponds to a reduction of over 40% in any height .
the height ( without shoes ) of each participant was measured to the nearest 0.1 cm with a wall - mounted stadiometer .
the weight of each participant ( without shoes and wearing only light clothing ) was measured to the nearest 0.25 kg using a double - beam balance scale .
the body mass index ( bmi ) was calculated by dividing the participant 's weight ( kilograms ) by her height squared ( square meters ) , and the subjects were categorized using the following cutoff points proposed by the world health organization ( who ) : normal weight = bmi<25 ; overweight = 25bmi<30 ; and obese = bmi30 ( 20 ) .
information regarding health , lifestyle and risk factors for osteoporosis / fractures was obtained through individual interviews .
women who had had two or more falls in the last 12 months were defined as chronic fallers ( 21 ) .
physical activity was classified as ( a ) low , does not even perform housework ; ( b ) moderate , performs regular housework , walks irregularly , and gardens ; and ( c ) high , performs regular physical activity aside from her daily routine at least twice a week for 30 min ( 22 ) . regarding concomitant diseases ,
those mentioned at the time of the interview were noted , as well as those diagnosed during the physical examination .
systemic arterial hypertension ( sah ) was defined as a history of hypertension with the use of antihypertensive drugs or a systolic blood pressure>140 mmhg and/or diastolic blood pressure>90 mmhg , which was measured with a standard sphygmomanometer with the subject seated for at least 5 minutes prior to the measurement ( 23 ) .
participants taking oral hypoglycemic agents or insulin or those with fasting blood glucose levels126 mg / dl were considered to be diabetic ( 24 ) .
the bmd was measured by dual x - ray absorptiometry ( dxa ) using hologic densitometry equipment ( hologic inc .
bedford , ma , usa , discovery model ) in the following regions : lumbar spine , femoral neck , and total femur .
anatomically abnormal vertebrae were excluded from the analysis of the lumbar spine only if they were clearly abnormal and were not assessable within the resolution of the system or if there was a difference in the t - score of more than 1.0 between the vertebra in question and adjacent vertebrae , as recommended by the international society for clinical densitometry ( iscd ) ( 25 ) .
the precision of the bmd measurements was determined based on standard iscd protocols ( 26 ) .
we calculated the least significant change with 95% confidence to be 0.033 g / cm for the spine , 0.047 g / cm for the femoral neck , and 0.039 g / cm for the total femur . according to the classification criteria of the iscd ( international society of clinical densitometry ) , the lowest t - score among the three sites ( lumbar spine , femoral neck , and total femur ) was used to classify each participant as having osteoporosis , osteopenia , or normal bone density .
thus , the individuals with t - scores that were 2.5 standard deviations or more below the scores for healthy controls for the peak bone mass were diagnosed with osteoporosis , individuals with t - scores between 2.5 and 1.0 standard deviations below the scores for healthy controls were diagnosed with osteopenia , and individuals with t - scores greater than 1.0 standard deviation below the scores for healthy controls were classified as normal ( 26 ) .
the sample size of 180 was based on a standard deviation of 15% ( 27 ) for the total qualeffo score and a two - sided 5% significance level .
the study had 95% power to detect a difference of 10 points in the total qualeffo score .
the results for the quantitative variables are expressed as the mean ( standard deviation ) , and results for the qualitative variables are described by the absolute and relative ( % ) frequencies .
demographic characteristics and the qualeffo results were compared between women with and without fractures using the mann - whitney - wilcoxon test for quantitative variables and the chi - square test for qualitative variables .
the associations between the qualeffo scores and potential determinants of quality of life were assessed using the wilcoxon rank - sum test or the kruskal - wallis test .
correlations between continuous variables and the qualeffo questionnaire data were tested using the spearman correlation coefficient ( rs ) .
generalized linear models with gamma distributions and logarithmic link functions were performed to determine the influence of the vertebral fractures on the qualeffo score .
variables with a statistical significance better than 0.1 ( p<0.1 ) in the bivariate tests were included in these models , and statistically significant variables ( p<0.05 ) were retained in the final model .
this study was conducted in compliance with the ethical principles of the helsinki declaration ( 2008 ) and local applicable laws and regulations .
this study was approved by the local ethics and research committee ( research protocol 1110/07 ) .
the demographic , anthropometric and clinical data for all participants in the study , grouped based on the presence ( vertebral fracture ) or absence of vertebral fractures ( no vertebral fracture ) , are shown in table 1 .
there were no significant differences with respect to the mean bmi or the percentage of caucasian individuals between the groups ( p>0.05 ) ( table 1 ) .
regarding the bmi classification , 22.8% and approximately 38% of the subjects were classified as normal and obese , respectively , with no significant difference between the two groups ( p>0.05 ) .
a tendency of older age in the vertebral fracture group was observed ( p = 0.057 ) .
the frequencies of hypertension ( p = 0.224 ) , diabetes ( p = 0.672 ) , hypothyroidism ( p = 0.723 ) , and two or more concomitant diseases ( p = 0.216 ) and the average number of medications used ( p = 0.497 ) were comparable between the groups .
interestingly , the vertebral fracture group contained a higher frequency of women defined as chronic fallers than the no vertebral fracture group ( 64.7 vs. 32.1% , p = 0.017 ) .
as expected , the vertebral fracture group had a higher frequency of osteoporosis ( 73.2 vs. 51.1% , p = 0.012 ) and a lower frequency of osteopenia ( 19.5 vs. 38.1% , p = 0.027 ) than the no vertebral fracture group .
the results for each domain of the qualeffo and the total qualeffo score in both groups are shown in table 2 .
the total qualeffo score was lower in the vertebral fracture group than in the no vertebral fracture group [ 61.4 ( 15.4 ) vs. 67.1 ( 14.2 ) , p = 0.031 ] .
likewise , the physical function domain score of the qualeffo was worse in the vertebral fracture group compared with the no vertebral fracture group [ 69.5 ( 20.1 ) vs. 77.3 ( 17.1 ) , p = 0.018 ] .
no difference was observed regarding the other qualeffo domains ( pain , social functioning , health perception , and mental functioning ) ( table 2 ) .
the total qualeffo score was inversely related to bmi ( rs = -0.21 ,
p = 0.005 ) and weight ( rs = -0.22 , p = 0.009 ) .
the total qualeffo score was worse in women classified as obese than in those classified as overweight or normal [ 61.7 ( 15.4 ) vs. 66.4 ( 13.8 ) vs. 70.8 ( 13.5 ) , respectively , p = 0.008 ] .
a lower total qualeffo score was also observed in women with low physical activity than in those with moderate or high activity [ 51.8 ( 19.1 ) vs. 64.8 ( 14.3 ) vs. 70.3 ( 13.2 ) , respectively , p = 0.010 ] ( table 3 ) .
similarly , the physical function domain of the qualeffo was inversely related to the bmi ( rs = -0.24 , p = 0.001 ) . in this domain , women classified as obese were found to have lower scores than women classified as overweight or normal [ 70.7 ( 19.3 ) vs. 76.1 ( 16.1 ) vs. 81.4 ( 17.4 ) , respectively , p = 0.002 ] . finally , lower scores for the physical function domain were found among women with low physical activity compared with those with moderate or high activity [ 49.6(24.9 ) vs. 75.1(18.1 ) vs. 80.7(12.6 ) , respectively , p = 0.002 ] ( table 3 ) . a generalized linear model with gamma distributions and logarithmic link functions was developed to identify patient characteristics that were related to the total and physical function domain scores of the qualeffo .
variables with a p<0.10 in the univariate analysis ( age , bmi classification , physical activity , diabetes , presence of at least one fracture ) were included as independent variables .
the presence of obesity was negatively associated with the total qualeffo score ( p = 0.001 ) .
a high physical activity level was positively associated with the total qualeffo score ( p = 0.001 ) .
the presence of at least one fracture was associated with a worse total qualeffo score , independent of age , bmi classification and physical activity level ( p = 0.030 ) .
likewise , the presence of at least one fracture was negatively associated with the physical function domain , independent of these same variables ( p = 0.041 ) ( table 4 ) .
this study was the first study conducted in brazil that specifically assessed the impact of vertebral fractures on the quality of life in older , community - dwelling women using a specific questionnaire . in this study
, we demonstrated that the presence of vertebral fractures in this population is related to worse hrqol , particularly with respect to physical functioning .
the major advantage of the present study is the homogenous selection of community - dwelling women , unlike previous studies in which individuals were recruited from clinics or from populations included in clinical trials .
studies showing worse hrqol in patients with vertebral fractures have been published in several countries ( 15,16 ) .
there are only two studies evaluating the hrqol in patients with vertebral fractures in brazil , but neither was specific to older community - dwelling individuals ( 17,18 ) .
the first of these two other studies was performed in 55 outpatient women divided into three groups : 1- women without osteoporosis , 2- women without osteoporosis and no vertebral fractures , 3- women with osteoporosis and vertebral fractures . in that study ,
the quality of life was assessed with the sf-36 , and no difference was found among the three groups .
one of the reasons for this finding was the inclusion of only women who were able to perform the spirometric tests , resulting in the exclusion of women in worse conditions who would most likely belong to the fracture group ( 17 ) .
later , de oliveira et al . evaluated the quality of life in ambulatory women with osteoporosis and found similar results for those who had vertebral fractures and those who did not .
however , that study was not designed to assess the impact of vertebral fractures on quality of life , and the number of women with fractures was too small to enable an accurate assessment ( 18 ) .
the assessment of quality of life in relation to the qualeffo pain domain was similar in women with and without vertebral fractures in our study .
some studies have found that the pain domain is worse in women with fractures ; however , the patients included in those studies were recruited based on clinical symptoms related to fractures and were compared with those without back pain ( 28 ) .
vertebral fractures do not always manifest with symptoms and are often diagnosed based on radiographs taken for other reasons .
evaluated the chest radiographs of older women who had been hospitalized for several causes , and they found that only a few of the vertebral fractures present had been previously identified by clinicians ( 29 ) .
indeed , in our previous study performed in brazil , 29.4% subjects had vertebral fractures , and none of these patients had prior knowledge of their vertebral fractures ( 9 ) .
as observed in other studies , the mental function , social function , and health perception domains were not significantly different between women with and without fractures ( 30 ) .
the relatively small differences between the groups with and without fractures may be the result of the acceptance of poor health conditions due to the natural expectation of physical decline in older women ( 13 ) . in our study
, we found that obesity and low physical activity were associated with lower quality of life .
other authors have reported that higher bmi and a sedentary lifestyle are factors that influence the quality of life in patients with osteoporosis ( 18,31 - 32 ) .
it is important to highlight the fact that both obesity and a sedentary lifestyle are preventable factors and can be controlled by a change in lifestyle .
some authors observed that physical exercise is associated with a better quality of life ( 33 ) and have demonstrated that a home exercise program for women with vertebral fractures ( 60 min / d , 3x / week for 12 months ) significantly improves the hrqol ( 34 ) . a study performed at our institution in osteoporosis patients showed that an exercise program improved the quality of life in these women ( 35 ) .
therefore , our data reinforce the need for all older women to be advised about the benefits of exercise .
these women should be encouraged to exercise regularly to reduce their bmi and improve their general welfare .
our study is the first in brazil to conduct a thorough assessment of the relationship between quality of life and vertebral fractures .
an important characteristic of this study was the use of standardized and reliable instruments for both the vertebral fracture assessment ( genant semi - quantitative method ) and the hrqol assessment ( qualeffo ) .
although the qualeffo has not been validated in brazil , which is a limitation of the present study , this tool is the most frequently used for assessing quality of life in osteoporosis and was recommended for the investigation of vertebral fracture subjects in multicentric studies that included centers in brazil ( 36 ) .
this questionnaire is more sensitive in detecting differences between groups , and it provides a better discrimination between individuals with and without vertebral fractures compared with generic hrqol instruments , such as the sf-36 , particularly with respect to physical functioning , which is significantly affected in these patients ( 8) . in conclusion , this study demonstrated the negative impact of vertebral fractures on the quality of life in older women , independent of other factors such as bmi and physical activity .
although the clinical relevance of vertebral fractures is well established , these results are important for assessing the burden of this disease and reinforce the need to reduce the underdiagnosis and undertreatment of these fractures . the results of this study also highlight the need for awareness of the importance of maintaining proper weight and promoting changes in lifestyle through physical activity and dietary control .
this study was supported by fundao de amparo e pesquisa do estado de so paulo ( fapesp ) # 03/09313 - 0 ; conselho nacional de cincia e tecnologia ( cnpq ) # 305691/2006 - 6 ( rmrp ) and # 119601/2009 - 5 ( lf ) ; federico foundation grants ( rmrp ) ; and coordenao de aperfeioamento de pessoal de nvel superior ( capes ) ( jbl , cpf ) .
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objectives : the aim of this study was to investigate the impact of asymptomatic vertebral fractures on the quality of life in older women as part of the sao paulo ageing & health study.methods:this study was a cross - sectional study with a random sample of 180 women 65 years of age or older with or without vertebral fractures .
the quality of life questionnaire of the european foundation for osteoporosis was administered to all subjects .
anthropometric data were obtained by physical examination , and the body mass index was calculated .
lateral thoracic and lumbar spine x - ray scans were obtained to identify asymptomatic vertebral fractures using a semi - quantitative method.results:women with asymptomatic vertebral fractures had lower total scores [ 61.4(15.3 ) vs. 67.1(14.2 ) , p = 0.03 ] and worse physical function domain scores [ 69.5(20.1 ) vs. 77.3(17.1 ) , p = 0.02 ] for the quality of life questionnaire of the european foundation for osteoporosis compared with women without fractures .
the total score of this questionnaire was also worse in women classified as obese than in women classified as overweight or normal .
high physical activity was related to a better total score for this questionnaire ( p = 0.01 ) . likewise , lower physical function scores were observed in women with higher body mass index values ( p<0.05 ) and lower physical activity levels ( p<0.05 ) .
generalized linear models with gamma distributions and logarithmic link functions , adjusted for age , showed that lower total scores and physical function domain scores for the quality of life questionnaire of the european foundation for osteoporosis were related to a high body mass index , lower physical activity , and the presence of vertebral fractures ( p<0.05).conclusion : vertebral fractures are associated with decreased quality of life mainly physical functioning in older community - dwelling women regardless of age , body mass index , and physical activity .
therefore , the results highlight the importance of preventing and controlling asymptomatic vertebral fractures to reduce their impact on quality of life among older women .
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INTRODUCTION
SUBJECTS AND METHODS
Population
Quality of life assessment
Assessment of vertebral fractures
Anthropometry
Other variables and definitions
Bone mineral density (BMD) assessment
Statistical analysis
Ethics
RESULTS
DISCUSSION
ACKNOWLEDGMENTS
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the geological and ( palaeo-)biological evolution of lowland amazonia during neogene and quaternary times remains fascinating since the early days of natural sciences ( for summaries of research history see e.g. , loczy , 1963 and wesselingh , 2008 ) .
many , but partly highly controversial models have been introduced to explain its historical development ( for recent compilations see e.g. , lundberg et al . , 1998 ;
, 2006 ; wesselingh and salo , 2006 ; rossetti and mann de toledo , 2007 ; haffer , 2008 ; hoorn and wesselingh , 2010 ; hoorn et al . , 2010 ;
generally , it is widely accepted that around the onset of the miocene ( 23 ma ) a mega - wetland ( pebas system , also called lake pebas ) developed in western amazonia due to the subsiding subandean foreland ( e.g. , hoorn , 1994 , 2006 ; wesselingh et al . , 2002 ; wesselingh and salo , 2006 ; shephard et al . , 2010 ) .
short - lived marine incursions or even a transcontinental seaway from the caribbean sea through the venezuelan / columbian llanos basin southwards to the argentinean paran basin are proposed ( e.g. , rsnen et al . , 1995 ;
2009 ) but heavily disputed ( e.g. , campbell et al . , 2006 ; cozzuol , 2006 ; westaway , 2006 ; latrubesse et al . , 2007 , 2010 ;
, 2003 ; hoorn and vonhof , 2006 ; wesselingh , 2006 ) . in the late miocene this mega - wetland disintegrated due to enhanced uplift of the northern / central andes .
the drainage pattern of northern south america started to reverse completely to today s easterly course and the modern amazon system became established during the early pliocene ( hoorn , 2006 ; figueiredo et al .
, 2009 ; hoorn et al . , 2010 ; latrubesse et al . ,
2010 ) . beside the vast size of amazonia and the still fragmentary regional coverage with field surveys ,
there are considerable inconsistencies in palaeoenvironmental reconstructions and , in particular , in the chronology and correlation of scattered outcrops .
wesselingh ( 2008 , p. 5 ) stated : the lack of geological data has led to the emergence of many grand theories about the origin of present - day amazon system and its highly diversity , often based on dubious interpretations of the little data available .
the present paper aims to contribute basic sedimentological data from a barely studied region ( eirunep , 2.000 km sw manaus ; fig .
1a ) , which is supposed to be placed at the south - eastern margin of the pebas system ( wesselingh and ramos , 2010 ) .
we demonstrate that there is no evidence for a long - lived lake ( sensu gorthner , 1994 ) or any marine influx .
conversely , we document a well - structured , aggrading fluvial system of late miocene age , which is in agreement with the sedimentation model and chronology proposed by latrubesse et al .
the studied sections are located along the juru and tarauac river , ne respectively se of the city eirunep ( state of amazonia , western brazil ; 245 km s of benjamin constant ; fig .
delineations and subdivisions of basins in western amazonia diverge notably and several authors attribute the region of eirunep to the solimes basin ( eirunep subbasin ; e.g. , caputo , 1991 ; roddaz et al . , 2005 ; ramos , 2006 ; wesselingh and salo , 2006 ; wesselingh et al . ,
however , based on subsurface information , obtained by extensive hydrocarbon and coal exploration campaigns during the 1970 s , this region is situated west of a basement high ( miura , 1972 ; iquitos arch ; corresponds to the carauari arch of caputo , 1991 ) on the eastern margin of the acre basin ( e.g. , del arco et al . , 1977 ; maia et al . , 1977 ; latrubesse et al . , 2010 ; fig .
beside quaternary deposits ( terraces , alluvium ) , the scattered outcrops along river banks expose sediments of the upper parts of the solimes formation ( del arco et al . , 1977 ;
maia et al . , 1977 ; paz et al . , in press ) . the solimes fm .
comprises more than 1000 m thick , largely pelitic sandy alternations with lignitic intercalations and covers a huge part of western amazonia ( fig .
. uncertainties in its definition , its stratigraphical and geographical extent as well as its depositional environments basically reflect the ongoing debate about amazonia s history through neogene times .
( 1977 ) , hoorn ( 1993 , 1994 ) , latrubesse et al .
the sections were vertically logged by visual inspection of the lithofacies ( including colour , grain size , bedding planes , sedimentary structures , macrofossil content ) . for lithofacies coding the scheme of miall ( 1996 )
was used : capital letters = dominant grain size ( g , gravel ; s , sand ; f , fine sand clay ) followed by a lowercase letter = sedimentary structures and/or biogenic features ( c , clast - supported ; m , massive / faint lamination ; h , horizontal bedding / lamination ; t and p , trough and planar cross bedding ; r , ripple bedding ; s , scour fill ; l , lamination ; r , rooted ; c , coal ; p , pedogenic overprint ) .
additionally the outcrops were mapped laterally as far as possible . due to poor outcrop conditions
an application of the architectural element concept ( miall , 1996 ) was only loosely possible .
for micropalaeontological investigations bulk samples ( 12 kg ) were taken from all outcrops .
500 g of dried sediment ( 40 c , 24 h ) were washed by using diluted hydrogen superoxide for disintegration through standard sieves ( h2o2 : h2o = 1 : 5 ; 63/125/250/500 m ) .
wet sieve residuals were washed with ethanol prior to drying ( 40 c , 24 h ) .
an in - depth taxonomic examination of the obtained microfossils ( including the sieve fractions < 250 m ) will be subject of further studies .
for preliminary stable isotope analyses ( o , c ; 18 samples ) two or three ostracod valves ( 4060 g ; cyprideis spp . ) from earlier sampling campaigns were used ( collected by m.i.r ) . for analyses
a thermo - finnigan kiel ii automated reaction system and a thermo - finnigan delta plus isotope - ratio mass spectrometer were used ( university of graz ; standard deviation = 0.1 relative to nbs-19 ; results in per mille relative to vpdb ) .
more detailed investigations are in preparation ( m. fonseca / belm , m. caporaletti / graz ) .
location : 12.6 km ne eirunep ( s 063355.5/w 0694611.7 , altitude : 108 m ) , left cutbank of the juru river ( fig . 2 ) .
description : the basal 1.8 m ( layers 19 ) of the section consists of partly ripple bedded sands and laminated silts ; some cm - thick clay intercalations were observed . above
follows a 0.65 m thick alternation of clay with incorporated calcareous nodules and laminated silty sand or ripple bedded sand ( 1017 ; fig .
the top is rich in vertebrate remains ( crocodiles , turtles ; 17 ; fig .
massive to finely laminated , occasionally bivalves - bearing ( silty ) clay with coaly and sandy layers forms the next 1.14 m ( 1823 ; fig .
beds 2425 ( 1.85 m thick ) comprise massive , horizontally or trough cross bedded sand and finely laminated or massive ( sandy silty ) clay / fine sand ( fig .
grey mottled , rooted and massive clay ( paleosol ) with a calcareous horizon on its top ( 26 ) .
up - section , a 4.2 m ( 2739 ) thick alternation of massive or laminated clay , silt and partly ripple bedded fine sand is developed ( a slight coarsening - upward trend is anticipated ) .
bed 32 displays a slightly erosive base with intraclasts in the lower part ( mud pebbles ) .
layers 3139 are cut by a channel ( 40 ) with a multiphase fill . large scale cross bedded sands ( -cross stratification ) with sporadic intraclasts ( mud pebbles ) on reactivation surfaces and trough cross bedded and ripple bedded fine
microfossil contents : samples from the basal part of the section ( pd2 ) yielded only scarce valves of cyprideis spp . and rare fish elements .
interpretation : the lithofacies of the basal part of the outcrop ( 117 ) refers to a deposition in a fluvial overbank environment .
the diffuse layers of calcareous nodules ( 17 ) are supposed to be largely a diagenetic feature ( groundwater caliche ) .
however , it may also hint to soil - forming processes at seasonally dry floodplain areas ( retallack , 2001 ) .
up - section the sediments become finer and contain several ( sandy silty ) coal - rich layers ( 1823 ) .
these beds are assigned to the formation of a floodplain pond , which was inhabited by opportunistic ostracods , freshwater bivalves and fishes .
however , crevasse splays influenced episodically that shallow lacustrine environment ( sandy and organic matter - rich layers ) .
later ( layers 2425 ) , the influx of crevasse splays into the pond , which is still populated by ostracod faunas and fishes increased ( more prominent sandy beds , partly scour - fills ) .
pedogenic processes are indicated by incipient soil formation in layer 26 ( mottled , roots ) .
floodplain pond conditions ended above ( beds 2739 ) due to enhanced clastic input from an approaching channel
. finally ( bed 40 ) , the overbank fines were intersected by a channel of at least 23 m in depth , which was filled up by large scale cross bedded sand .
more or less perpendicular to the main accretion surfaces oriented ripple bedding indicates deposition by lateral accretion of a point bar .
location : 17.9 km ne eirunep ( s 063251.1/w 0694239.4 , altitude : 107 m ) , left cutbank of the juru river ( fig . 4 ) .
description : the lower 5 m of the outcrop are formed by ( section 1 ) : ( 1 ) greenish grey , mottled clay with ferran cutans and cm - sized carbonate nodules ( fig .
5a ) ; ( 2 ) dark grey , massive or indistinctly laminated clay with cm - sized carbonate concretions ( fig .
5b ) , thin , pyrite - rich coaly layers , plant fragments , root traces and vertebrate remains ( crocodiles , turtles ) ; ( 3 ) greenish yellowish , mottled clay with ferran cutans ( passes gradually into bed 4 ) ; ( 4 ) violet - red green - grey , mottled clay / paleosol ( fig .
5c ) ; and ( 5 ) green grey , mottled clay with vertebrate remains ( reptiles ) on top .
a trough cross bedded sandy layer with coaly interlayers in the se part of the site mentioned by paz et al .
( in press ) was not exposed during our observation ( section 4 ) . in the nw part ( section 1 ) , above layer 5 , ripple bedded fine sand with intraclasts ( mud pebbles ) at its base is recorded , which contains bivalves and coalified wood remains ( 6 ) .
laminated sand alternations ( 7 ) and massive sandy clay , which is rich in molluscs in its lower and upper part ( 8) . up - section ( 9 ) , an alternation of clay and coal layers , rich in molluscs ( i.e. , mycetopoda sp . ) , as well as a thinly bedded succession of laminated sand and thin clay interlayers ( 10 ) follow . towards the se ( section 24 )
an up to 2 m thick , large scale cross bedded sandy unit is developed ( fig .
occasionally , intraclasts and vertebrate fragments are found at the base of this element or on accretion surfaces , which dip around ne .
the dip of ripple foresets is approximately perpendicular oriented to these surfaces ( sw ) .
this unit is overlain by a layer of calcareous nodules ( 11 ; section 3 ) and laminated clay ( silt)coal alternations with bivalve and vertebrate remains ( 13 and 14 = equivalent to 9 ) .
bed 12 ( plastic , strongly weathered clay ) is an equivalent of layer 10 .
microfossil contents : samples mn1 and mn2 lack microfossils , in mn8a and mn8b only scattered valves of cypria sp . and heterocypris ?
mn9 and mn 14 yielded many fish , gastropod and bivalve ( sphaeriids ) remains as well as abundant ostracod valves ( cyprideis spp . ) .
earlier reports on fossil material : celestino and ramos ( 2007 ; see also wesselingh and ramos , 2010 ) documented the following ostracod taxa : cyprideis graciosa , cyprideis lacrimata , cyprideis longispina , cyprideis machadoi , cyprideis pebasae , cyprideis olivencai , cyprideis sp . 1 and 2 .
further findings ( plants , fishes , reptiles ) are mentioned in del arco et al .
the later authors also reported the occurrence of cyathecidites annulatus , echitricolporites spinosus and grimsdalea magnaclavata pollen .
greenish to yellowish and dark grey colouration might indicate more or less water - logged conditions , whereas reddish colours may hint to rather well - drained , oxidizing environments .
however , diagenetic alteration can hamper such an interpretation significantly ( retallack , 2001 ) .
layer 2 is less affected by pedogenic processes ( faint lamination , coaly layers , plant and reptile fragments ) and perhaps represents floodplain lake / backswamp deposits .
the paleosols ( overbank fines ; 15 ) are overlain by a sandy channel fill ( point bar ) with lag deposits at its base and repeatedly occurring intraclasts at its lateral accretion surfaces ( channel depth > 2 m based on the preserved height of this unit ) . in the nw part of morada nova layers
68 represent an abandoned channel fill , which could be the fill of a subordinate channel or the fill of a chute channel .
up - section , the influx of the active channel decreased and a floodplain lake developed ( 9 , 13 , 14 ) , which enabled the establishment of plentiful ostracod and aquatic molluscs faunas .
the pond itself or its surroundings were richly vegetated ( coaly layers ) and settled by semi - aquatic reptiles . finally , enhanced input of sandy matter points to an increasing influence of the channel , probably due to crevasse splays .
( s 063140.8/w 0693952.0 ; altitude : 106 m ) , left cutbank of the juru river ( fig .
description : section 1 ( w part ) starts with a 1.6 m thick succession of laminated , ripple and cross bedded silty sand with coaly intercalations ( layers 16/1 ) .
up - section ( 714/1 ) , a 2.5 m thick sequence of ripple bedded silty fine sand layers with commonly erosive basal boundaries ( incl .
convolute bedding and m - thick concretions are frequent in its upper part ( 1114/1 ) .
silty clay ; 1520/1 ) and comprise coal and mollusc - rich pelitic ( 16/1 , 19/1 ) and coaly layers ( 18/1 ) as well as carbonate - cemented clay ( 20/1 ) .
layer 21/1 ( 0.75 m thick ) is composed of greenish violet , mottled or poorly laminated clay with scattered bivalve shells .
intercalated are several layers ( 10 cm - thick ) , which consist of 12 mm large , rounded clay clasts ( fig .
it is overlain by 1 m of massive clay with abundant mollusc ( e.g. , sheppardiconcha septencincta ; fig .
leaves ; 22/1 ) and a > 0.5 m thick ( weathered ) coaly layer ( 23/1 ) with mollusc fragments ( e.g. , anodontites ?
section 2 ( e part ; 120 m downstream of section 2 ) starts with > 0.35 m thick , laminated fine sand with coaly interlayers ( layer 1/2 ) .
it is cut by a 4 m wide and 0.8 m deep channel fill ( 25/2 ; fining - upward , from base to top : laminated , coal - rich fine sand with rare mollusc and plant remains ; ripple bedded fine sand , rich in plant fragments ; massive clay , rich in bivalves ( sphaeriids ) , several wood remains ; fig .
this channel fill is topped by a 0.5 m thick , massive poorly laminated clay with thin fine sand interlayers ( 6/2 ) and 0.2 m of massive , partly indurated ( cemented ) clay ( 7/2 ) .
coalified wood remains are rare ; bivalves ( sphaeriids ) are abundant ( especially upper part of 6/2 ) .
layer 6/2 and 7/2 taper off towards the w. up - section ( 1 m thickness ) , follow massive , silty sandy clays ( 89/2 ) and ripple bedded silty fine sand with silt interlayers ( 1014/2 ) .
especially layer 8/2 is rich in molluscs ( sphaeriids ) , 1314/2 contain coaly fragments ; clay intraclasts are observed at the base of 13/2 .
these beds are topped by 2.7 m of mottled silty fine sand with coaly fragments in the lower 0.3 m ( 15/2 ) .
. layer 16/2 ( > 1.2 m thick ) comprises coal coaly fine sand finely laminated clay alternations ( fig .
7f ) and is rich in bivalves ( sphaeriids ) in the lowermost 57 cm .
whereas samples aq5/1 and aq6/1 contain only rare ostracod remains , aq16/1 yields , beside fish fragments , some valves of cytheridella sp . and darwinulids , accompanied by rare specimens of cypria sp . , cyprideis spp . and
, darwinulids , cypria sp . , ilyocypridids ) and fish bones . several ( aq22a/1 ) respectively rare ( aq22b/1 ) cyprideis spp .
valves are recorded in the samples of section 1 above , together with fish remains and gastropods ( aq22b/1 ) .
sample aq3/2 of section 2 delivered only rarely cyprideis spp . and some fish remains . in the samples aq 5 - 6 - 8 - 13/2 plentiful ostracod faunas ( cytheridella sp . , ilyocyprinids , darwinulids , cyprideis spp .
are documented along with fish remains and sphaeriid bivalve shells ( aq8/2 and aq13/2 ) . aq15/2 and aq16/2 are almost barren of ostracods but contain fish and bivalve ( sphaeriids ) fossils . earlier reports on fossil material : celestino and ramos ( 2007 ; see also wesselingh and ramos , 2010 ) recorded the following ostracod taxa : alicenula ( darwinula ) fragilis , cypria aqualica , c. longispina , c. pebasae , cyprideis sp . 3 , cytheridella purperae , heterocypris ?
( 2006b ) re - examined mollusc material studied by roxo ( 1937 ) from aquidab and described : ampullariidae sp .
sheppardiconcha septemcincta . additional findings ( including vertebrates and plants ) are mentioned by del arco et al .
the basal , sandy silty layers ( 16/1 = 1/2 ) possibly represent crevasse splay deposits , which are followed by a series of crevasse splay and/or crevasse channel sediments ( 714/1 respectively 25/2 ) .
alternatively , these layers could represent channel deposits of an avulsive river arm . in section 2 a subordinate abandoned channel fill ( floodplain pond ; 67/2 ) is developed .
afterwards ( 1523/1 and 816/2 ) the influx of crevassing respectively the active channel successively decreased and led to the formation of a floodplain lake , which was only influenced by flash floods in more proximal settings ( section 2 ; e.g. , 12/2 ) . aquatic faunas ( bivalves , ostracods ,
fishes ) and semi - aquatic crocodilians inhabited that lake , which was surrounded by a densely vegetated backswamp .
these beds display a mottled appearance and contain ( 21/1 ) layers of crumb peds ( clast - supported layers of rounded but not interlocked tiny mud balls ) , which indicate bioturbation due to invertebrate activity ( retallack , 2001 ) .
finally , the lake became replaced by a swampy environment ( 23/1 and 16/2 ) .
location : 27.4 km ne eirunep ( s 063122.0/w 0693542.8 ; altitude : 105 m ) , left cutbank of the juru river ( fig .
description : at the base > 2 m of slightly southwards inclined ( < 10 ) rippled bedded silty fine sand ( layer 1 ) are followed by 2.7 m thick violet - brown ( 2 ) and yellowish ( 3 ) mottled paleosols with calcareous nodules ( between 2 and 3 a pedogenically overprinted clay plug is intercalated ) .
layers 13 are cut by a channel , whose fill starts ( 4 ) with > 1.5 m thick , massive poorly laminated silty clay with gastropods , rare bivalves and plant ( leaves ) remains .
bed 4 grades into a 0.8 m thick , brownish - grey , blocky structured silty clay ( 5 ) with a calcareous crust on its top .
it is overlain by 2.4 m of violet - brown , mottled clay ( 6 ) with intercalated layers of calcareous nodules .
strata 13 as well as the channel fill ( 46 ) are topped by a 0.4 m thick alternation of laminated silty clay and laminated or rippled bedded fine
medium sand with a high amount of plant detritus ( also wood remains ) and abundant vertebrates ( turtles ; 7 ) .
the badly exposed , 7.2 m thick beds up - section ( 8) consist of cross and ripple bedded fine sand with intercalations of clayey lenses ( 2 m lateral extension , 20 cm thickness ) , which are formed by clay intraclasts ( mud pebbles , 1 cm diameter ) .
repeatedly coaly layers are intercalated and rarely vertebrate ( crocodiles ) remains were found in the intraclast conglomerate .
layer 9 represents a 1.2 m thick alternation of horizontally or ripple bedded fine
the topmost 1.2 m thick layer 10 consists of ripple bedded silty fine sand and silty clay alternations , which form cm - thick , fining - upward couplets .
microfossil contents : sample re4 yielded only a few valves of cyprideis spp . as well as some fish and gastropod remains .
interpretation : layer 1 could represent a crevasse splay or levee deposit , which is overlain by pedogenically altered floodplain fines ( 23 ) with intercalated minor channel fills .
these overbank deposits are cut by a channel ( ? 20 m wide and 5 m deep ) , which became filled by pelitic , afterwards pedogenically overprinted sediments .
the rare ( a matter of preservation ? ) mollusc and ostracod record indicates at least at the beginning ( 4 ) aquatic life in the abandoned channel . above , a crevasse splay ( 7 ) points to rising proximity of the active channel .
large wood remains and vertebrate fragments were accumulated by this splay . the sandy succession up - section (
8) can only be attributed to a sandy bedform sensu miall ( 1996 ) due to insufficient outcrop conditions .
intercalated shallow scours , filled up with mud balls indicate deposition of reworked material and variations of the flow regime .
the more or less rhythmically stratified , sandy - pelitic alternations at the top of remanso ( 910 ) mark a shift towards overbank environment and might represent a bar - top assemblage . location : 17.5 km se eirunep ( s 064923/w 0694704 , altitude : 117 m ) , left cutbank of the tarauac river ( fig .
description : the basal 1.7 m ( layers 13 ) consist of massive or laminated pelite- and fine sand with rare bivalve remains .
they are topped by 0.2 m of reddish - brown , massive clay ( 4 ) .
the sandy silty layers 59 display a general coarsening - upward trend and contain only rarely bivalve fragments .
up - section ( 10 ) , a 0.25 m thick intraclast conglomerate ( mudstone pebbles ) with abundant mollusc ( e.g. , ampullariidae ?
this stratum is overlain by thin sand and clay layers ( 1117 ) with abundant mollusc remains ( 12 , 14 , 16 , lower part of 17 ) .
layer 14 is rich in coal fragments ( wood , leaves ) at its top .
the 2.57 m thick succession above ( 1823 ) starts with thin , ripple bedded fine sand beds ( 1819 ) , followed by massive silty / sandy clay with coaly fragments ( 20 ) and cm - thick alternations of fine sandy silt and silty fine sand ( 21 ; forming couplets of small - scale fining - upward cycles ) .
beds 22 and 23 comprise alternations of massive , fine sandy silt and ripple bedded , silty fine sand .
24 consists of 10 cm - thick alternations of indistinctly ripple bedded silty fine sand and massive or poorly laminated pelite . the upper part ( 1.5 m ) is mottled and primary sedimentary structures are obscured due to pedogenic processes .
these samples contain several fish remains and in to12 rare characean gyrogonites were found additionally . the samples to20 and to 23 delivered only rare ostracod ( cypria sp .
earlier reports of fossil material and facies : ramos ( 2006 ) mentioned the following ostracod taxa : c. aqualica , c. graciosa , c. lacrimata , c. longispina , c. pebasae , c. purperae , cytheridella sp . , darwinula fragilis as well as additional vertebrate and crab findings .
interpretation : the lowermost sandy - pelitic layers ( 14 ) might be deposited in a floodplain pond / lake , which was influenced by crevasse splays .
the coarsening - upward trend of layers 59 hints to a progressive influx of a crevasse channel ( layer 10 ) .
it remains unclear if the ostracods of this layer are allochthonous or if they are incorporated in that layer due to subaquatic inflow of the crevasse splay ( crevasse delta ) into a floodplain pond / lake .
up - section ( 1124 ) , plentiful ostracod and mollusc faunas ( especially in the lower part : 1117 ; compare ramos , 2006 ) indicate the return to an aquatic environment and the abandonment of the crevasse channel .
small - scale fining - upward alternations of ripple bedded sand and pelite document individual flooding events or surges .
location : 22.1 km sse eirunep ( s 065218.3/w 0694705.1 ; altitude : 120 m ) , left cutbank of the tarauac river ( fig .
description : layers 16 represent a 3.2 m thick succession of greenish ( 1 ) , reddish ( 2 ) , yellowish ( 3 , 5 , 6 ) or violet grey ( 4 ) coloured , mottled , frequently calcareous nodules - bearing paleosols . in layer 6 vertebrate remains ( crocodile teeth ) and gastropods were found .
above follows a 1.1 m thick , indistinctly bedded clay to fine sand ( 7 ) , which contains in its lower half coaly plant fragments and becomes towards the top and laterally progressively mottled ( paleosol ) .
the 0.65 m thick sediment column up - section ( 811 ) is composed of massive clay ( 8) , ripple bedded fine sand ( 9 ) , laminated silty fine sand to silt ( 10 ) and fine sand ( 11 ) .
each of these layers displays a slightly erosive base and an internal fining - upward trend .
the beds 8 , 9 and 11 each start with a basal layer of concretionary mud clasts in which ( layer 8 and 11 ) abundant vertebrate remains were found .
layer 12 is formed by 1.2 m thick , ripple bedded fine sand with cm - thick , laminated silty clay interlayers .
up - section a 0.7 m thick , orange - grey , mottled paleosol with mud clasts and rare vertebrate remains at its base ( 13 ) as well as a > 1.5 m thick , violet - purple coloured , mottled paleosol are developed ( 14 ) .
microfossil contents : the samples ba7a and ba7b contained several ostracod valves ( mainly cyprideis spp . , some specimens of cypria sp .
interpretation : the basal layers ( 16 ) represent a succession of paleosols , which could be seriously altered floodplain deposits . due to the occurrence of ostracods , probably bed 7 was deposited in a floodplain lake environment .
afterwards , a series of crevasse splay ( maybe partly also crevasse channel ) deposits follows ( 813 ) , which is topped by pedogenically overprinted floodplain fines ( 14 ) .
the sedimentary record of the investigated outcrops documents vertically as well as laterally highly variable fine - grained clastic successions ( predominantly clay fine sand , along with fine medium sand ) . based on the lithofacies and , as far as possible , lateral observations , these sediments represent fluvial deposition within active channels as well as in overbank environments .
macroforms , generated within the active channel include lateral accretion deposits ( sandy point bars ; figs . 2 and 4 ) and undifferentiated sandy bedforms ( stacked dune fields ? ; fig .
8) , which are probably partly intersected by chute channels or are overlain by overbank fines .
these comprise pelite sand - alternations of levee , crevasse splay and crevasse channel deposits .
frequently , a lag of mud pebbles and vertebrate fragments is found at the base of crevasse channels / splays . in part
, some splays may have entered floodplain ponds / lakes via crevasse deltas ( e.g. , figs . 6 and 9 ) .
abandoned , fine - grained channel fills ( mud plugs ) were observed ( fig .
decimetre - thick , clay silt beds with rich ostracod and mollusc faunas indicate the presence of short - lived , shallow ponds or lakes within the floodplain ( floodbasin ; e.g. , fig .
6 ) point to partly swampy , poorly - drained conditions and a densely vegetated floodplain , which is , however , influenced by high clastic input .
intensively mottled paleosols with root casts and calcareous nodules are ubiquitous ( e.g. , fig .
differences in colour and occasional calcic horizons may reflect diverging water - logging due to e.g. , local topography and/or seasonal fluctuations in discharge or climate ( kaandorp et al .
nevertheless , diagenesis may have altered significantly primary features and more specific investigations are required ( retallack , 2001 ) .
we are aware that restricted outcrop conditions and the limited areal and stratigraphical extent ( 30 m stratigraphical thickness ) of the current investigation hamper conclusions about the nature of fluvial style .
reconstructions of fluvial geomorphology , based solely on lithofacies assemblages and fragmentary known fluvial elements , remain to some degree tentative ( miall , 1996 ; bridge , 2003 )
. however , it seems obvious that we are dealing with a suspended load river system due to the high amount of overbank fines , probably within a tropical to subtropical wet dry climate ( kaandorp et al . , 2005 ; latrubesse et al .
we observed : ( a ) a large proportion of overbank deposits , ( b ) laterally extensive , heterogeneous , fine - grained avulsion deposits ( including crevasse splays ) , ( c ) predominantly sandy channel deposits , which change laterally and vertically rapidly into overbank deposits as well as ( d ) lacustrine and coaly deposits . some sandy point bars ( of secondary channels ? ) indicate subordinate lateral sediment accretion .
these features are not exclusive to anastomosing river deposits , however , they largely coincide with the standard model of an anastomosing river sensu miall ( 1996 ) as well as with characteristics of such a system as reviewed by makaske ( 2001 ; compare also smith , 1983 ) .
the general setting ( subsiding foreland basin , extensive sediment input from the andes , possibly incompletely framed by tectonically active basement highs towards the east ) as well as a wet dry seasonal climate ( seasonal floods , channel banks stabilized by vegetation ) would provide the conditions for anastomosing river systems ( smith , 1983 ; nanson and knighton , 1996 ; makaske , 2001 ; latrubesse et al .
( late miocene ) in the state of acre ( sw brazil ) led to similar assumptions ( latrubesse et al .
these authors proposed a megafan system , which originates in the andes and encompass a complex mosaic of avulsive rivers and associated wetlands ( including abandoned channels , floodplain lakes , floodbasin deltas , backswamps and well - drained floodplains ; see also wilkinson et al .
this system is suggested to reach to the east at least as far as 67 w ( iquitos arch sensu del arco et al . , 1977 ) and probably also beyond ( purus arch ; latrubesse et al . , 1997 , 2010 ; fig
according to that , the sections around eirunep were located in a more distal position , which could be the reason for a more pronounced anastomosing - anabranching pattern due to a lower gradient ( compare also interpretations of the late miocene tariquia formation , southern bolivia ; uba et al .
( 2007 , 2010 ) compared the depositional environment of the late miocene solimes fm . in acre with i.e. , the quaternary megafans of the chaco plain and the pantanal wetland ( iriondo , 1993 ; horton and decelles , 2001
the mollusc fauna , recorded from floodplain pond / lake deposits , is dominated by sphaeriids and the pachychilid sheppardiconcha ( fig .
7b ) and indicates freshwater conditions within a fluvio - lacustrine environment ( wesselingh et al . , 2006b ; wesselingh and ramos , 2010 ) .
typical freshwater ostracods ( darwinulids , ilyocyprinids , cypria , cytheridella ) are associated with diverse species of cyprideis .
this genus is a holoeuryhaline taxon , able to cope with fluctuating salinities , aberrant water chemistries , variable temperatures and oxygenation ( e.g. , meisch , 2000 ; keyser , 2005 ) . as it could adapt to freshwater conditions ( e.g. , lake tanganyika ; wouters and martens , 2001 ) and co - occurs here with exclusively freshwater taxa
, there is no constraint to conclude a brackish water environment ( ramos , 2006 ) .
preliminary stable isotope data ( o , c ) measured on cyprideis valves from aquidab , morada nova and torre da lua , yielded very negative values ( o : 5.7 to 9.7 , c : 10.3 to 12.5 ) , which exclude any marine influx .
there is still intensive debate on definition and timing of palynological biozonation concepts ( e.g. , hoorn , 1993 , 1994 ; latrubesse et al . , 2007 , 2010 ; jaramillo et al . , 2010 ; silva - caminha et al . ,
however , palynostratigraphy remains a cornerstone for correlation across amazonia up to now and provides at least a rough stratigraphical framework .
( 2006a ) are highly linked to the concept established by hoorn ( 1993 ) .
based on the presence of some index taxa ( c. annulatus , e. spinosus , g. magnaclavata ) a late miocene age ( probably asteraceae zone sensu lorente , 1986 ) is proposed by paz et al .
( in press ) for the outcrops morada nova and torre da lua recently . the vertebrate fauna from torre da lua ( ramos , 2006 ) is related to the so - called acre fauna , which is one of the best documented faunas of northern south america ( cozzuol , 2006 ; latrubesse et al .
the acre vertebrate assemblage displays significant affinities with the mesopotamian faunas of argentina and uruguay as well as with the urumaco assemblages ( venezuela ) and is dated to the late miocene ( huayquerian south american land mammal age ; cozzuol , 2006 ; latrubesse et al .
is still poor and has to be extended by later works ( wesselingh and ramos , 2010 ; see roxo , 1937 ; wesselingh et al .
, 2006b ; wesselingh , 2008 , and outcrop descriptions herein ) . from a stratigraphical point of view the mollusc faunas of aquidab and torre da lua
( 2006b ) to be of late miocene origin and post - date the pebas fauna
( compare celestino and ramos , 2007 ; wesselingh , 2008 ; wesselingh and ramos , 2010 , which suggested a late middle miocene age based on the ostracod record ) .
a late miocene age seems to be in agreement with the lack of typical endemic pebasian molluscs ( wesselingh and ramos , 2010 ) . the recorded cyprideis - species from aquidab , morada nova and torre da lua ( ramos , 2006 ; celestino and ramos , 2007 ; wesselingh and ramos , 2010 ) have generally long ranges according to muoz - torres et al .
( 1998 , 2006 ; see also whatley et al . , 1998 ) .
the occurrence of c. lacrimata and c. pebasae might be indicative for an age not younger than the ( early ) late miocene .
nonetheless , due to some contradictions ( e.g. , c. lacrimata is supposed to appear first after the last appearance of c. longispina but both are co - occurring at morada nova and torre da lua ) and the lack of key taxa , the current ostracod zonation needs some adjustment and is not further stressed here .
however , to date , a late miocene age seems most plausible for all outcrops described herein by considering the palaeontological record .
moreover , this fits well to the palaeogeographic context of this region as proposed by latrubesse et al .
sedimentological observations derived from outcrops around eirunep ( south - western part of the state of amazonia ) document various subenvironments of a fluvial system ( upper part of the solimes formation , late miocene ) . beside sandy channel deposits ,
the main part comprises overbank sediments of levees , crevasse splays / channels / deltas , abandoned channels , backswamps and floodplain paleosols .
based on the lithofacies and the general geological setting , this system can be possibly related to an anastomosing river style .
there is not any indication for a long - lived lake ( lake pebas ) or any marine influx in this region during the late miocene .
( 1997 , 2007 , 2010 ; compare also cozzuol , 2006 ; westaway , 2006 ) .
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in miocene times a vast wetland existed in western amazonia .
whereas the general development of this amazing ecosystem is well established , many questions remain open on sedimentary environments , stratigraphical correlations as well as its palaeogeographical configuration .
several outcrops located in a barely studied region around eirunep ( sw amazonas state , brazil ) were investigated to obtain basic sedimentological data .
the observed deposits belong to the upper part of the solimes formation and are biostratigraphically dated to the late miocene .
vertically as well as laterally highly variable fine - grained clastic successions were recorded .
based on the lithofacies assemblages , these sediments represent fluvial deposits , possibly of an anastomosing river system .
sand bodies formed within active channels and dominant overbank fines are described ( levees , crevasse splays / channels / deltas , abandoned channels , backswamps , floodplain paleosols ) .
lacustrine environments are restricted to local floodplain ponds / lakes .
the mollusc and ostracod content as well as very light 18o and 13c values , measured on ostracod valves , refer to exclusively freshwater conditions . based on palaeontological and geological results the existence of a long - lived lake ( lake pebas ) or
any influx of marine waters can be excluded for that region during the late miocene .
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Introduction
Working area and geological background
Field and laboratory work
Results sedimentology of the outcrops
Discussion
Conclusions
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ribonucleotide reductase ( rr ) inhibitors have been studied as radiation sensitizers for over 30 years in both the lab and the clinic .
the first of these , hydroxyurea , has been studied in both cervical and head and neck cancers , among others .
although initially promising , many of the clinical trials produced negative results , or those that were difficult to interpret . there has recently been a significant advance in our understanding of this pathway from a number of well - done pre - clinical studies .
in addition , the discovery of new rr inhibitors with increased potency and improved binding characteristics has produced a significant increase in interest in this area .
this review will synthesize the data detailing the response of rr to ionizing radiation ( ir ) and will provide a perspective on the use of rr inhibitors as radiosensitizers in the treatment of human cancers . over the years , many groups have explored the use of rr inhibitors as chemotherapeutics in their own right , or as adjuncts to dna damaging molecules , particularly in hematologic malignancies . while this area looks promising , this subject will not be reviewed here .
ribonucleotide reductase is the rate limiting enzyme in the synthesis and repair of dna and is the only enzyme responsible for the conversion of ribonucleoside diphosphates to deoxyribonucleotide diphosphates , the fundamental building blocks of dna synthesis and repair .
r1 ( also called rrm1 or m1 ) is the larger , regulatory subunit that is constitutively expressed throughout the cell cycle .
it binds allosteric modulators , ribonucleoside diphosphates , and the nucleoside analogs gemcitabine and fludarabine .
there are currently two known smaller subunits that bind r1 to form the active enzyme ; r2 ( also called rrm2 or m2 ) and a more recently discovered p53 inducible homolog of the r2 subunit , known as p53r2 .
both contain a tyrosine free radical stabilized by a non - heme iron complex that is critical in the reduction of ribonucleotides .
r2 is known to be cell cycle regulated , with the highest levels during s phase , however the precise roles of the r2 and p53r2 subunit in the response to ir are an area of active debate , as outlined below ( figure 1 ) .
given the pivotal role of rr in dna synthesis and repair , many studies have investigated the effect of dna damaging agents , including ir , on rr and its subunits .
even so , there is currently still a great deal of controversy surrounding the exact mechanism of rr response to ir .
the first is that the small subunit r2 is up - regulated and provides dntps for dna repair in addition to its usual role in dna synthesis during s phase .
this is supported by a number of studies , including one by kuo et al . , who characterized the response of rr in the human cervical cancer cell line , caski .
they demonstrated an increase in r2 protein levels after treating cells with ir , which was correlated with an increase in rr activity .
however , there was no increase in the transcription of r2 , implying that the protein increase was due to post - transcriptional regulation ( kuo and kinsella , 1998 ) .
this finding was reinforced by studies that demonstrated dna damage dependent stabilization of the r2 protein without any change in mrna after ir exposure in mouse balb/3t3 cells ( chabes and thelander , 2000 ) ; however it was in contrast to earlier work showing transcriptional activation of the r1 and r2 promoters after exposure to ir in the same cell line ( filatov et al . , 1996 ) . even though the precise mechanism of r2 response to ir damage is unclear , work has shown that human nasopharyngeal cancer cells overexpressing r2 demonstrate a significant increase in radioresistance and fewer dna double strand breaks ( dsb ) after exposure to ir ( kuo et al . , 2003 ) , confirming the concept that r2 is important in the cellular response to ir
the second theory involves the p53 inducible subunit , p53r2 , first reported by tanaka et al .
they showed that p53r2 was not cell cycle regulated ( unlike r2 ) , but was significantly induced after exposure of normal fibroblasts to ir ( tanaka et al . , 2000 ) and was found to translocate to the nucleus , the proposed site of most important dntp production ( r2 did not ) . in cells that lacked wild - type ( wt ) p53 ,
there was no induction , indicating that functional p53 was necessary for p53r2 up - regulation .
cells transfected with p53r2 were resistant to dna damage induced cell death . in agreement with other studies , they found no increase in r2 mrna , but did not measure protein levels .
additional studies have subsequently shown that p53r2 forms an active complex with r1 ( guitett et al . , 2001 ) and
rr activity increased in correlation with the increase in p53r2 , however the response of r2 was variable , with decrease in some lines and a moderate increase in the others ( yamaguchi et al . , 2001 ) .
finally , other models are possible , including one described by xue and colleagues . in their study , both subunits were found to bind p53 in human oropharyngeal carcinoma cells , and in response to ir , were released to bind r1 in the nucleus ( xue et al . , 2003 ) , highlighting a third possibility in contrast to those previously presented .
clearly , there is still work to be done in elucidating the response of rr to ir .
many of the differences seen in the studies discussed can likely be attributed to the use of different techniques , materials , and especially cell lines .
what should be clear is that rr is involved in the cellular response to ir and targeting it is both rational and likely desirable in enhancing the treatment of cancers with ir .
it is likely that both r2 and p53r2 are involved to a different degree in different cancers , with cellular phenotype likely playing a key role in determining their relative significance .
hydroxyurea ( hu ) is a hydroxylated analog of urea and was the first rr inhibitor to be extensively studied .
it has directed activity at the tyrosine radical moiety of r2 and was first found to be active against cancer cells in 1963 ( stearns et al . , 1963 ) .
subsequent experiments in vitro showed that in addition to its direct inhibition of dna synthesis , it was also a sensitizer of cell killing by x - rays , particularly if given before or after ir ( sinclair , 1968 ) .
later experiments showed that this was also the case in in vivo animal tumor models using isotransplants of spontaneous c3h / he mouse mammary carcinomas ( piver et al . , 1972 ) .
the total dose of ir to cure 50% of tumors was reduced when hu was combined with fractionated ir , although this effect was nt seen with single fraction ir treatments . given these encouraging pre - clinical results during the 19601970s ,
hu was subsequently examined in a number of clinical trials in a variety of human cancers .
the majority of these trials have occurred in cervical cancer , most commonly in locally advanced disease . in particular , there were a number of prospective randomized controlled trials in the 1970s and 1980s that examined the effect of hu plus radiotherapy vs. radiotherapy alone .
and the gynecologic oncology group ( gog ) enrolled 190 women with figo stage iiib or iva cervical carcinoma .
hu was administered orally at 80 mg / kg starting on the first day of irradiation and every 3 days thereafter for 12 weeks .
patients received at least 50 gy minimum tumor dose to the whole pelvis followed by a single brachytherapy treatment of 20 gy to point a. in spite of the large number of patients enrolled , only 90 were eligible for assessment of response .
this was due to ineligibility ( wrong stage , wrong cell type , etc . ) and those that were inevaluable ( refused treatment , periaortic node irradiation , improper field , etc . ) .
the data were impressive , with a complete response ( cr ) of 68.1% in the hu group vs. 48.8% in the placebo ( p = < 0.05 ) , and a median progression free survival ( pfs ) of 13.6 vs. 7.6 months ( hreshchyshyn et al . , 1979 ) . however , myelosuppression was more common in the hu group , with seven grade iii or iv myelotoxicities .
they recruited 148 women with figo iib or iiib cervical carcinoma and again compared hu to placebo in the setting of conventional radiotherapy , with hu given every 3 days for 12 weeks .
of the stage iib patients , 74% receiving hu had no evidence of disease at the completion of therapy compared with 43.5% of the placebo ( p = < 0.01 ) .
of the stage iiib patients , the cr rates were 52.5 vs. 33% ( p = 0.22 ; piver et al . , 1977 ) .
again , 78% of patients in the hu group developed leucopenia vs. 11% in the placebo group indicating a significant toxicity in addition to the improved clinical effect . at the time these studies were published
, it was felt that hu added significant benefit to the treatment of locally advanced cervical carcinoma , and hu plus radiotherapy became the standard of care for the gog .
however , over time , much of the data in these studies has been challenged , particularly in a systematic review by symonds et al .
they found a number of methodological problems with the studies such as small sample size , large numbers of exclusions post randomization , subgroup analyses of already small groups and questionable censoring .
they concluded that hu appears to add to acute toxicity and probably increases late complications and that there is no convincing evidence of sufficient quality to suggest a therapeutic effect of this drug
although the gog initially used hu as its standard of care in the treatment of locally advanced cervical cancer , it was nt long before this combination was supplanted by a new adjunct to radiation therapy . one of the most important studies prompting this paradigm shift was the gog 120 trial , first reported in 1999 ( rose et al . , 1999 ) and recently updated ( rose et al . , 2007 ) .
gog 120 was a randomized phase iii study comparing cisplatin alone vs. cisplatin , fluorouracil , and hu vs. hu alone , in conjunction with pelvic irradiation for patients with locally advanced cervical cancer and pathologically negative para - aortic nodes .
they reported a significant improvement in pfs and overall survival ( os ) in both cisplatin containing arms ( p < 0.001 ) , with relative risks for progression of disease or death of 0.57 and 0.51 for cisplatin alone and cisplatin , fluorouracil and hu , respectively , when compared with hu alone . in addition , toxicities were similar in all groups .
further , a similar study by whitney et al . compared the efficacy of standard radiotherapy ( rt ) plus hu with standard rt plus fluorouracil ( 5-fu ) and cisplatin in a randomized controlled trial . in 368 women with figo stage iib , iii , or iva cancer of the uterine cervix , there were significant improvements in pfs and os with the 5-fu and cisplatin combination ( whitney et al . , 1999 ) .
adverse effects included leucopenia : 4% of 5-fu / cisplatin patients and 24% of hu patients experienced grade iii or iv toxicity .
given the findings of these studies , concurrent cisplatin and radiotherapy became the standard of care in locally advanced cervical cancer , spelling the end of hu use in the treatment of cervical cancer .
in addition to its study in cervical carcinoma , hu has also been extensively investigated in other cancers , particularly head and neck .
while early studies had significant flaws in methodology , a phase i study showed a 90% response rate in patients treated with hu , 5-fu , and palliative dose fractionated ir ( vokes et al .
, 1989 ) , prompting a series of trials by the same group and others .
this included work by mantz et al . , who examined the benefit of hu when added to a more extensive chemoradiation protocol including cisplatin , 5-fu , leucovorin , and interferon-2b induction chemotherapy followed by 5-fu and hu with fractionated radiotherapy in 32 laryngeal cancer patients .
median follow up was 44.5 months , and , after completion of all therapy , the cr rate was 94% .
median os was 44.5 months , and median pfs was 86 , 78 , and 78% at 1 , 3 , and 5 years , respectively , which compared favorably with other published data and for the first time saw patients with stage iv head and neck cancer failing distantly more often than locally ( mantz et al .
the increased local control , unfortunately , was associated with increased toxicity , which has also been seen in other studies .
one phase i study with prolonged infusion of hu with hyperfractionated , accelerated , external radiation in patients with advanced squamous cell cancer of the head and neck showed an increase in the severity of swallowing toxicity compared with previous trials , with severe edema , and reductions in motility and mobility of pharyngeal and laryngeal structures ( beitler et al . , 1998 ) .
the same group later published a study examining the long term impact on swallowing that showed persistent , severe swallowing dysfunction ( smith et al . , 2000 ) .
interestingly , these studies examined continuous hu infusions based on pre - clinical data that suggested that hu should always be given concurrently with ir to maximize effect . even though local control was excellent , with just 4 of 26 patients experiencing recurrence ,
quality of life is of great importance in these patients , and the late follow up reported esophageal strictures for the first time after chemoradiotherapy , suggesting that this particular regimen may be too aggressive .
the state of hu in head and neck cancer is well reviewed by argiris et al .
( 2003 ) and while the authors are optimistic about the future of chemoradiation in head and neck cancer , it remains to be seen where hu and other rr inhibitors will fit in the future treatment of this disease site . even as hu has slowly progressed in the clinic
, work has continued on examining the mechanism by which it sensitizes cells to the effects of ir . in particular , work by kuo et al .
has shown that the sequence with which cells are treated with hu plays an important role .
they demonstrated that in the caski cervical cancer cell line , clinically relevant concentrations of hu had a significant interaction with ir , with post - ir exposure > pre - ir ( kuo et al . , 1997 ) .
this was associated with increased g2 delay , suggesting a decrease in the repair of damaged dna .
in addition , in cells overexpressing the r2 subunit , hu is able to return ir sensitivity to baseline ( kuo et al . , 2003 ) , demonstrating that r2 inhibition is the likely mechanism for hu radiosensitization in these cells .
these findings are potentially informative about the failure of hu to become established as a radiosensitizer in cervical cancer . in the majority of the early trials ,
given that hu works best in vitro when dosed immediately after ir exposure , one could conclude that these trials were not optimized for best effect .
in addition , hu has recently been shown to have a significant effect on the mechanism of dna dsb repair employed by cells after exposure to ir .
burkhalter et al . showed that cells pre - incubated with hu were unable to use homologous recombination ( hr ) to repair dsb , and instead relied on non - homologous end joining ( nhej ) .
in addition , cells that were nhej deficient had significantly more dsb after hu treatment ( burkhalter et al . , 2009 ) .
given that nhej is thought to be the dominant dsb repair mechanism in cells treated with hu , rr inhibitors are likely to have enhanced activity in tumors that are nhej deficient .
even with new studies on its mechanism of action , hu will likely remain consigned to history due to the many inadequacies it has as a drug molecule .
while its oral absorption is almost complete and it is completely distributed in the water compartments of the body , hu has a short half - life ( between 1.6 and 4.45 h ; gwilt and tracewell , 1998 ) and its effectiveness is limited by relatively low affinity for rr and by the development of resistance .
one area where it could potentially find use in the future is in cns neoplasms , as it does cross the blood
recent studies have examined its use in progressive meningioma in combination with 3d - conformal radiotherapy and adjuvant chemotherapy . in one trial ,
pfs at 1 and 2 years was 84 and 77% , which is similar to other adjuvant studies ( hahn et al .
in spite of the mixed clinical data for hu , there is sufficient proof of concept to suggest that a rr inhibitor can be efficacious as a radiosensitizer in human cancers .
thus , there has been a concerted effort to discover more potent molecules with more favorable pharmacokinetics and pharmacodynamics for this purpose .
one of the more promising of these is triapine , a thiosemicarbazone that destroys the tyrosyl radical in r2/p53r2 by forming a redox active complex with iron , producing reactive oxygen species . in studies comparing it with hu in vitro
, triapine was shown to have significantly higher potency in both enzyme and cell assays .
in addition , it was fully active against hu and gemcitabine resistant cells and was equally potent against r2 and p53r2 , whereas hu was approximately threefold less potent at binding p53r2 ( zhu et al . , 2009 ) .
in addition , in in vivo models , triapine was active against hu resistant l1210 and kb cell lines and caused significant inhibition of solid tumor growth in mouse xenograft models ( finch et al .
further studies have examined the radiosensitizing properties of triapine in a number of human cell lines .
used a panel of three human tumor cell lines , including glioma , pancreatic , and prostate cancer cells , with triapine enhancing radiosensitivity when delivered 16 h before or immediately after ir by 1.5- to 2-fold .
ir interaction was associated with a reduction in the repair of dna dsb as evidenced by a persistence of h2ax foci at 24 h ( barker et al . , 2006 ) .
a similar effect was seen in mouse tumor xenografts , again , with greater effect if triapine was dosed just after ir .
the most effective temporal relationship between triapine dosing and ir is similar to that seen with hu in earlier pre - clinical studies .
interestingly , normal human fibroblasts were only sensitized when triapine was given before , not after ir , suggesting a potential for an improved therapeutic index for ir triapine sequencing that may be incorporated into future clinical studies .
are ic50 ( concentration of compounds producing 50% inhibition of recombinant rr activity ) values for both compounds in an in vitro rr activity assay with r2 or p53r2 bound to r1 ( zhu et al . , 2009 ) .
also shown are elimination half - lives ( t1/2 ) for both compounds ( gwilt and tracewell , 1998 ; kunos et al . ,
2010b ) . of note , many cancers have virally or mutationally silenced p53 that allows rr activity to continue unchecked . in these cancers , it is potentially of increased importance to inhibit the r2 and p53r2 subunits that have lost p53 regulatory control .
this is the case in cervical cancer , where the vast majority have dysfunctional p53 due to hpv infection . in one study , three cervical cancer cell lines with mutated or dysfunctional p53
were irradiated 6 h after triapine exposure . in all cases , the cell lines were sensitized to ir and sustained dna damage as measured by persistence of h2ax foci . in addition , by measuring dctp levels , the investigators were able to show reduced rr activity in cells with and without functional p53 , demonstrating that the inhibition of rr is p53 independent ( kunos et al . , 2009 ) .
these findings were reinforced by further work of the same group that also showed a synergistic effect when cisplatin was added in addition to triapine and ir ( kunos et al . , 2010a ) .
these promising pre - clinical data have prompted the initiation of a number of clinical trials .
indeed , triapine has so far been studied in 27 clinical trials in the usa at various stages of recruitment ( www.clinicaltrials.gov ) , including those in both solid and hematologic malignancies .
in particular there are three trials investigating the radiosensitizing potential of triapine , with data from one phase i study being published by kunos et al .
the purpose of their study was to assess the safety / tolerability , pharmacokinetics , and clinical activity of triapine three times weekly in concert with once weekly cisplatin and pelvic radiation in 11 patients with gynecologic malignancies .
triapine was dosed in 2 h infusions and the half - life was found to be 2 h. all 10 patients with advanced stage ib to ivb cervical cancer achieved complete clinical response , with a median 18 month follow up showing no disease progression in any of the patients .
five of the 10 patients had pet / ct evident pelvic or para - aortic lymphadenopathy before treatment , with complete resolution on follow up imaging after treatment ( kunos et al .
in addition to the clinical data , the authors also examined objective markers of disease response .
late responders had a significantly higher rr activity on day 10 vs. day 1 and there was no temporal change in rr activity in the early responders , indicating that the expected spike in rr activity after ir was suppressed by triapine .
the fact that the late responders experienced durable responses in spite of elevated rr activity at day 10 suggests that there may be other mechanisms of triapine activity that require further study .
the promising results from this phase i trial prompted a phase ii trial which is now underway .
over the last 10 years , there have been a number of major leaps forward in the field of radiation oncology that allow us to deliver higher doses of radiation in ever more specific ways to our patients .
still , in the vast majority of cases , we are constrained by dose limiting toxicities that must be accounted for when designing treatment plans and balanced against the therapeutic benefit realized .
therefore , the field of radio modulation and specifically radiosensitization will continue to grow in importance in the coming years as we search for ways to maximize the therapeutic index of our treatments .
while there are many different radiosensitizers currently being studied , the rr inhibitors are among the oldest of targets , and are getting a new lease of life in recent times with the development of more modern drug molecules .
as outlined in the review above , the story began with the study of hu in combination with ir in a number of pre - clinical trials in the 1960s , leading to a large amount of interest in the clinic , culminating with the gog adopting hu and ir as its standard of care in locally advanced cervical cancer . while its success in this arena was short
lived , many parallel studies investigating the mechanism of action of hu were carried out , resulting in a far clearer understanding of the biological interactions between ir and rr inhibitors .
even as hu was fading from view , investigators were working on successors including the intriguing molecule , triapine .
as shown above , this drug works in a similar fashion to hu , but has significantly improved potency , pharmacokinetics , and pharmacodynamics .
the phase i study in cervical cancers that was recently published includes some very encouraging data , and the field waits in anticipation for the publication of further trials .
it remains to be seen what the best dosing regimen for triapine will be , however the weight of pre - clinical data , including that with hu , suggests that daily dosing shortly after radiation therapy will be most effective and provide the greatest therapeutic window .
these authors would encourage studies incorporating this type of dosing schedule in the clinic , although this must obviously be weighed carefully against the potential for increased toxicity .
in addition to triapine , there are other new rr inhibitors being tested at various stages , including trimidox and motexafin gadolinium .
( 1994 ) , in a paper that reported 100-fold more potency at rr than hu in a cell based assay .
subsequent in vitro studies have demonstrated that while it acts as a radiosensitizer in panc-1 human pancreatic cancer cells ( ahmed and hassan , 2000 ) , it is less potent than hu and further work is ongoing to fully assess its potential .
motexafin gadolinium is a texaphyrin molecule that targets thioredoxin reductase in addition to rr . in in vitro experiments , it was shown to inhibit rr with an ic50 of 26 m ( hashemy et al . , 2006 ) , although given that it has a number of other cellular effects , it is unclear how much of its potency as a radiosensitizer is due to rr inhibition alone .
it is likely that the coming years will continue to see the emergence of new rr inhibitors with unique properties .
the recent advances in the understanding of rr biology and the development of new inhibitors places us at an important crossroads in the story of rr inhibitors .
there are sufficient data to provide proof of concept for the target from a biological standpoint , however clinical trials to this point have not been wholly convincing .
it will be interesting to follow the development of the field in the next 510 years , particularly with the clinical trials of triapine in cervical cancer , and hopefully at least one of the many rr inhibitors being studied will eventually bring additional therapeutic benefit to patients in the near future .
the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest .
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ribonucleotide reductase ( rr ) , the rate limiting enzyme in the synthesis and repair of dna , has been studied as a target for inhibition in the treatment of cancer for many years .
while some researchers have focused on rr inhibitors as chemotherapeutic agents , particularly in hematologic malignancies , some of the most promising data has been generated in the field of radiosensitization .
early pre - clinical studies demonstrated that the addition of the first of these drugs , hydroxyurea , to ionizing radiation ( ir ) produced a synergistic effect in vitro , leading to a large number of clinical studies in the 19701980s .
these studies , mainly in cervical cancer , initially produced a great deal of interest , leading to the incorporation of hydroxyurea in the treatment protocols of many institutions .
however , over time , the conclusions from these studies have been called into question and hydroxyurea has been replaced in the standard of care of cervical cancer . over the last 10 years
, a number of well - done pre - clinical studies have greatly advanced our understanding of rr as a target .
those advances include the elucidation of the role of p53r2 and our understanding of the temporal relationship between the delivery of ir and the response of rr . at the same time
, new inhibitors with increased potency and improved binding characteristics have been discovered , and pre - clinical and early clinical data look promising . here
we present a comprehensive review of the pre - clinical and clinical data in the field to date and provide some discussion of future areas of research .
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Introduction
Hydroxyurea
Triapine
Conclusion
Conflict of Interest Statement
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embryos generated through assisted reproductive technologies ( art ) are deemed surplus when , for some reason , they are not used for reproductive purposes ( svendsen and koch 2008 ; haimes and taylor 2011 ) ; instead , they are then disposed of or , where this is allowed by a country s art regulation , may be donated for a variety of research purposes .
thus , the generation and subsequent fate of embryos that are designated surplus are determined as much by legislation as by embryo biology or technological constraints .
for example , legislatures differ in whether cryopreservation of embryos is permitted , meaning that in some countries any ivf embryos not immediately transferred for pregnancy are surplus , while in others they may be frozen for later transfer . in some countries , the law considers surplus embryos as the primary source of material for human embryonic stem cell ( hesc ) research , while in others this use of embryos is expressly forbidden .
although hesc research has been subjected to exhaustive ethical scrutiny , the lack of consensus on the ontological or ethical status of surplus embryos means that the uses to which they may then be put remain contested ( deckers 2007 ; guenin 2008 ; moller 2009 ) .
even if using human embryos for stem cell research is deemed permissible , ethical issues continue to emerge as new social practices and roles develop around the act of embryo donation .
the relative novelty of these practices and roles , complicated by the pace of change in technical possibility and regulation , mean that there is still uncertainty about how to conceptualise embryo donation , not just in terms of systematic ethics but as a sociomoral practice in everyday social life and morality ( banks , scully , and shakespeare 2006 ) . from a sociological perspective , and also to understand more generally how everyday morality deals with unprecedented ethical dilemmas , it is important to examine which conceptualisations emerge as salient and how they are stabilised and used .
for example , one way of thinking about the donation of embryos to research is as a bodily gift relationship , one of the many established by modern biomedicine such as the donation of eggs and sperm for reproductive purposes , organs or tissues for transplantation , blood for transfusion , and other biological tissues for research .
drawing analogies to existing practices in this way has proved helpful in other instances of bioethical novelty ( hofmann , solbakk , and holm 2006 ) .
however , analogies can equally well be misleading , if there are morally relevant but unacknowledged differences between situations .
the contexts in which different organs and tissues are donated vary significantly , and these differences influence the sociomoral understanding of donation in each case and make them noncomparable in ethical terms
. reproductive tissue , for example , is generally distinguished from other types of donated tissue because eggs , sperm , and embryos have the potential to give rise to new individuals , not just to prolong the lives of existing individuals , or to be used for research . because embryos are generally considered to have a different moral status from other tissues , the use of surplus embryos in research raises moral unease about the instrumentalization of human life that is not raised in quite the same way by the donation of either ova or sperm .
it is therefore unclear whether an embryo can meaningfully be treated as a gift ( donation ) without blurring the morally relevant differences between embryos and other tissues .
similarly , hesc research , to which the embryo may be given , is a domain of biomedical practices and not an entity . as such
, hesc research is not a subject with which a gift relationship can be established ( unlike , for instance , an organ recipient ) .
understanding the social and ethical meanings that are emerging for the practices associated with embryo donation calls for a detailed empirical examination of people s reasoning behind donation decisions .
however , most empirical studies of embryo donation have not focused on people s donation rationales in depth ( with some exceptions ; for example , haimes et al . 2008 ; de lacey 2003 ) .
the scarcity of data on donor rationales means there is a corresponding lack of information on contextual and other factors that influence donor decisions and how these can be related to the evaluations of theoretical ethics .
the authors of this paper have been involved in a series of linked qualitative studies of practices of embryo donation , first in the united kingdom ( researcher haimes and colleagues ) , then switzerland ( scully , rehmann - sutter , and porz ) , and in a smaller pilot project in china ( mitzkat , rehmann - sutter , and haimes ) .
all three studies shared an interest in understanding reasons for the donation or non - donation of embryos . while the studies in switzerland and china drew upon the original u.k .
design , each study was conducted independently , and the details of each project , including interview design , differed in light of the varying regulatory , clinical , and cultural contexts . however , by looking across the three data sets , we hope to gain cross - cultural insights into donation and non - donation rationales and the moral understandings on which they are based .
the three studies all used open - ended , semi - structured , one - off interviews between the researcher and people who had been in the position of deciding whether to donate their surplus embryos to research .
interviews were designed to explore in depth not just the interviewees decision about donation but also the background to that decision , such as their ivf story , their family and other relationships , their relationship with the clinic and its staff , and so on .
the interviews were transcribed , coded , and initially analysed to identify the reasons for donation decisions given by participants in each study , using an interpretative approach that all of us have previously found useful for identifying key themes around decision - making and implicit or explicit ethical judgements ( charmaz 2006 ; smith , flowers , and larkin 2009 ) .
study ran for more than three years ; after some familiarisation and observation in the collaborating clinic , 44 in - depth interviews were conducted with couples who had been asked to donate their surplus fresh embryos , generated through ongoing ivf treatment , to hesc research ( haimes and taylor 2009 ) . in the united kingdom , it has been possible for some years to donate unused embryos to stem cell and other kinds of research and also to other couples .
the swiss study was carried out shortly after a change in the law that permitted fresh or cryopreserved surplus ivf embryos , under strictly defined conditions , to be donated to stem cell research only . in this study , 17 individuals who had variously chosen to donate or not to donate were interviewed ; thus , the decision concerned the fate of cryopreserved embryos some time after the ivf treatment that had produced them ( scully , rehmann - sutter , and porz 2010 ) . the chinese work involved a much smaller three - month pilot study carried out in a large art hospital .
it was designed as a pilot to provide a comparison with the u.k . and swiss material and is , therefore , included here despite the low number of participants , but with no attempt to draw general conclusions from it .
legislation in china does not allow the donation of embryos to other couples , but consent can be given for a surplus embryo to be used in stem cell research or for it to be discarded .
the study included participant observation of the information and consent procedures and qualitative interviews with five ivf patients , three choosing to donate to stem cell research and two refusing ( mitzkat , haimes , and rehmann - sutter 2010 ) .
for the purposes of this paper , the authors involved in the studies jointly compared the rationales for donation decisions given by their participants .
we also independently reexamined interview transcripts for material relevant to the discussion of gratitude . in the rest of this paper , we first identify and compare the main reasons given by participants in these three studies for their decisions to donate or to refuse to donate their surplus embryos to research .
we then look in more detail at the implications of our findings for one area of potential ethical concern : the possible role of gratitude in making embryo disposition decisions . in this way
, we not only collect empirical data to help understand the emerging moral meaning of a new practice , but also give an example to show how empirical data can be used to question and then refine the conceptual basis of bioethical theory .
participants who chose to donate their surplus embryos to research had a background premise that donation is fundamentally permissible because embryos do not have the sort of ontological or moral status that would forbid it .
the swiss study discovered people stating this explicitly : [ embryos ] are not , they are not yet people ; they are nt beings with souls as far as i m concerned , as one swiss participant said.1 but this did not mean that the surplus embryos were considered as morally equivalent to any other tissue , and participants in both the swiss and u.k .
interviewees stopped themselves midway through sentences referring to the left over embryos , which they clearly felt , on reflection , was an inappropriate phrase .
so the embryos did not have the sort of moral status that would have made it wrong to donate them , but neither were they completely disposable .
to some of our participants , embryos had a value ; they were a precious resource ( haimes et al .
2008 ) that made simply throwing them out an unjustifiable waste . as another u.k .
[ i]t just really seemed a waste , really , not to have them used .
it was not always clear , however , quite how interviewees were using this notion of precious : whether with reference to economic bio - value ( waldby 2000 ; waldby and mitchell 2006 ) or because they had been obtained at intense personal and emotional cost , or because they had high moral value , or possibly a combination of all these aspects
. particularly in the swiss study , which involved cryopreserved embryos stored for some time , participants referred to embryos moral status in a nuanced way that was highly sensitive to the developmental stage of the embryo , its physical location ( whether inside or outside the body ) , and its state ( whether cryopreserved or not ) ( see scully , rehmann - sutter , and porz 2010 ) .
studies seemed less interested in explicitly defining the moral status of an abstract embryo than working out what their own particular embryos meant for them at defined points in their own story , and what this meaning then indicated it was morally permissible for them to do with that embryo .
across all three studies the commonest rationale for opting to donate was a willingness to contribute to potentially curative medical research .
such research was seen as a valuable endeavour by those like the swiss participant who said , i feel that , fundamentally , research has to go on , and i support that .
donation of their spare embryos to research was therefore seen as a morally good act , based on a kind of ethical arithmetic in which simple disposal produced zero good from the surplus embryo , while donation had the potential to do good by supporting research . as one u.k .
interviewee said , it was quite simply that we ve been helped by the system , we should help the system back. not because of some sort of martyrdom or heroics or anything like that , just quite simply that without such efforts things do nt progress . in the u.k .
study there was an added imperative to donate surplus embryos to research as there had been a lot of press coverage about the hoped - for curative outcomes of stem cell science :
i think the research is very important , like it was in the news a while ago to say that [ the senior clinician ] was successful in stem cell research and that s what you need in order to provide medical assistance in the future , so i m all for it .
this observation raises a separate ethical concern about the patients understanding of the information they were given about the research goals . here
in the swiss study , the notification from the clinic storing the frozen embryos stated clearly that donation was to stem cell research , yet the majority of donors we interviewed who referred to research did so in terms that were directly relevant to infertility .
a lot of preparatory work , research work , went on before medicine was advanced enough that it was possible for us to have children .
[ t]his is now maybe a tiny tiny contribution , that we can give back , if we now donate a , er , fertilised egg , perhaps for further research .
note that in the chinese pilot study none of the participants spoke in precise terms about the research involved ; however , they described it broadly as
one chinese interviewee said she did not really know what the research was about , but trusted the doctors and hoped it would help other infertile women . in switzerland ,
the donors were former ivf patients donating cryopreserved embryos ; in china , they were current ivf patients donating either fresh or cryopreserved embryos . in neither of these studies
were participants asked outright if they thought their donated embryo was being used in infertility research .
however , the fact that they used phrases such as helping others as we have been helped ( italics added ) indicates a potential misunderstanding .
it raises the possibility that if they had been fully aware that the research undertaken with their embryos did not concern infertility , participants might have chosen against donation . the u.k .
participants had much clearer ideas about the research to which they were being asked to donate ; they knew that it was not for research on fertility issues .
these participants , as in the chinese study , were current patients donating fresh embryos .
the differences in comprehension are therefore unlikely to be due to the request context ( former vs. current patients or fresh vs. frozen embryos ) in itself . however , in the u.k .
study participants were asked to decide whether or not to donate to a number of specific projects , for each of which the goal and methodology was explained in detail , orally and in writing .
it is possible that this level of engagement with the detail of the research underlies the difference .
it will be important to clarify this , and to identify best practices for maximising patient comprehension of the research goals , because if ( as our material indicates ) the type of research to which people donate is a relevant factor in their moral evaluation , then the question of whether they fully understand its nature is ethically crucial . as we have seen , some of our participants reasoned that if their embryo was not going to be used for pregnancy , then donating it to a good endeavour ( research ) would be more meaningful than simply discarding it .
for example , one interviewee said : if we ve created life that we , or i , do nt want any more , then maybe it s least sinful , or however you want to put it , if something meaningful happens to it .
i feel a little bit guilty sometimes , not very much , but a little bit like if we can at least give these embryos for a good reason , then it was nt completely in vain . using similar wording , another said , if i ve already gone a bit astray , and in a sense we ve produced life , erm , then at the very least something meaningful should come out of it .
our interpretation of their statements is that they felt they had ( inadvertently ) done something wrong in creating life but then not using that life as intended in pregnancy , and that donation to a good endeavour would in some way make up for this wrong .
( we want to be quite clear here that we are not arguing that failure to use an ivf embryo in pregnancy actually is a moral wrong , only that some of our participants said that in their case they felt so . )
they did not indicate that they felt their use of ivf in itself had been wrong , but articulated the sense that in ending up with surplus embryos they had done something less than ideal .
for these swiss participants , then , their donation decision appears to have been driven in part by what we could call a reparative urge .
no indication of any similar reparative urge was observed in either the u.k . or the chinese interviews : we discuss this difference further below .
across all three studies the commonest rationale for opting to donate was a willingness to contribute to potentially curative medical research .
such research was seen as a valuable endeavour by those like the swiss participant who said , i feel that , fundamentally , research has to go on , and i support that .
donation of their spare embryos to research was therefore seen as a morally good act , based on a kind of ethical arithmetic in which simple disposal produced zero good from the surplus embryo , while donation had the potential to do good by supporting research . as one u.k .
we ve been helped by the system , we should help the system back. not because of some sort of martyrdom or heroics or anything like that , just quite simply that without such efforts things do nt progress . in the u.k .
study there was an added imperative to donate surplus embryos to research as there had been a lot of press coverage about the hoped - for curative outcomes of stem cell science :
i think the research is very important , like it was in the news a while ago to say that [ the senior clinician ] was successful in stem cell research and that s what you need in order to provide medical assistance in the future , so i m all for it .
this observation raises a separate ethical concern about the patients understanding of the information they were given about the research goals . here
in the swiss study , the notification from the clinic storing the frozen embryos stated clearly that donation was to stem cell research , yet the majority of donors we interviewed who referred to research did so in terms that were directly relevant to infertility . for instance , one participant said ,
a lot of preparatory work , research work , went on before medicine was advanced enough that it was possible for us to have children .
[ t]his is now maybe a tiny tiny contribution , that we can give back , if we now donate a , er , fertilised egg , perhaps for further research .
note that in the chinese pilot study none of the participants spoke in precise terms about the research involved ; however , they described it broadly as scientific research for the ivf treatment .
one chinese interviewee said she did not really know what the research was about , but trusted the doctors and hoped it would help other infertile women . in switzerland ,
the donors were former ivf patients donating cryopreserved embryos ; in china , they were current ivf patients donating either fresh or cryopreserved embryos . in neither of these studies
were participants asked outright if they thought their donated embryo was being used in infertility research .
however , the fact that they used phrases such as helping others as we have been helped ( italics added ) indicates a potential misunderstanding .
it raises the possibility that if they had been fully aware that the research undertaken with their embryos did not concern infertility , participants might have chosen against donation .
participants had much clearer ideas about the research to which they were being asked to donate ; they knew that it was not for research on fertility issues .
these participants , as in the chinese study , were current patients donating fresh embryos .
the differences in comprehension are therefore unlikely to be due to the request context ( former vs. current patients or fresh vs. frozen embryos ) in itself . however , in the u.k .
study participants were asked to decide whether or not to donate to a number of specific projects , for each of which the goal and methodology was explained in detail , orally and in writing .
it is possible that this level of engagement with the detail of the research underlies the difference
. it will be important to clarify this , and to identify best practices for maximising patient comprehension of the research goals , because if ( as our material indicates ) the type of research to which people donate is a relevant factor in their moral evaluation , then the question of whether they fully understand its nature is ethically crucial .
as we have seen , some of our participants reasoned that if their embryo was not going to be used for pregnancy , then donating it to a good endeavour ( research ) would be more meaningful than simply discarding it .
for example , one interviewee said : if we ve created life that we , or i , do nt want any more , then maybe it s least sinful , or however you want to put it , if something meaningful happens to it .
i feel a little bit guilty sometimes , not very much , but a little bit like if we can at least give these embryos for a good reason , then it was nt completely in vain . using similar wording , another said ,
if i ve already gone a bit astray , and in a sense we ve produced life , erm , then at the very least something meaningful should come out of it .
our interpretation of their statements is that they felt they had ( inadvertently ) done something wrong in creating life but then not using that life as intended in pregnancy , and that donation to a good endeavour would in some way make up for this wrong .
( we want to be quite clear here that we are not arguing that failure to use an ivf embryo in pregnancy actually is a moral wrong , only that some of our participants said that in their case they felt so . )
they did not indicate that they felt their use of ivf in itself had been wrong , but articulated the sense that in ending up with surplus embryos they had done something less than ideal . for these swiss participants ,
then , their donation decision appears to have been driven in part by what we could call a reparative urge .
no indication of any similar reparative urge was observed in either the u.k . or the chinese interviews : we discuss this difference further below .
approximately half of the swiss participants who were interviewed turned out to have decided against donation and instead have their surplus cryopreserved embryo(s ) destroyed .
none of these explicitly used the moral status of the embryo as the fundamental basis for rejecting donation to research .
the scientists would do with their embryo , which was articulated either as disapproval of specific types of research ( e.g. , not if it s for cloning ) or as a more general unwillingness to relinquish responsibility of the embryo to unknown others . in both sets of reasoning , therefore , participants were expressing unease about the loss of control over what would happen to their embryo .
the two chinese interviewees who had opted against donation also did so because of reservations about lack of knowledge of exactly what the embryos would be used for , explicitly mentioning the fear that they would be donated to ( i.e. , used to generate pregnancy in ) other women .
in addition , two of the swiss participants gave financial reasons , that is , they expressed anger that they would not be compensated for giving up their embryo and that through donation researchers were getting something for nothing .
study , speculation about why some people might refuse to donate raised concerns about what might be done with the embryos and whether a baby would be developed through experimentation ; others wondered if refusers were worried that another infertile couple would receive their embryos and have a baby when they , the embryo providers , failed to do so .
sample contained only two actual refusers , who objected on the grounds of possible use in animal research ( haimes and taylor 2009 ; hug 2008 ) .
these observations provide material for an empirically grounded bioethics of the ivf stem cell interface ( franklin 2006 ) . in the rest of this paper , we consider a single aspect in detail : the potential problem of gratitude influencing informed consent .
empirically , the main reasons given by our participants for donation were : ( 1 ) to avoid the waste of a precious resource , ( 2 ) to give something back to research , and ( 3 ) to compensate for a perceived moral wrong . in none of the three countries
did our participants indicate that their decision arose out of any sense of gratitude to the physician who had given them ivf treatment .
this is an important observation , because the possible impact of gratitude on informed consent in embryo donation has previously been raised by bioethical commentators .
it has been suggested that if there is any institutional or procedural link between ivf treatment and hesc research , the potential donors gratitude toward the clinicians who have helped them to conceive may steer them toward opting for donation ( haimes and luce 2006 ; parry 2006 ; mcleod and baylis 2007 ; also discussed in roberts and throsby 2008 ) .
the bioethical concern is that parents who receive the gift of a baby will feel a sense of gratitude ; feeling grateful might then cause parents to feel that they should respond with a gift in return ; and if a request for donation of surplus embryos is made in that context , potential donors might inadvertently be encouraged to see donation as an appropriate form of return gift . in this way
the moral emotion of gratitude could compromise the potential donor s freedom to weigh the pros and cons of the request , which in turn means compromising the capacity to give fully voluntary consent .
even where the clinicians and researchers themselves are scrupulous about not using persuasion , gratitude might be persuading potential donors to do something they would not otherwise choose to do . to avoid this possibility , countries that allow embryo donation for research may attempt a strict separation of ivf treatment and the stem cell research domain .
( not all legislatures do this , however : china is one country that does not . ) in the united kingdom , for example , it is accepted good practice that requests for donation of spare embryos should not be made by the physician(s ) who delivered the original ivf treatment . in switzerland ,
a predominant interpretation of the current law on embryo donation is that a couple undergoing ivf treatment should not be told even of the theoretical possibility that a surplus embryo might be generated unless it actually is ( porz et al .
regulators hope to rule out any conflict of interest on the part of the treating physician as well as the possibility of gratitude on the part of the patient .
there are several theoretical questions that can be asked about a claim that the risk of gratitude affects potential donors decisions .
for example , moral psychologists might want to examine the emotional exceptionalism in which gratitude is seen as potentially compromising , while other emotions , or even the absence of emotion , is not .
there is also an obvious empirical question which does not seem to have been closely examined : whether potential embryo donors actually do experience a sense of personal gratitude to the physician or one strong enough to sway their donation decision .
however , they do indicate that participants rationales for donation indicate a desire to give back in a more complex way .
first , our empirical results show the importance of distinguishing between the three moral emotions of gratitude , indebtedness , and solidarity .
what we normally mean by gratitude is an emotion primarily associated with gifts or with help that is undeserved .
gratitude has been defined as a feeling of thankful appreciation for favours received ( guralnik 1971 , 327 ) and is experienced as a positive emotion .
( 2006 ) provide a basis from experimental psychology for distinguishing between indebtedness and gratitude .
one important distinction is that indebtedness is an emotion of exchange , whereas gratitude is not ( watkins et al .
even if a debt of gratitude is felt , it does not appear to be analogous to an economic debt
indebtedness , however , is , literally , a debt : a state of obligation to repay another ( mauss 2001 , 2 ) .
a further feature associated with indebtedness but not with gratitude is the inherent imbalance of power , so that a hierarchy is created in which those who are indebted are rendered more vulnerable . the empirical social psychological work of watkins et al .
indicates that , in experimental settings at least , a person s feelings of gratitude diminish as the expectation of return ( indebtedness ) increases . the more reciprocity is expected or demanded ,
importantly for considering the effects of these social emotions on donation decisions , watkins et al . found that the greater the expectation that something will be given back in return , the less motivated the participants were actually to comply with the norm of reciprocity ( watkins et al .
these results suggest that if patients were to sense any expectation of return by an individual physician or researcher , it would if anything lessen any gratitude they might have felt . in the ivf context , what people have received ( the thing for which they might feel grateful ) is their baby or pregnancy , or at the very least , treatment . donors who achieve this have not received a gift , but a medical service , which will have been paid for in one way or another . in switzerland , where ivf is not covered by the mandatory health insurance
, this will often be direct payment from the patients to the clinic , as it will also often be in china . in the united kingdom ,
patients who do not already have children commonly receive two free cycles of nhs treatment ( paid for through taxes on the population ) , and then they pay for future cycles directly through fees to the clinics . whatever the system of payment , however , the point is that the ivf physician or clinic has already been paid , directly or indirectly , for the treatment which produced the outcome .
so in the case of embryo donation , it could be argued that neither gratitude nor indebtedness should be anticipated .
gratitude would not be expected , because what patients have received was not a favour or unexpected gift ; and indebtedness would not be expected , because there is no debt if payment has already been made . in our three studies , although we found appreciation and high esteem were expressed for the work of the treating hospital or team , we found no evidence of gratitude being expressed toward an individual physician .
this is despite the fact that in both the united kingdom and china mention was made of the well - known head physicians who led each clinic ; the mentions were in appreciation of their reputations , not as a debt of gratitude .
participants wanted to do something with the surplus embryo that would be of benefit to research and medical treatment for infertility , but this was not directly associated with the physician who provided it originally . indeed , some of our interviewees were critical of aspects of their own ivf treatment , while still wanting to support the infertility research enterprise overall . in this context , it should be remembered that about half the swiss participants interviewed in fact chose not to donate , primarily out of anxiety about losing control over the fate of their embryos , but also in some cases because it meant researchers were getting something for nothing .
study , a few patients were suspicious that research was being prioritised over treatment and that embryos might have been kept back for the research rather than frozen .
although only a small number of participants expressed this view , it has particular relevance here as it indicates clearly the absence of either gratitude or indebtedness : indeed , it suggests that these participants felt that , if anything , donation would mean the researchers were receiving something beyond their entitlement or were indebted to the patients rather than the reverse .
social exchanges may entail giving back not to an individual , because the individual may be unknowable or because the benefit may not have come from a single identifiable person , but to the pattern of social life
( becker 1986)for example , giving back to the institutional biomedical research that had helped the participants . in serial reciprocity , person a receives something from person b , but pays back not to person b but to some other third party ( moody 2008 ) . serial reciprocity accounts for the way that altruistic blood donors often do describe their action in terms of reciprocity , even though they have not received anything from the person to whom their blood eventually goes .
what drives serial reciprocity is not gratitude or indebtedness to an individual or an organisation but a sense of solidarity with unknown others in the community . in the case of embryo donation , what appears to be a directly reciprocal act of gratitude a surplus embryo in return for treatment may thus be understood as something quite different .
participants in our studies used a rationale for donation based not on reciprocal exchange between individuals , but on giving back to a research enterprise from which they had benefited and which they hoped would benefit unknown others in the future . for the swiss and chinese donors who choose to donate because they think they are supporting infertility research , these unknown others are not completely anonymous : they are known because of imaginatively shared experience . giving something back to the research that had helped them ( as they think they are doing )
was not considered an obligation but , rather , a form of return that was meaningful and seemed especially fitting to them because of the experiences they had gone through . in the u.k .
study , although there was greater clarity about the purpose of the research being contributed to , there was still a sense among embryo providers that they were benefiting from the fact that others had participated in research in the past which had improved ivf ; they were clear in expressing solidarity with this imagined community of previous ivf research contributors by making their own contributions to research , albeit research in another domain .
at least some of our participants , then , also see their donation to research as an indirect way of passing on the benefit they have received from research to someone else , whose situation in some way resembles their own .
the social meaning of this indirect reciprocity is neither gratitude nor indebtedness , but solidarity with other present and future patients .
to summarise : our interpretation of the material suggests that donors reasons for donating were not connected in any straightforward way to either gratitude or indebtedness . for some people
the desire not to waste a valuable ( in more than one sense ) resource is foremost .
a generally positive stance toward biomedical science means that donation is a good use of a valuable resource . with some of the swiss participants
, we also identified a reparative urge coming from a sense of moral unease , and here donation seems to offer a route through which the urge to make reparation can be satisfied .
all of this suggests that neither gratitude nor indebtedness per se are present in decision - making at the ivf stem cell interface , at least not in terms of undue inducement or of compromising the capacity for informed consent .
the reparative urge foregrounds a different set of ethical questions about the sociomoral meaning of the generation of spare embryos and the act of donation .
for example , the perceived need for reparation that appears to drive some of the swiss participants donation choices arises because people felt some sense of wrongdoing at creating embryos that are not used for pregnancy .
the spirit of the swiss legal framework strongly reinforces the sense that the occurrence of embryos not used for pregnancy is to be considered as wrong . from this point of view
, one possible conclusion could be that the felt desire for reparation is in fact an ethically appropriate response : if the act of deliberately creating an embryo is a morally significant one , which needs to be justified by the good of its intended outcome ( pregnancy ) , then not using it for the purpose that justifies it does indeed present a genuine ethical difficulty .
on the other hand , the novelty of surplus embryos in the swiss legal and social context ( scully and rehmann - sutter 2006 ) meant that many patients would have been unaware at the time of their ivf of the possibility of generating a surplus embryo .
moreover , in most cases there were perfectly valid clinical or legal reasons why the embryos could not be transferred , which fully justified the failure to use them as originally intended . that these factors were not in the participants control
the swiss participants were quite aware of the valid reasons why their embryos could not be transferred , and yet some of them still said that they had fallen short of some moral standard .
we suggest that this persistent unease ( which does not appear so prevalent in the u.k .
these are captured in swiss legislation s highly defensive attitude toward embryos in general and the generation of surplus embryos in particular .
swiss law operates from the presumption that a surplus embryo is an exceptional event that will happen only through the failure of regulation and practice specifically set up to prevent it ( porz et al .
in addition , as we discussed earlier , the generation of surplus embryos is still an unfamiliar social practice and the role of the embryo donor is one that lacks widespread cultural recognition . in the swiss context of legal exceptionalism , then , producing one of these culturally unfamiliar entities may more readily be understood as something
wrong for which reparation is in order . in the more permissive cultural and legislative context of the united kingdom , although some interviewees expressed guilt about other aspects of the process ( for example , inappropriate styles of speech about embryos ) , there was no sense that not using the embryos as intended in itself constituted a moral failing .
if our interpretation is correct , as the creation of surplus embryos becomes normalised in swiss society and the role of embryo donor becomes normalised in both the u.k . and
swiss ( and other ) societies , there will be a shift in ideas about the moral meaning of surplus embryos , and this should be empirically testable . for the majority of the participants in switzerland and the united kingdom who chose to donate their surplus embryos , donation decisions were not driven by a reparative urge but by the feeling that donation expressed solidarity with research and/or other patients .
what our empirical work shows is that gratitude to an individual must be distinguished from a solidarity - based desire to give something back to medical research .
however , this does not necessarily mean that solidarity - based reciprocity has no relevant impact on the capacity for voluntary consent .
one possible consequence , for example , is that if potential donors feel solidarity with the research enterprise , or with present and future patients , then they might also experience an obligation to support it .
however , in all three studies there were people who chose not to donate ( about half of the swiss interviewees did not donate , while in the united kingdom approximately 46 percent of those asked have been shown to opt against donation [ choudhary et al .
2004 ] ) , which suggests either that there is no sense of obligation , or that it is neither universal nor irresistible .
there is a second problem if solidarity - based reciprocity is taken as something to be prevented .
if there is a risk that gratitude felt by an individual could influence donation decisions , then the risk can be minimised by separating ivf and stem cell research physically and procedurally , as bioethicists have suggested and some clinics and guidelines have implemented .
but if instead there is a desire to benefit research or to show solidarity with collective others rather than an individual , then what sort of physical or procedural barriers could be set up to prevent it having an influence
? there are further theoretical questions to be explored here about the assumption that contextual emotions such as gratitude or solidarity compromise the decision to donate .
the situation in which donation of a surplus embryo is a possibility comes about as a result of a variety of social , cultural , political , legal , and emotional features that combine to make the situation what it is .
it will be important to identify carefully the features that are constitutive of the situation of having a surplus embryo ( including a sense of solidarity to similar others , emotional bonds with the embryo that change over time , regret over the failure to use all embryos for pregnancy , and so on ) and to consider whether these constitutive features can or should also be treated as factors that compromise the responsible , autonomous decision - making capacity of the people involved .
this introduces the question of the extent to which any decision to donate can be detached from its context and the contextual factors that shape how people respond to donation requests .
finally , we should not forget that the possibility that some donors misunderstand the kind of research they are donating to raises some ethical difficulty .
it means that donors may be deciding for donation out of a misplaced sense of solidarity a different ethical problem .
the deliberative processes of the donor participants in our three studies incorporated their appreciation of the particular moral value of their own embryos and of the value of biomedical research .
these deliberations are complex , and the participants own dependency on pre - existing medical research and their affiliation with others in similar situations must be recognised , we suggest , in order to understand how they might configure donation as a means to cope with the moral ambivalence of the situation in which they find themselves .
their decisions are not driven by some unacknowledged sense of debt , but rather are responsive to the emerging social and moral reality of embryo donation in the first decade of the 21st century .
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this paper is based on linked qualitative studies of the donation of human embryos to stem cell research carried out in the united kingdom , switzerland , and china .
all three studies used semi - structured interview protocols to allow an in - depth examination of donors and non - donors rationales for their donation decisions , with the aim of gaining information on contextual and other factors that play a role in donor decisions and identifying how these relate to factors that are more usually included in evaluations made by theoretical ethics .
our findings have implications for one factor that has previously been suggested as being of ethical concern : the role of gratitude .
our empirical work shows no evidence that interpersonal gratitude is an important factor , but it does support the existence of a solidarity - based desire to give something back to medical research .
thus , we use empirical data to expand and refine the conceptual basis of bioethically theorizing the ivf stem cell interface .
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Embryo Donation as a Sociomoral Practice
Empirical Exploration of Donation Decisions
Reasons for Donating
Contribution to Research
Reparation
Reasons Not to Donate
The Problem of Gratitude
Concluding Comments on Reparation, Solidarity, and Consent
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neurosurgery is characterized by the delicate balance between surgical success and potential for devastating side effects . thanks to multiple technological improvements
, the morbidity of neurosurgical interventions has substantially decreased over the last decades , allowing for the resection of previously inoperable lesions . in particular , image - guided neurosurgery ( igns ) devices allow the use of coregistered and fused multimodality 3d images to guide the surgeon 's hand and help define preoperatively the boundaries of pathological and predefined functional structures . meanwhile
, new modes of medical imaging have also improved the localization of pathological lesions and their characterization .
medical imaging nowadays includes a wealth of different techniques , such as computed tomography ( ct ) , structural and functional magnetic resonance imaging ( smri and fmri ) , diffusion tensor imaging ( dti ) , and positron emission tomography ( pet ) .
although the overall accuracy of igns is estimated to be 12 mm , current neuronavigation systems can not , however , adapt to changing conditions over time .
skull - opening brain shift , brain retraction , cerebrospinal fluid suction , lesion resection , perfusion , and pharmacological manipulation during surgery indeed all alter the 3d morphology of the structures [ 25 ] .
these changes can lead to localization errors that are one order of magnitude larger that igns accuracy [ 1 , 2 , 6 ] and may result in incomplete resections or unexpected damage to normal brain .
such inaccuracies could be reduced if one could acquire , throughout surgery , fresh images of the same modalities and quality as the preoperative ones
intraoperative images such as intraoperative mr ( imr ) images are with the exception of a handful surgical facilities usually acquired using low - field mri scanners that provide lower resolution and contrast than their preoperative counterparts , and , to this date , several useful imaging modalities , such as pet and possibly meg , can not be acquired intraoperatively .
one solution is to bring over the high - quality preoperative multimodality images into the intraoperative configuration of the brain using a nonrigid registration technique [ 710 ] .
one category of nonrigid registration techniques uses physics - based models , where landmarks are tracked in successive reduced - quality intraoperative images , and their displacement fields drive the deformation of a biomechanical model .
so far , most of the mechanical conditions of the brain can not be estimated in the operating room , such as the volume of cerebrospinal fluid flowing out of the skull cavity , intercellular fluid volume changes that result from mannitol injection , or changes in blood volume and vessel permeability .
the fact that an intraoperative image can provide the knowledge of the current state of the brain after some deformation partly eliminates the need for a complete evaluation of these mechanical conditions .
the nonrigid registration technique replaces them with the landmark displacements evaluated from successive intraoperative images . using a nonrigid registration technique based on a biomechanical model , three types of brain deformations
have been identified that require specific modeling , although they depend on common parameters , such as csf suction , perfusion , or pharmacological manipulation .
the first deformation is the brain shift , which appears at the beginning of surgery with the opening of the skull and dura .
the suction or leakage of csf , as well as the release of intracranial pressure caused by tumor growth , generally cause such shift of the brain ( note that in this work , we name brain shift the specific shift of the brain that occurs after the opening of the skull and dura , before any other surgical act has happened ) .
however , for these deformations , we will consider that the shift is a part of these two deformations .
the second deformation is the retraction ; when target tissues are located deep inside the brain , the surgeon incises brain tissues and inserts a retractor to spread out the tissues , and to create a path towards the target .
the third deformation is the resection , that is , the removal , of lesion tissues .
three deformations can thus be defined in terms of the two elemental actions that change the topology of the brain : the introduction of a discontinuity and the removal of some tissues .
most studies of brain deformation based on biomechanical models have focused on shifts ( the topology of the brain is not modified ) , that occurs just after the opening of the skull and dura [ 1125 ] .
a good review of these different studies can be found in [ 24 , 2628 ] .
resection and retraction are more complex to model than ( brain ) shift . until recently ,
their modeling for the specific application of preoperative image update has received much less attention .
one of the difficulty for modeling resection and retraction is that both induce a topological change of the brain because some tissue are cut .
a method of mesh adaptation [ 2931 ] or remeshing [ 3235 ] must be used in conjunction with fem if an accurate representation of the location of the cut , for example , the resection cavity or retraction path , is needed to deform the model . indeed , fem can not directly handle discontinuities that go through the fes , and requires to realign the discontinuity with fe boundaries . in the field of fracture mechanics , which studies the growth and propagation of cracks in mechanical parts
, some methods were developed to avoid using fem in conjunction with mesh adaptation or remeshing .
the extended finite element method ( xfem or x - fem ) appeared in 1999 and has been the object of considerable research since then .
xfem works by allowing the displacement field to be discontinuous within some fes of the mesh .
the mesh does not have to conform to the discontinuities , so that these can be arbitrarily located with respect to the underlying fe mesh . because xfem allows an accurate representation of the discontinuities
while avoiding mesh adaption or remeshing , and because of the similarity between cracks in mechanical parts and cuts in tissue , we proposed the use of xfem for handling cut , resection , and retraction in the updating of preoperative images .
this paper presents a complete 3d framework for updating multimodal preoperative images with respect to surgical brain deformations , due to brain shift and successive resections , followed and quantified using imr images .
our approach is modular , and is applied iteratively each time a new intraoperative image is acquired .
we take into account successive deformations based on a linear elastic biomechanical model which is deformed using fem or xfem , depending on the type of deformation occurring between the pair of imr images under consideration , namely , brain shift or resection .
while some 3d results have already been presented for brain shift , and initial 3d results for resection modelings , this paper is the first complete and detailed account of the generalization to 3d of our 2d previous work .
we present the state - of - the - art of resection modeling for preoperative image update . in section 3
, we describe our basic strategy for updating preoperative images based on successive intraoperative images . in section 4 , we give detail about our methods and algorithms . in section 5 ,
we consider two patient cases that illustrate our approach for handling brain shift followed by successive resections .
among studies that take into account resection for preoperative image update , one should distinguish two categories .
the first category of studies models brain deformation using two time - point images , the first image being acquired before surgery has started , the second image being acquired after resection . in this category ,
the methods that existed for a second image showing some brain shift are adapted for a second image showing some resection .
the second category of studies models brain deformation using more than two time - point images , and models at least two successive resections . among the first category of studies ,
hagemann et al . developed a 2d method for modeling brain deformation between a preoperative mr image and a postoperative mr image , the postoperative image showing a complete resection .
the 2d mesh of the biomechanical model corresponded to the underlying pixel grid of the 2d image .
the biomechanical model included four different linear elastic laws for the skull / skin region , the whole - brain region , the csf region , and the image background .
they computed the correspondence of the skull boundary , the whole - brain region boundary in the neighborhood of the tumor , and the posterior midline between the two images .
they also computed the correspondence between the internal tumor region boundary visible in the preoperative image , and the resection cavity boundary visible in the postoperative image , both boundaries corresponding under the assumption that the resection is complete .
the displacements fields of these landmarks drove the deformation of the biomechanical model . as a result , the biomechanical model presented high deformation in the tumor region , which is not physically plausible .
however , the resection was complete , and , thus , they were not interested by the displacement field of the biomechanical model in the tumor region itself .
clatz et al . developed a 3d method for modeling the brain deformation between a preoperative mr image and an imr image , the latter showing partial or complete resection .
the biomechanical model was deformed based on a sparse volume displacement field evaluated from the two images , using a block matching algorithm . in their algorithm , blocks of voxels that presented discriminant structures were selected in the preoperative image .
the blocks were then matched to blocks in the imr image using a similarity criterion , for example , a coefficient of correlation .
the value of the similarity criterion was used as a value of confidence in the displacement measured by the block matching algorithm .
the biomechanical model was then deformed iteratively , driven by the sparse displacement field of the matched blocks , where a block rejection step was included for measured block displacements initially selected but considered as outliers . in the imr image , a part , or the totality , of the tumor tissues
the blocks were thus selected and matched in only the healthy - brain region of the two images .
they tested their algorithm on six patient cases , and used for validation nine landmarks picked up manually in each image .
they found a mean and maximum error on displacements of 0.75 mm and 2.5 mm , respectively .
they explained this phenomenon by the fact that a substantial number of block matchings were rejected in the tumor neighborhood .
the deformation of the biomechanical model in the tumor neighborhood was thus essentially governed by the linear elastic law , and the result might show the limitation of this model .
archip et al . also tested the nonrigid registration method presented in on eleven patient cases , and used the 95% hausdorff distance for evaluating the alignment of the nonrigidly registered images . as a result
, they obtained a mean error of 1.82 mm . among the second category of studies , miga et al .
they built a linear poroelastic biomechanical model and preoperatively tagged the tetrahedron fes that were going to be removed to simulate the brain deformation due to successive resections .
second , a boundary condition was applied to the new boundary of the resection cavity , in order to model the relaxation of strain energy , induced by preoperative tumor growth or surgery acts , stored in the resected tissues , and released after their removal . in this approach ,
the tissue discontinuity was represented as best as possible with a jagged topology defined by the fe facets defining the boundary of the resection cavity .
[ 45 , 46 ] also modeled the removal of tetrahedra in order to model the action of an ultrasonic aspirator in the context of real - time surgery simulation .
[ 13 , 47 , 48 ] modeled successive resections based on several time - point imr images . between two successive images
, they deformed the biomechanical model , in its current state of update , to take into account the ( partial ) resections(s ) that took place between these two images .
first , the biomechanical model , in its current state of update , was deformed in accordance with the displacement field of the healthy - brain boundary between the pair of images under consideration .
second , the fes that fell into the resection cavity in the second image of the pair were removed , while the fes that laid across the resection - cavity boundary were cut . to ensure the link between the successive deformed configuration of the biomechanical model , their algorithm kept track of the topology modification between fes and nodes of the mesh before and after the removal of fes .
they tested their algorithm on one patient case including five imr images ( the first two imr images being used for brain shift modeling ) , and used for validation thirty - two landmarks picked up manually in each image .
they found a mean and maximum error on the displacements of 0.9 0.7 mm ( mean standard deviation ) and 3.7 mm , respectively .
they explained this phenomenon by the limited accuracy in the process of picking landmarks in that region , and because the retraction occurring between the second and third images was modeled as a resection , that is , a removal of tissues , even though the tissues were not removed but simply spread out .
the methods described above have been all developed using an fem - based biomechanical model for intraoperative image registration .
the objective of a surgical simulator is to provide an interactive manipulation with force feedback of the anatomical part to be operated using various surgical instruments . in order to model a large range surgical procedures ,
jebkov and kuhlen have applied nonlinear xfem for simulating cut , and have shown that xfem is successfully efficient for such purpose .
the block diagram of figure 1 shows our global approach for updating preoperative images using successive imr images acquired at different critical points during surgery .
although the principles of the approach are quite general , they are tailored for use based on images acquired with a 0.5 tesla intraoperative ge signa scanner , which guarantees that the full volume of brain tissues is included in the image field of view . in our present strategy ,
the preoperative images are updated incrementally . at the end of each update , the preoperative images should be in the best possible alignment with the last imr image acquired .
prior to surgery , a patient - specific biomechanical model is built from the set of preoperative images . because the patient does not necessarily lie in the same position during the acquisition of each of the preoperative images
, one may need to perform a rigid registration ( involving translations , rotations , and scales ) to bring these images into correspondence , assuming , in first approximation , there is no local , that is , nonuniform , brain deformation between preoperative images .
once the 1st imr image has been acquired prior to the opening of the skull , the set of registered preoperative images and the biomechanical model are registered to the 1st imr image via a rigid transformation . in the present situation , it is assumed that the patient 's brain imaged in the 1st imr image has the same physical shape as the brain imaged in the preoperative images ( note that in the following , when an imr image is defined by a number , this number is the index of the imr image in the series for a specific patient case .
the 1st imr image thus corresponds to the very first imr image of the series ) . as each imr image
is acquired , this new image and the preceding imr image are used to estimate the deformation of the brain .
the update of the preoperative images is done incrementally with each new pair of successive imr images . for each pair , we proceed as follows . a set of common anatomical landmarks
the use of surface structures rather than volume structures seems more appropriate given the reduced - quality of typical intraoperative images , and would be more easily adapted to intraoperative modalities other than imr , such as ius .
the landmark surface displacement fields resulting from the matching are then applied to the biomechanical model , which is deformed using fem or xfem , depending on the type of deformation occurring between the acquisition times of the imr images in the pair under consideration , namely , brain shift , or resection .
the resulting displacement field of the biomechanical model is finally used to warp the set of preoperative images in their current state of updating .
note that , for each deformation modeling , the biomechanical model is deformed in accordance with the landmark displacements tracked between the pair of successive imr images under consideration . because intraoperative deformation can follow a reverse direction , it is important to track the landmarks between the next - to - last and the last acquired imr images , rather than track the landmarks between the first and the last acquired imr images . for the patient cases treated in section 5 , we assume that the brain undergoes relatively small deformations ( small strains and small displacements ) , and we use a linear finite - element formulation in the biomechanical model .
a consequence of using this linear formulation ( linear elasticity ) is that the equations of solid mechanics can be solved based on the initial configuration of the solid .
actually , knowing the displacement field increment un = u u at the anatomical landmarks between configuration n and increment n + 1 , one can apply this constrained displacement field increment un to the initial configuration , and the finite element analysis will lead to the deformation tensor increment n between the configuration n and n + 1 .
the final deformation tensor or the body is thus simply obtained from = k=0k .
remark that rigorously , the increment of constrained displacement field at the landmark should be applied to the balanced solution of the solid reached after increment n , but as we are using a linear elasticity model , this step can be skipped owing to the superposition principle : if = c , then = k=0k = ck=0k = c , where c is the hooke tensor . as a summary , with this approach ,
the process of deformations is modeled as a succession of deformations k , for example , brain deformation composed of shift followed by successive resections and the current configuration of the brain biomechanical model , after a specific deformation can then be recovered by adding the computed volume displacements for all successive incremental deformations . remark
that this is not a limitation of the method as we could easily extend it to nonlinear model by simply keeping in memory the previous deformed configuration n and adding the constrained displacement field increment un to compute the new deformed configuration at increment n + 1 , simply this would be less computationally efficient . because we use a linear formulation ( and , thus , the incremental volume displacement fields can be added to recover the current configuration of the biomechanical model ) , we could theoretically obtain an identical deformed configuration of the biomechanical model using the two following approaches .
the first one would consist of computing and adding the successive incremental deformations of the biomechanical model based on the landmarks tracked between the next - to - last and the last acquired imr images .
the second approach would consist in computing directly the deformed configuration of the biomechanical model based on the landmarks tracked between the first and the last acquired imr images .
however , the landmarks selected to drive the deformation of the biomechanical model vary depending on the type of deformation , namely , brain shift or resection .
in addition , part of the biomechanical model is cut , using xfem , to model resection .
consequently , we would not get an identical deformed configuration of the biomechanical model by these two approaches .
in order to use a maximum of information from the imr images , we track , as explained for the first approach , the landmarks between the next - to - last and the last acquired imr images .
the problem of updating preoperative images between more than two critical points during surgery , that is , based on more than two imr images , is addressed in only a small number of studies . in our previous work [ 39 , 41 ] , and in
, the biomechanical model was successively deformed , and this was done using a linear formulation .
the framework proposed here , where the initial biomechanical model is always used , instead of using it in its successive states of deformation , has the important advantage of using a good quality mesh for each deformation modeling rather than using a mesh whose quality progressively deteriorates with each successive deformation modeling , and which would require remeshing or mesh adaptation for getting back good fe quality . to summarize ,
for each deformation , the landmarks are tracked between the two successive imr images under consideration . because we use a linear formulation ,
the displacement fields of these landmarks are applied to the initial , rather than current , configuration of the biomechanical model .
the resulting volume displacement field corresponds to the deformation that the brain undergoes between the two imr images .
this volume displacement field is used to deform the preoperative images in their current state of update , that is , registered ( at the previous step , if any ) to the first imr image of the pair .
after the deformation , the preoperative images are thus in as good as possible registration to the second imr image of the pair . in all the rest of this work ,
we make a simplification of the approach just presented , by using the 1st imr image as a substitute for the preoperative images .
the biomechanical model is thus built based on structures visible in the 1st imr image , instead of in the preoperative images , and the structures used in the model are limited to the ones visible in the intraoperative image . except for the rigid registration between the preoperative images , the biomechanical model , and the 1st imr image , this simplified approach allows us to discuss , illustrate , and test all key aspects of the system .
the above strategy allows us to focus on the main issue of this paper , that is , the estimation and handling of 3d deformations .
even though the issues involved in the update of preoperative images will need to be addressed in a operational image update system , the present strategy of deforming the imr images remains useful for calibration purpose , even in the operating room .
this section details the different methods that are commonly used for updating preoperative images in presence of brain shift and resection .
more specifically , the block diagram of figure 2(a ) shows the building of the biomechanical model from the preoperative images .
specific regions from the preoperative images are segmented , meshed , and assigned appropriate constitutive laws .
the block diagram of figure 2(b ) shows , for any pair of successive imr images , a detailed view of the calculation of the volume displacement field of the initial biomechanical model that corresponds to the deformation that has occurred between the acquisition time of these images . all along surgery , the patient is lying inside the 0.5 tesla intraoperative ge signa scanner .
although the patient 's head is fixed , one can not totally rule out the possibility of slight head motion .
imr images thus have to be rigidly coregistered to take into account this potential rigid motion .
the rigid registration that we use is the point - based landmark transform available in vtk ( http://www.vtk.org/ ) .
the segmentation of imr images into specific regions , for example , healthy - brain and tumor regions , is first performed manually using 3d slicer ( http://www.slicer.org/ ) and then smoothed to minimize the dependance of the results on segmentation roughness .
it is clear that performing a manual segmentation in the operating room is not acceptable , and that this process needs to be automated as completely as possible to test the feasibility of our framework online .
however , while there exist sophisticated segmentation algorithms that could be used [ 5052 ] , in particular for extracting the whole - brain region ( skull and external cerebrospinal fluid masked out ) , the segmentation of the tumor region is still challenging . as mentioned above , the biomechanical model is built , in the present context , from the 1st imr image rather than from the preoperative images . thanks to the use of xfem instead of fem for modeling discontinuities , this biomechanical model can be built offline before the operation starts and does not need to be repeated ( through remeshing ) during the surgery . with respect to fem - based approaches ,
the execution time thus ceases to be a limiting parameter , which is a remarkable advantage of our approach .
it thus requires specific techniques , and we use the meshing software tool isosurf ( http://svr-www.eng.cam.ac.uk/~gmt11/software/isosurf/isosurf.html ) . our goal is to model the boundaries of healthy - brain and tumor regions as two connected surfaces meshes .
however , isosurf can only mesh the boundaries of one or several separate regions , and , thus , does not allow one to mesh connected region boundaries with common nodes at their intersections .
we thus start by building two separate surfaces meshes that we connect using our own routines based on vtk .
the two connected triangle surfaces are then jointly meshed into a single volume mesh of tetrahedra that conform to the two surface meshes using gmsh ( http://www.geuz.org/gmsh/ ) .
further details on the building of the biomechanical model , in particular the building of the connected surface meshes , can be found in .
a linear elastic law is assigned to the biomechanical model , with young modulus e = 3000 pa and poisson ratio = 0.45 . because displacements , rather than forces , are applied to the model using a linear formulation , the fem or xfem solution is independent of young modulus e .
we choose as surface landmarks the whole - brain and internal tumor region boundaries . to evaluate the surface deformations of these region boundaries between two imr images , we use an active surface algorithm [ 55 , 56 ] .
because these region boundaries to match must be closed surfaces , we thus use as surface landmarks the whole - brain and healthy - brain region boundaries .
the surface deformation of the internal tumor region boundary will be derived from the active surface algorithm of the healthy - brain region boundary . in our active surface algorithm coming from [ 13 , 47 , 48 ] , the external forces f(x ) are computed using a gradient descent on a distance map of the region boundary . with such external forces , the active surface algorithm is not able to take correctly into account local rigid motion due , as an example , to lateral or tangential movement depending on the head orientation . for the whole - brain region , any rigid transformation that could have occurred
has already been taken into account by the rigid registration of the imr images ( section 4.1 ) . however , for the healthy - brain region , it can happen that the internal tumor region boundary moves partly in a rigid way .
therefore , the active surface , initialized from the healthy - brain region boundary in the first imr image , is first locally transformed in a rigid way along the internal tumor region boundary using the iterative closest point transform available in vtk .
then , this resulting surface is deformed using the active surface algorithm as explained above .
further details on the local rigid registration of the healthy - brain region boundary can be found in . before applying the displacements whole - brain and internal tumor region boundaries to the biomechanical model nodes ,
the two surface displacement fields are smoothed based on a weighted - distance average , that is , the displacement of each node is averaged with the displacements of its n closest neighbor nodes .
this smoothing will make them consistent with each other , and compatible with the volume mesh in order to avoid element flipping , in particular at the intersections between whole - brain and internal tumor region boundaries .
the displacement fields of the surface landmarks are applied to the biomechanical model , which deforms according to the laws of solid mechanics .
the equations of solid mechanics are solved using fem or xfem , depending upon the type of circumstances , namely , brain shift or resection .
we use the fem - software tool metafor ( http://metafor.ltas.ulg.ac.be/ ) developed in our mechanical - engineering department , to which we have added an xfem module .
the initial stress state of the brain is unknown and is thus set to zero for each fem or xfem computation , as in [ 10 , 13 ] .
fem discretizes the solid of interest into a mesh , that is , into a set of fes interconnected by nodes , and approximates the displacement field u(x ) by the fem displacement field u(x ) defined as
( 1)ufem(x)=iii(x)ui ,
where i is the set of nodes , the i(x ) 's are the nodal shape functions ( nsfs ) , and the ui 's are the nodal degrees of freedom ( dofs ) .
each i(x ) is defined as being continuous on its compact support i , which corresponds to the union of the domains of the fes connected to node i . in our approach , we use linear nsfs .
in contrast , xfem handles a discontinuity by allowing the displacement field to be discontinuous within fes [ 37 , 5860 ] .
the xfem displacement field generalises the fem displacement field ( 1 ) with
( 2)uxfem(x)=iii(x)ui+iji(x)j=1neigj(x)aji .
the first term corresponds to the fem displacement field ( 1 ) , where i is the set of nodes , the i(x ) 's are the fem nsfs , and the ui 's are the nodal fem dofs . the heart of xfem is the enrichment that adds a number , n , of dofs aji to each node i of the set j , which is the subset of nodes of i whose support is intersected by the discontinuity of interest .
these dofs are multiplied by the nsfs i(x ) and the discontinuous functions gj(x ) .
the use of specific xfem enrichment functions gj(x ) for a node i j depends on the type of discontinuity , for example , crack , hole , material interface , and so forth , to be modeled .
suppose that our goal is to model a crack , characterized by a discontinuity in the displacement field ( as opposed to a material interface for instance , characterized by a discontinuity in the derivative of the displacement field ) .
when the crack fully intersects the support of the node , a simple choice is a piecewise - constant unit function that changes sign at the boundary across the crack , that is , the heaviside function
( 3)h(x)={1for ( xx)en>0,1for ( xx)en<0 ,
where x is again the position of a point of the solid , x * is the position of the point on the crack that is the closest to x , and en is the outward normal to the crack at x * . in case of resection deformation , the goal is to model a discontinuity such that the part of tissues corresponding to tissue removed by the resection has no influence on the deformation of the remaining part of the tissues .
one is actually interested in the deformation of the remaining part of the tissues only . in that sense ,
the hole function as the following equation :
( 4)v(x)={1for ( xx)en>0,0for ( xx)en<0 ,
could be used as xfem enrichment function , instead of the heaviside function , and would be totally sufficient .
the results that we would obtain on the remaining part of the tissues would be identical .
however , because the heaviside function is necessary for retraction modeling , we have used the same function for the resection modeling even if it was not strictly necessary . when minimizing the total deformation energy , the resulting xfem equations remain sparse and symmetric as for fem .
whereas fem requires a remeshing and the duplication of the nodes along the crack to take into account any discontinuity , xfem requires the identification of the nodes whose support is intersected by the crack and the addition of dofs : ( 1 ) any node whose support is not intersected by the discontinuity remains unaffected and thus possesses three dofs ; ( 2 ) any node whose support is fully intersected by the discontinuity is enriched with three heaviside dofs and thus possesses six dofs . to qualitatively estimate the similarity between two images , we compare the edges extracted from these images using the canny edge detector available in itk ( http://www.itk.org/ ) .
indeed , although potentially useful for the sake of comparing methods on a mathematical basis and defining unique correspondences , landmark - based target analysis presents several relevant limitations in the present setting . having experts picking landmarks introduces significant intra- and interobserver variability . picking landmark points , as ferrant et al .
did , is rather difficult when it comes to define enough visible landmarks especially in the tumor region on the 5 different images ( and not 2 images only , as majority of studies focusing on brain shift are using ) . rather than point targets , linear tumor contours , and limits between structures and potential eloquent structures matter most in the practical case of tumor ablation neurosurgery .
these are the reason why we chose to use the canny edges in order to evaluate the registration . besides
, while it is true that these edges do not necessarily physically correspond between the successive imr images , these images have been acquired with the same image protocol ( mr sequence , voxel size , grayscale value range ) , which should limit this problem .
to quantitatively estimate the similarity of the two edge maps , we compute the modified hausdorff distance between the sets of edge points , that is , voxels representing the edges , in these two images .
the modified hausdorff distance (a , b ) between two sets of points a and b is defined as
( 5)(a , b)=max(h(a , b),h(b , a ) ) with h(a , b)=1naaad(a , b ) ,
where the directed hausdorff distance h(a , b ) is a measure of the distance of the point set a to the point set b , na is the number of points in set a , and d(a , b ) is the distance of point a a to the closest point in b , that is , d(a , b ) = minbb||a b|| , where ||a b|| is the euclidean distance .
the directed hausdorff distance h(a , b ) thus computes the average distance of points of a to points of b. the averaging minimizes the effects of outlier points , for example , due to image noise .
the value of the modified hausdorff distance (a , b ) increases with the amount of difference between the two sets of edges points . in the following ,
we denote by (ia , ib ) the modified hausdorff distance of the edges extracted from the whole - brain region of the images ia and ib , that is , with the skull and external cerebrospinal fluid masked out from them .
in this section , we apply our methods , respectively , of brain shift and resection ( imr images are acquired with the 0.5 tesla intraoperative ge signa scanner of the brigham and women 's hospital , boston , usa .
imr image size is 256 256 60 voxels , and voxel size is 0.9375 0.9375 2.5 mm ) .
two patient cases , each including five imr images , are treated to illustrate our modeling and brain shift followed by successive resections . in both cases , the 1st imr image was acquired prior to the opening of the skull ; the 2nd imr image was acquired after the opening of the skull and dura , and shows some brain shift ; the 3rd , 4th , and 5th imr images were acquired after successive resections .
the modelings of brain shift , 1st , 2nd , and 3rd resection are performed using different techniques , as detailed below . except where otherwise noted
, the following discussion applies to both patient cases ( the result of each deformation modeling is shown for the two patient cases at the end of section 5.2.3 ) . to model brain shift based on the 1st and 2nd imr images
, we estimate the surface displacement fields of the whole - brain region boundary and the internal tumor region boundary from the two imr images .
no tissue discontinuity is involved in the brain shift deformation , so the biomechanical model is deformed using fem .
this results in the volume displacement field of the biomechanical model , which is illustrated in figure 3 for the first patient case .
this volume displacement field is used to warp the part of the 1st imr image corresponding to the whole - brain region . in the following sections ,
the three successive resections are modeled separately , because they require different types of processing .
matching two region boundaries to get a displacement field makes sense only if they correspond to the same physical entity .
once the resection has started , we can no longer rely on the entirety of the whole - brain region boundary , since a part of it is now missing . for modeling the successive resections , we thus evaluate the displacement field for the boundary of the healthy - brain region only .
the 1st resection occurs between the times the 2nd and 3rd imr images are acquired .
however , since the corresponding removal of tissues is most likely accompanied by deformation , one can not exactly determine what tissue is removed based just on the two imr images .
we thus decided to model the 1st resection by still relying on the displacement fields of key surfaces , here the healthy - brain region boundary , to deform the biomechanical model .
this indeed appears to be the only reliable information concerning the deformation due to resection that we can extract from the 2nd and 3rd imr images .
consequently , we do not model explicitly the removal of tissue , but we model directly the deformation resulting from it , without introducing any tissue discontinuity . using the surface displacement field of the healthy - brain region boundary , we compute the deformation of the biomechanical model via fem . then , using the resulting volume displacement field , we warp the part of the 2nd imr image corresponding to the whole - brain region , in the same way as we did in the case of for brain shift .
the image resulting from the 1st resection modeling is now registered to the 3rd imr image , except outside of the healthy - brain region boundary , that is , for the tumor region . finally , we alter the resulting image to reflect the effect of resection . for this
, we assign the background color to the voxels corresponding to the resected tissue volume
the significant feature of the 2nd resection is that some tissue has already been removed by the 1st resection , which means that this tissue can not have any physical influence on subsequent brain deformations because it does not
recall that the biomechanical model has been deformed to model the brain shift and the 1st resection and is thus registered to the 3rd imr image .
so , using the 3rd imr image , we can define the boundary of the 1st resection , that is , the tissue discontinuity to include in the deformed biomechanical model ( figures 4(a ) and 4(b ) ) .
we then enrich the nodes whose supports are intersected by the discontinuity with heaviside dofs .
consequently , when the xfem - based biomechanical model deforms , the part corresponding to tissue removed by the 1st resection has no influence on the deformation of the remaining part of the brain .
for the first patient case illustrated in figure 4 , the tetrahedron mesh consists of 3,317 nodes , which corresponds to 9,951 fem dofs .
the biomechanical model is deformed in accordance with the displacement field of the healthy - brain region boundary evaluated from the 3rd and 4th imr images .
the bottom part of the mesh , representing the tissue remaining after the 1st resection , has been deformed according to the displacement field of the healthy - brain region boundary , while the top part , representing the tissue removed by the 1st resection , has been subjected to a translation , but only for visualization purposes . even though the mesh is displayed as two separate parts , it is , in fact , a single entity .
indeed , a main feature of xfem is its ability to handle the effect of a discontinuity without modifying the underlying mesh , that is , without remeshing . for modeling the 2nd resection , the edges of fes straddling the discontinuity
have been made discontinuous and their nodes moved apart . using the xfem volume displacement field , we warp the part of the 3rd imr image corresponding to the whole - brain region .
the resulting image is then masked out with the whole - brain region segmented out from the 4th imr image .
one significant feature of the procedure described for modeling the 2nd resection is that it can be applied repetitively for each subsequent resection visible on successive imr images , no matter how many there are .
the modeling of the 3rd resection is thus identical to the modeling of the 2nd resection .
the tissue discontinuity due to the 2nd resection is defined from the 4th imr image , and used to appropriately enrich the nodes of the biomechanical model .
then , this biomechanical model is deformed using xfem , in accordance with the displacement field of the healthy - brain region boundary evaluated from the 4th and 5th imr images . for the first patient case ,
a simplification for the modeling of the 3rd resection can be made because , by the time the 5th imr image is acquired , the resection is complete .
this means that we only need to compute the volume displacement field of the healthy - brain region .
since we apply displacements exactly to the boundary of the healthy - brain region , the results obtained with fem and xfem will be identical .
using the fem ( for the first patient case ) or xfem ( for the second patient case ) volume displacement field , we warp the part of the 4th imr image corresponding to the whole - brain region . the resulting image is then masked out with the whole - brain region segmented out from the 5th imr image .
figures 5 and 6 show the results of warping the imr images , as well as the edges extracted from them , after brain shift and each successive resection modeling for the two patient cases . as explained in section 5.2.3 , since we apply displacements exactly to the boundary of the healthy - brain region , the results obtained with fem and xfem are identical in the healthy - brain region .
one can deduce that using xfem for modeling resection is interesting when the neurosurgeon needs to have an accurate displacement field of the remaining tumor tissues . in this case
, it is interesting to evaluate the impact of using fem , instead of xfem , to model the resection as if no resection was performed before .
using fem for modeling resection is equivalent to ignoring the presence of resection on intraoperative images . to illustrate the comparison between fem and xfem results , we choose the 3rd resection modeling of the second patient case
indeed , it is the deformation with remaining tumor tissues that shows the largest magnitude , and , thus , that is likely to give a maximum difference between the two computations .
the healthy - brain and tumor regions segmented out from the 4th and 5th imr images are respectively shown in figures 7(a ) and 7(b ) .
the volume displacement fields of the biomechanical model using xfem and fem are respectively shown in figures 7(c ) and 7(d ) .
the part of the 4th imr image corresponding to the whole - brain region is warped , first with the volume displacement field obtained via fem , and then with that obtained via xfem .
the difference between the two warped images is shown in figure 7(e ) . as expected
however , the difference between the two volume displacement fields is smaller than the image resolution ( although the difference between the two volume displacement fields is smaller than the image resolution , the difference between the images resulting of the warping using these two volume displacement fields is nonzero .
this is explained by the fact that the ( gray ) value of each voxel of the warped image is defined as a weighted - value of voxels of the original image .
the weights are defined based on the overlapping ratio of the voxel of the warped image , with voxels ( determined using the volume displacement field ) of the original image ) .
in addition , the deformed 4th imr images , using the xfem- and the fem - based deformations of the biomechanical model , show the same similarity , computed based on the modified hausdorff distance , with the 5th imr .
two reasons explain that the differences between the fem and xfem results are so small .
first , the brain deformation itself due to the 3rd resection is small , and , thus , it is expected to obtain small differences between the two resulting brain deformations .
second , in the case the remaining tumor tissues are close to the healthy - brain region boundary , it implies that they are close to the boundary where surface displacement fields are applied to drive the deformation of the biomechanical model .
although this comparison between fem and xfem should be done on more patient cases , we suggest that , in first approximation , fem could be used for modeling resection cases with small brain deformations .
nevertheless , the presentation of the successive resections using xfem shows the generality of our framework , and details how xfem is implemented .
note that in section 6 devoted to validation , the warped images are the ones deformed with xfem .
for each deformation modeling based on a pair ( ik , ik+1 ) of two successive imr images that are already rigidly registered , we compare the similarity between these ik and ik+1 images , as well as the similarity between the ik and ik+1 images , where ik is the result of warping ik .
this gives us an estimate of how well we are able to capture , and compensate for , the local deformations between ik and ik+1 .
the goal of the nonrigid registration is , however , to deform the preoperative images . by warping ik for each deformation modeling
, we do not take into account the fact that an error of alignment after each deformation modeling could propagate and amplify through the successive deformation modelings . to evaluate the effect of this error amplification on the results
, we also perform the required succession of warpings on i1 , and we denote the resulting image by i1,k .
we then compare , for each deformation modeling , the similarity between i1 and ik+1 , together with the similarity between i1,k and ik+1 .
this allows one to evaluate the propagation , that is , the amplification , of alignment error on the results .
the modified hausdorff distance computed for each pair of imr images are given in tables 1 and 2 .
table 1 shows , for each deformation modeling based on a pair ( ik , ik+1 ) of two successive imr images , the values of the modified hausdorff distances (ik , ik+1 ) and (ik , ik+1 ) .
these values are computed using the canny edges extracted from the pair of images ( ik , ik+1 ) ( figures 5 ( d ) and 6 ( d ) ) and ( ik , ik+1 ) ( figures 5 ( e ) and 6 ( e ) ) .
we observe that the values for the images nonrigidly registered are relatively constant , that is , 1 mm , for each deformation modeling .
six out of eight deformation modelings give smaller modified hausdorff distances when the imr images are ( rigidly and subsequently ) nonrigidly registered .
however , the modified hausdorff distance increases for the 3rd resection modeling of the first patient case , as well as for the brain shift modeling of the second patient case . to understand
if the nonrigid registration is responsible for the increase of the misalignment of the two imr images everywhere in the whole - brain region , or if this effect is localized , we compute the modified hausdorff distance in the region and neighborhood of the tumor only ( volume region that extents by 25 mm the tumor region segmented in i1 for both patient cases ) .
the modified hausdorff distance decreases from (i4 , i5 ) = 1.70 mm to (i4 , i5 ) = 1.37 mm for the first patient case , while it decreases from (i1 , i2 ) = 1.36 mm to (i1 , i2 ) = 1.28 mm for the second patient case .
this indicates that the nonrigid registration enhances the alignment of the two imr images within the tumor region and its neighborhood , which is in fact the location requiring the best modeling accuracy .
this behavior could be explained by the fact that a maximum of information from the imr images is used in this region , that is , one or two ( in case of brain shift modeling ) surface displacement fields are applied around it .
the increase of misalignment elsewhere in the brain volume could be explained by two reasons .
first , the landmarks tracked from the imr images are surfaces . as a consequence , the nonrigid registration is expected to give better results near the tracked surfaces than far from them in the volume .
the volume misalignment could point out the need for better parameters values and/or other constitutive laws .
table 2 shows , for each deformation modeling based on a pair ( ik , ik+1 ) of two successive imr images , the values of the modified hausdorff distances (i1 , ik+1 ) and (i1,k , ik+1 ) .
so far , igns systems allow one to rigidly register preoperative and successive imr images .
(i1 , ik+1 ) thus represents the navigation accuracy that we can obtain with an igns system at the present time .
the comparison of (i1 , ik+1 ) with (i1,k , ik+1 ) gives the improvement that could be practically achieved in the alignment with our approach .
as expected , table 2 shows that the igns accuracy decreases through the successive deformations .
indeed , the modified hausdorff distance increases from (i1 , i2 ) = 1.24 mm to (i1 , i5 ) = 1.78 mm for the first patient case , and from (i1 , i2 ) = 1.01 mm to (i1 , i5 ) = 1.68 mm for the second patient case .
six out of eight deformation modelings give smaller modified hausdorff distances when the imr images are nonrigidly registered .
to understand if the modified hausdorff distance increases everywhere in the whole - brain region for the brain shift and 1st resection modeling of the second patient case , we compute the modified hausdorff distance in the neighborhood of the tumor region ( in the same way as explained for table 1 ) , and observe the improvement of the alignment within the tumor region and its neighborhood . as opposed to the values of the modified hausdorff distances in table 1 , the values for the images nonrigidly registered in table 2 increase through the successive resection modeling .
this amplification error is due to the fact that , after having modeled brain deformation between a pair of imr images , the deformed biomechanical model is not in perfect alignment with the second image of the pair .
since , for the subsequent deformation modeling , the surface landmarks are initialized based on the deformed biomechanical model , this can thus ampliy a misregistration error .
we developed a complete 3d framework for serial preoperative image update in the presence of brain shift followed by successive resections .
the nonrigid registration technique used an homogeneous linear elastic biomechanical model , driven by the deformations of whole - brain and internal tumor region boundaries for brain shift modeling , and healthy - brain region boundary for resection modelings , tracked between successive imr images .
the biomechanical model was deformed using fem for brain shift modeling , and fem or xfem for resection modeling , depending upon whether some brain tissues were previously resected or not .
we showed that our approach was modular , and could be applied each time a new imr image is acquired .
we used a linear formulation to characterize the deformation of the brains of both patients because the brains underwent relatively small deformations and displacements .
while nonlinear biomechanical models have proven effective to decrease yet do not abolish the inaccuracies of fem - based modeling methods of large brain deformations , the deformations observed in our patients during surgery remained moderate ( 47 mm ) , thus reducing the theoretical benefit of using nonlinear models .
this allowed us to use simpler linear models and focus on the added value of xfem to simultaneously account for surgical deformations , namely , shift and resection . using a linear formulation
implied that , for each new deformation modeling , one could use the initial configuration rather than the last - deformed configuration of the biomechanical model .
this had the important advantage of using a good quality mesh for each deformation modeling rather than using a mesh whose quality progressively degraded with each successive deformation modeling .
this also had the advantage that we did no longer need to reconnect the deformed mesh for each new xfem calculation , which was one drawback of our previous method , presented in [ 39 , 41 ] , where the biomechanical model was successively deformed .
we also showed how xfem could handle a discontinuity for modeling resection without any remeshing or mesh adaptation while the representation of the discontinuity remained accurate , that is , the representation of the discontinuity was not based on a jagged topology using fe facets .
xfem thus also avoided making the mesh resolution richer in the neighborhood of the resection - cavity boundary for improving the accuracy of the representation of the discontinuity for that purpose only .
we showed that our nonrigid registration technique improved the alignment of the successive imr images for most of the deformation modeling of both patient cases . when our nonrigid registration failed , it still improved the alignment locally , that is , within the tumor region and its neighborhood .
we tested the explicit modeling of the lateral ventricles ' region with a soft , compressible law in addition to the whole - brain region law used in the homogeneous biomechanical model .
in addition to the validation that is usually performed for successive deformation modelings , that is , validation between pairs of successive intraoperative images , shown in table 1 of section 6 or in the work of ferrant et al .
[ 13 , 47 , 48 ] , we also evaluated the fact that an error of alignment after each deformation modeling could propagate and amplify through the successive deformation modelings . as a result , shown in table 2 of section 6
, we showed that our approach suffered from the propagation of misregistration through the successive deformation modelings .
we expected that this was due , at least partly , to the algorithms used to evaluate intraoperative surface displacements fields from the whole - brain and healthy - brain region boundaries .
the surface displacement fields were computed using active surface algorithms , and smoothed to make them compatible with the biomechanical model .
because of these two smoothings , the deformed biomechanical model was likely to not be in a perfect alignment with the imr image to which it was registered .
because the surface displacement fields evaluated for the next deformation modeling were initialized based on the deformed biomechanical model , we expected to observe an amplification of the misregistration , which was confirmed by our quantitative evaluation . at the present time
though , commercial igns systems allow one to register preoperative images and successive imr images , but in a rigid way only .
consequently , although the effect of error amplification exists , our technique still enhances the current capabilities of commercial igns systems .
future work on modeling of brain shift followed by successive resection is required in five main areas .
first , the effect of error amplification through the successive brain deformation modelings calls for further research .
consequently , the segmentation , and the subsequent smoothing , as well as the evaluation of surface displacement fields , should be improved to minimize the effect of error amplification .
second , further research is required to include additional structures in the biomechanical model in general , and to study the best way to include the lateral ventricles in particular .
the use of a poroelastic model in order to model the cerebrospinal fluid filling the ventricles could be considered [ 17 , 18 ] .
indeed , these images provide volume information ( rather than surface information only ) , are of good quality in comparison to other intraoperative modalities , and possess a field of view that includes the full volume of brain tissues ( for the 0.5 tesla ge signa scanner ) .
these images thus allow one to evaluate what , and how , new structures of the brain could be used , to enhance the modeling of brain shift .
some regions , for example , the lateral ventricles ' region , could be extracted from the two imr images , and used as surface landmarks to drive the deformation of the biomechanical model [ 13 , 62 ] . indeed , the workflow presented in this paper has the advantage of being easily adaptable . in case
the tumor region would not be visible ( enough ) on the imr images , these new structures , easier to segment , could also adequately replace the tumor for driving the deformation .
fourth , our global approach should no longer be based on the 1st imr image used as a substitute for preoperative images , but on the preoperative images themselves .
fifth , we should implement , for the surgery cases involving large deformations of the brain , a nonlinear formulation of fem [ 63 , 64 ] , and , particularly , a nonlinear formulation of xfem , which is the subject of recent research [ 65 , 66 ] .
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current neuronavigation systems can not adapt to changing intraoperative conditions over time . to overcome this limitation
, we present an experimental end - to - end system capable of updating 3d preoperative images in the presence of brain shift and successive resections . the heart of our system is a nonrigid registration technique using a biomechanical model , driven by the deformations of key surfaces tracked in successive intraoperative images .
the biomechanical model is deformed using fem or xfem , depending on the type of deformation under consideration , namely , brain shift or resection .
we describe the operation of our system on two patient cases , each comprising five intraoperative mr images , and we demonstrate that our approach significantly improves the alignment of nonrigidly registered images .
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1. Introduction
2. State-of-the-Art
3. Basic Strategy for Serial Preoperative Image Update
4. Methods
5. Results
6. Validation
7. Conclusions and Future Work
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the contribution to human health of the specific fatty acid ( fa ) composition of the diet has received considerable attention in the literature .
fatty acids are key nutrients that affect early growth and development as well as the prevention of chronic disease in later life.1 among the fas , omega-3 polyunsaturated fatty acids ( n-3 pufa ) and omega-6 polyunsaturated fatty acids ( n-6 pufa ) have been suggested to decrease and increase several human diseases , respectively .
pufa that contains more than one carbon double bond consists of two major classes such as n-6 and n-3 ( fig .
linoleic acid ( la ) is a representative n-6 pufa , serving as a substrate to be converted into an arachidonic acid ( aa ) affecting the prevalence and severity of inflammation .
n-6 pufa can induce cardiovascular disease , diabetes , cancer , and age - related disease.1
-linolenic acid ( ala ) , eicosapentaenoic acid ( epa 20:5 ) and docosahexaenoic acid ( dha 22:6 ) are important n-3 pufa involved in human .
they are essential fatty acids that can not be synthesized by mammals , by which must be obtained from dietary sources such as cold - water fish , certain seeds ( flax ) and nuts ( walnuts ) .
a lot of studies suggest that n-3 pufa , as diet - dependent factors , may be critical to preventing disease backed up with authentic antioxidative and anti - inflammatory actions . especially , n-3
pufa has been shown to exert beneficial effects on some chronic degenerative diseases such as cardiovascular disease,2,3 rheumatoid arthritis,4 diabetes,5 other autoimmune diseases,6,7 and cancer.8,9 increased fat consumption by western diet has been associated with the development of cancer such as breast , colon , pancreatic , and prostate cancers with the notable exception of n-3 pufa , which show to have multiple beneficial anti - tumor actions that affect the essential alterations that dictate malignant growth in a number of studies.10 a diet rich in n-3 pufa may protect from cancer , at least at certain sites .
studies on the fatty acid status of patients with several cancer types including bladder , pancreatic , lung and esophageal cancer show low concentrations of plasma phospholipid n-3 pufa , ranging from 55 to 88% of amounts in healthy individuals.1113 recent studies have found a positive association between n-6 pufa and cancer risk , whereas in the same model , n-3 pufa were shown to reduce the development of cancer .
epidemiological studies suggest that a high n-3 pufa to n-6 pufa ratio may be the optimal strategy to decrease breast cancer risk.14 solid epidemiological study shows that consumption of n-3 pufa appears to protect against the development of hepatocellular carcinoma , even among patients with hepatitis b virus ( hbv ) and/or hepatitis c virus ( hcv ) infection.15 recently , zhennan et al .
has reviewed prospective studies investigating the possible protective effects of the dietary intake of n-3 pufa on prostate cancer development.16 fasano et al also has reviewed a lot of in vivo and in vitro experimental studies providing strong indications of the anti - tumor action of n-3 pufa against lung cancer.17 the purpose of this review is to discuss the potential role of n-3 pufa in gastrointestinal ( gi ) cancer development .
we believe that increased consumption of n-3 pufa may lower the risk of gi cancer development via various chemopreventive activities .
future studies should include combination treatment of n-3 pufa and nutrients with different and complementary mechanisms of chemopreventive action .
among various cancers , most of the gi cancers including esophageal cancer , stomach cancer , and colon cancer , have a natural history of multi - step transition from precursor lesions to malignant lesions , inflammation , adenoma formation , dysplastic changes.18 therefore , gi cancers usually have premalignant lesions before developing invasive cancers , for instances , barrett s esophagus for esophageal cancer , chronic atrophic gastritis accompanied with intestinal metaplasia for gastric cancer , and adenoma or dysplasia originating from chronic ulcerative colitis for colon cancer .
because the western diet contains disproportionally high amounts of n-6 pufa and low amounts of n-3 pufa , denoted as a high n-6 to n-3 pufa ratio , n-3 pufa may feasibly play a role in several stages of gi cancers management .
esophageal cancer is ranked as the sixth leading cause of cancer death worldwide . according to the increasing incidence of gastroesophageal reflux disease ( gerd ) ,
esophageal cancer is a tumor that has increased in incidence more than 7-fold over the past several decades .
suggests that negative associations between n-3 pufa intake and the risk of esophageal adenocarcinoma.19 higher in - takes of n-3 pufa [ cases vs. population controls ; or=0.46 , 95% ci=0.220.97 , 4th vs. 1st quartiles of intake ] was associated with a lower risk of barrett s esophagus .
in contrast , higher trans - fat intakes were associated with increased risk ( or=1.11 ; 95% ci=1.031.21 per g / day ) .
moreover , it is reported that n-3 pufa supplemented parenteral nutrition can reduce inflammation and improve immune function in patients following esophageal cancer surgery20 and n-3 pufa - containing diet may be beneficial to patients with esophageal cancers who receive chemoradiation therapy ( crt ) by reducing crt toxicity.21 in contrast to other gi cancer , little work has so far been performed on the influence of n-3 pufa in oesophageal adenocarcinogenesis and neoplastic progression.22 there is a need for appropriately powered randomized - controlled studies to assess the long - term benefit of n-3 pufa . gastric cancer ( gc ) is the fourth most common cancer worldwide , and almost two thirds of affected individuals will die of their disease .
some studies about the association of n-3 pufa and gastric disease suggests a protective effect of n-3 pufa on gastric cancer .
recently , correa et al . suggests that docosahexanoic acid ( dha ) inhibits helicobacter pylori ( h. pylori ) growth in vitro and mice gastric mucosa colonization.23
h. pylori are recognized as a major etiological factor in chronic active gastritis , gastric duodenal ulcers and gastric cancer .
it has been proposed that pufa hold an inhibitory effect on bacterial growth via disruption of cell membrane leading to bacteria lysis.24 mohamed also shows that n-3 pufa reduced iodoacetamide - induced gastritis in rats through decrease of malondialdehyde ( mda ) , gastrin , and nitric oxide ( no ) and normalization of mucosal glutathione.25 especially , it is reported that the erythrocyte composition of dha was found to be negatively linked to risk of gastric cancer , of well - differentiated adenocarcinoma.26 application of a diet enriched with n-3 pufa delayed tumor growth in a mouse xenograft model.27
in vitro studies have shown that n-3 pufa inhibited macrophage - enhanced gastric cancer cell migration and attenuated matrix metalloproteinase ( mmp)-10 expression through erk and stat3 phosphorylation28 and inhibited the growth of human gastric carcinoma cell via apoptosis and combination with 5-fluorouracil has synergetic effect in inhibiting the proliferation of gastric cancer cells.29 moreover , n-3 pufa are beneficial for preventing oxidative stress - induced apoptosis by inhibiting apoptotic gene expression and dna fragmentation of gastric epithelial cells.30 on the other hand , dha induced apoptosis of gastric cancer cells by inducing the expression of apoptotic genes in gastric cancer cells.31 although a large body of literature spanning numerous cohorts from many countries and with different demographic characteristics does not provide evidence to suggest a significant association between n-3 pufa and stomach cancer incidence,32 further studies are needed to investigate action of n-3 pufa relevant to antitumor effects in the stomach .
colorectal cancer ( crc ) is the second leading cause of cancer - related death in men after lung cancer and third in women behind lung and breast cancers in the united states . among the gi tract cancer
, crc cancer has raised the most attention over the past decades , as they share a long precancerous stage ( the adenoma in crc ) which provides a window of opportunity to intervene and prevent development of cancer . recently
there is also evidence suggesting improved efficacy and/or tolerability of conventional colon cancer chemotherapy when administered with n-3 pufa.9 epidemiological studies about the association of dietary fat and cancer suggests a protective effect of n-3 pufa and a promoting effect of n-6 pufa on cancer .
although epidemiological studies of the association between fish intake , n-3 pufa intake or blood n-3 pufa levels and crc risk have not consistently suggested beneficial effects of n-3 pufa on crc and other gi cancer risk .
dietary administration of one or both of the main n-3 pufa in rodent models of colorectal carcinogenesis has been demonstrated to reduce colorectal tumor size and multiplicity , compatible with crc chemopreventive activity.33 in meta - analyses of prospective cohort studies that evaluated the association between fish consumption or n-3 fatty acids and colorectal cancer incidence or mortality , the pooled relative risks for colorectal cancer incidence were 0.960.97 ( 95% confidence interval : 0.92 , 1.00 ) for each extra occurrence of fish consumption per week.34 in a population - based prospective study on the association of n-3 pufa and cancer , there was an inverse relationship between marine n-3 pufa intake and the risk of colorectal cancer , but this association was only statistically significant in the proximal site of the large bowel.35 cockbain et al.9 has reviewed a lot of in vitro and in vivo experimental studies and epidemiological observations providing strong indications of the cancer treatment and prevention of n-3 pufa against colorectal cancer .
kim et al.36 provided a significant dose - dependent reduction in crc risk for total n-3 pufa intake ( or=0.61 for the highest vs lowest quartile ) , as well as for epa and dha intake individually in a case - control study of 1,872 patients ( 929 cases of distal crc and 943 controls ) . pot measured and compared serum n-3 pufa levels in 861 patients ( 363 cases of colorectal adenoma and 498 controls ) .
there was a significant reduction in colorectal adenoma risk ( or=0.67 ) between low level of n-3 pufa ( < 1.8% ) and high level of n-3 pufa ( > 2.3%).37 recently , sorensen et al.38 reported that n-3 pufa is incorporated rapidly into colonic mucosa and colonic muscular layer in patients given 3 g of n-3 pufa daily for 7 days before surgery for colorectal cancer .
the preventive effect of n-3 pufa has been demonstrated in a number of carcinogen - induced models like aom and dimethylhydrazine ( dmh)-induced rat model and apc mouse models relevant to prevention of crc including ours .
studies of rodents fed an n-3 pufa - supplemented diet versus a control diet have consistently reported a 20 - 50% reduction in chemically induced tumor incidence , together with a 3070% reduction in tumor multiplicity , in both carcinogen and apc mouse studies .
similar findings have been reported for studies of growth of human crc cell lines such as hct26 , ht-29 , hct116 , and dld-1 grown as xenograft tumors in immunecompromised mice.9 n-3 pufas are likely to have multifaceted roles in both prevention and treatment of crc .
the excellent tolerability and safety profile of n-3 pufas combined with other health benefits , particularly cardiovascular , make n-3 pufas an attractive candidate for prevention and treatment of crc.9
esophageal cancer is ranked as the sixth leading cause of cancer death worldwide . according to the increasing incidence of gastroesophageal reflux disease ( gerd ) ,
esophageal cancer is a tumor that has increased in incidence more than 7-fold over the past several decades .
suggests that negative associations between n-3 pufa intake and the risk of esophageal adenocarcinoma.19 higher in - takes of n-3 pufa [ cases vs. population controls ; or=0.46 , 95% ci=0.220.97 , 4th vs. 1st quartiles of intake ] was associated with a lower risk of barrett s esophagus .
in contrast , higher trans - fat intakes were associated with increased risk ( or=1.11 ; 95% ci=1.031.21 per g / day ) .
moreover , it is reported that n-3 pufa supplemented parenteral nutrition can reduce inflammation and improve immune function in patients following esophageal cancer surgery20 and n-3 pufa - containing diet may be beneficial to patients with esophageal cancers who receive chemoradiation therapy ( crt ) by reducing crt toxicity.21 in contrast to other gi cancer , little work has so far been performed on the influence of n-3 pufa in oesophageal adenocarcinogenesis and neoplastic progression.22 there is a need for appropriately powered randomized - controlled studies to assess the long - term benefit of n-3 pufa .
gastric cancer ( gc ) is the fourth most common cancer worldwide , and almost two thirds of affected individuals will die of their disease . some studies about the association of n-3 pufa and gastric disease suggests a protective effect of n-3 pufa on gastric cancer .
recently , correa et al . suggests that docosahexanoic acid ( dha ) inhibits helicobacter pylori ( h. pylori ) growth in vitro and mice gastric mucosa colonization.23
h. pylori are recognized as a major etiological factor in chronic active gastritis , gastric duodenal ulcers and gastric cancer .
it has been proposed that pufa hold an inhibitory effect on bacterial growth via disruption of cell membrane leading to bacteria lysis.24 mohamed also shows that n-3 pufa reduced iodoacetamide - induced gastritis in rats through decrease of malondialdehyde ( mda ) , gastrin , and nitric oxide ( no ) and normalization of mucosal glutathione.25 especially , it is reported that the erythrocyte composition of dha was found to be negatively linked to risk of gastric cancer , of well - differentiated adenocarcinoma.26 application of a diet enriched with n-3 pufa delayed tumor growth in a mouse xenograft model.27
in vitro studies have shown that n-3 pufa inhibited macrophage - enhanced gastric cancer cell migration and attenuated matrix metalloproteinase ( mmp)-10 expression through erk and stat3 phosphorylation28 and inhibited the growth of human gastric carcinoma cell via apoptosis and combination with 5-fluorouracil has synergetic effect in inhibiting the proliferation of gastric cancer cells.29 moreover , n-3 pufa are beneficial for preventing oxidative stress - induced apoptosis by inhibiting apoptotic gene expression and dna fragmentation of gastric epithelial cells.30 on the other hand , dha induced apoptosis of gastric cancer cells by inducing the expression of apoptotic genes in gastric cancer cells.31 although a large body of literature spanning numerous cohorts from many countries and with different demographic characteristics does not provide evidence to suggest a significant association between n-3 pufa and stomach cancer incidence,32 further studies are needed to investigate action of n-3 pufa relevant to antitumor effects in the stomach .
colorectal cancer ( crc ) is the second leading cause of cancer - related death in men after lung cancer and third in women behind lung and breast cancers in the united states . among the gi tract cancer ,
crc cancer has raised the most attention over the past decades , as they share a long precancerous stage ( the adenoma in crc ) which provides a window of opportunity to intervene and prevent development of cancer .
there is also evidence suggesting improved efficacy and/or tolerability of conventional colon cancer chemotherapy when administered with n-3 pufa.9 epidemiological studies about the association of dietary fat and cancer suggests a protective effect of n-3 pufa and a promoting effect of n-6 pufa on cancer .
although epidemiological studies of the association between fish intake , n-3 pufa intake or blood n-3 pufa levels and crc risk have not consistently suggested beneficial effects of n-3 pufa on crc and other gi cancer risk .
dietary administration of one or both of the main n-3 pufa in rodent models of colorectal carcinogenesis has been demonstrated to reduce colorectal tumor size and multiplicity , compatible with crc chemopreventive activity.33 in meta - analyses of prospective cohort studies that evaluated the association between fish consumption or n-3 fatty acids and colorectal cancer incidence or mortality , the pooled relative risks for colorectal cancer incidence were 0.960.97 ( 95% confidence interval : 0.92 , 1.00 ) for each extra occurrence of fish consumption per week.34 in a population - based prospective study on the association of n-3 pufa and cancer , there was an inverse relationship between marine n-3 pufa intake and the risk of colorectal cancer , but this association was only statistically significant in the proximal site of the large bowel.35 cockbain et al.9 has reviewed a lot of in vitro and in vivo experimental studies and epidemiological observations providing strong indications of the cancer treatment and prevention of n-3 pufa against colorectal cancer .
kim et al.36 provided a significant dose - dependent reduction in crc risk for total n-3 pufa intake ( or=0.61 for the highest vs lowest quartile ) , as well as for epa and dha intake individually in a case - control study of 1,872 patients ( 929 cases of distal crc and 943 controls ) . pot measured and compared serum n-3 pufa levels in 861 patients ( 363 cases of colorectal adenoma and 498 controls ) .
there was a significant reduction in colorectal adenoma risk ( or=0.67 ) between low level of n-3 pufa ( < 1.8% ) and high level of n-3 pufa ( > 2.3%).37 recently , sorensen et al.38 reported that n-3 pufa is incorporated rapidly into colonic mucosa and colonic muscular layer in patients given 3 g of n-3 pufa daily for 7 days before surgery for colorectal cancer .
the preventive effect of n-3 pufa has been demonstrated in a number of carcinogen - induced models like aom and dimethylhydrazine ( dmh)-induced rat model and apc mouse models relevant to prevention of crc including ours .
studies of rodents fed an n-3 pufa - supplemented diet versus a control diet have consistently reported a 20 - 50% reduction in chemically induced tumor incidence , together with a 3070% reduction in tumor multiplicity , in both carcinogen and apc mouse studies .
similar findings have been reported for studies of growth of human crc cell lines such as hct26 , ht-29 , hct116 , and dld-1 grown as xenograft tumors in immunecompromised mice.9 n-3 pufas are likely to have multifaceted roles in both prevention and treatment of crc .
the excellent tolerability and safety profile of n-3 pufas combined with other health benefits , particularly cardiovascular , make n-3 pufas an attractive candidate for prevention and treatment of crc.9
n-3 pufa , especially , epa and dha , have been shown to have multiple anti - tumor actions .
current knowledge of the anti - tumor activity of n-3 pufa has been comprehensively reviewed elsewhere.9,16,39 recently , stephenson et al.10 has reviewed more recent underlying mechanisms providing strong indications of the anti - tumor actions of n-3 pufa on the hallmarks of cancer .
n-3 pufa appear to down - regulate epidermal growth factor receptor ( egfr ) , protein kinase c ( pkc ) , ras , and nf-b , insulin like growth factor ( igf ) , which are important cell signaling mediators often found to be elevated in carcinogenesis .
secondly , n-3 pufa induces cancer cell apoptosis via modulation of peroxisome proliferator - activated receptors ( ppars ) , the bcl-2 family , and nf-b cell signaling .
thirdly , n-3 pufa decreases sprouting angiogenesis by suppressing vascular endothelial growth factor ( vegf)- and platelet derived growth factor ( pdgf)-stimulated endothelial cell proliferation , migration , and tube formation and by inhibition of mmps via no production and nf-b and -catenin cell signaling .
fourthly , n-3 pufa also decreases cell - cell adhesion via down regulation of rho - gtpase , which inhibits cytoskeleton reorganisation , and reduction in intercellular adhesion molecule ( icam)-1 and vascular cell adhesion molecule ( vcam)-1 expression .
cockbain et al.9 has also proposed the four main anti - tumor actions of n-3 pufa ( i ) modulation of cox activity ; ( ii ) alteration of membrane dynamics and cell surface receptor function ; ( iii ) increased cellular oxidative stress and ( iiii ) derived anti - inflammatory lipid mediators .
firstly , n-3 pufa can act as an alternative substrate for cox-2 , instead of aa , leading to a reduction in formation of pro - tumorigenic
secondly , incorporation of n-3 pufa into cell membranes alters the fluidity , structure and/or function of lipid rafts or calveolae .
especially , the localization of cell surface receptors , such as g protein - coupled receptors ( gpcrs ) , toll - like receptors ( tlrs ) , and epidermal growth factor receptor ( egfr ) , in lipid rafts is believed to be crucial for downstream receptor signaling , controlling proliferation and apoptosis .
thirdly , n-3 pufa may have an anti - tumor effect through alteration in the cellular redox state .
n-3 pufa can increase reactive oxygen species ( ros ) because it is highly peroxidisable .
therefore , n-3 pufa can induce cancer cell apoptosis via elevation of intracellular ros levels .
fourthly , n-3 pufa can be metabolized novel anti - inflammatory lipid mediators including resolvins , protectins and maresins .
it is known that resolvins exhibit antineoplastic activity via anti - inflammatory and inflammation resolution activity in animal models of acute inflammation . besides these multiple anti - tumor actions
, it is also reported recently that n-3 pufa can activate nrf2 and induced nrf2-directed gene expression40,41 and can suppress lipopolysaccharide - induced inflammation through induction of nrf2 expression.42 nuclear factor erythroid 2-related factor 2 ( nrf2 ) is a redox - sensitive master regulatory transcriptional factor that plays an important protective role in cells by regulating cellular redox balance.43 moreover , n-3 pufa significantly reduces oxidative stress - induced endothelial cell ca influx .
this effect might be associated , at least in part , with altered lipid composition in membrane lipid rafts.44 recently , the engineered n-3 pufa desaturase transgenic mice ( fat-1 mice ) , which can endogenously synthesize n-3 pufa in their tissues , allows carefully controlled studies to be performed in the absence of potential confounding dietary factors.45 the synthesis of n-3 pufa is achieved through the expression of the fat-1 transgene encoding for an n-3 desaturase , which utilizes n-6 pufa as substrate .
this allows production of high n-3/n-6 ratios in the animals , thus eliminating the potential diet variations .
hence , the fat-1 transgenic mouse is a valuable in vivo system for elucidating the role of n-3 pufa in carcinogenesis .
since fat-1 mice were generated , xia et al.46 showed that melanoma formation and growth are reduced in fat-1 transgenic mice .
nowak and jia47,48 reported that lower incidence and growth rate of colon tumors induced by dss ( dextrane sodium sulfate ) plus aom ( azoxymethane ) in the fat-1 transgenic mice via its anti - inflammatory properties .
song et al.49 also reported that the growth of pancreatic cancer in vivo was significantly reduced when mouse pancreatic cancer cells , panc02 cells , were inoculated into the fat-1 transgenic mice .
recently , mohammed et al.50 suggested the beneficial effects of n-3 pufa for chemoprevention of pancreatic cancer using fat-1 mice .
they generated compound fat-1-kras transgenic mice and showed a dramatic reduction in incidence of pancreatic ductal adenocarcinoma ( 84% ; p<0.02 ) in fat-1-kras mice compared to kras mice . besides of gi cancers ,
the growth of hepatocellular carcinoma ( hcc ) in vivo was significantly reduced in the fat-1 transgenic mice.5153 mice expressing mmtv - neu(ndl)-yd5 and fat-1 , which were bred with mouse mammary tumor virus ( mmtv)-neu(ndl)-yd5 mice ( an aggressive breast cancer model ) , displayed significant ( p<0.05 ) reductions in tumor volume ( 30% ) and multiplicity ( 33%).54 recently , in our laboratory an in vivo study has shown a suppressive effect of fat-1 mice in gi cancer development ( unpublished data ) .
fat-1 mice were bred with the apc mouse colon cancer transgenic mice to generate compound fat-1-apc transgenic mice .
a dramatic reduction in incidence of aom - dss induced colon adenocarcinoma in fat-1-apc mice compared to apc mice was shown . moreover , in the case of stomach cancer development , h. pylori initiated- , salt diet - promoted - gastric tumor was also reduced in fat-1 mice . in conclusion , several studies using fat-1 mice model indicate that balancing the tissue n-6/n-3 ratio could exert a significant effect on gi cancer development .
the fat-1 mouse model allows carefully controlled studies to be performed in the absence of restricted diets , which can create confounding factors that limit studies of this nature.55
recently , the engineered n-3 pufa desaturase transgenic mice ( fat-1 mice ) , which can endogenously synthesize n-3 pufa in their tissues , allows carefully controlled studies to be performed in the absence of potential confounding dietary factors.45 the synthesis of n-3 pufa is achieved through the expression of the fat-1 transgene encoding for an n-3 desaturase , which utilizes n-6 pufa as substrate .
this allows production of high n-3/n-6 ratios in the animals , thus eliminating the potential diet variations .
hence , the fat-1 transgenic mouse is a valuable in vivo system for elucidating the role of n-3 pufa in carcinogenesis .
since fat-1 mice were generated , xia et al.46 showed that melanoma formation and growth are reduced in fat-1 transgenic mice .
nowak and jia47,48 reported that lower incidence and growth rate of colon tumors induced by dss ( dextrane sodium sulfate ) plus aom ( azoxymethane ) in the fat-1 transgenic mice via its anti - inflammatory properties .
song et al.49 also reported that the growth of pancreatic cancer in vivo was significantly reduced when mouse pancreatic cancer cells , panc02 cells , were inoculated into the fat-1 transgenic mice .
recently , mohammed et al.50 suggested the beneficial effects of n-3 pufa for chemoprevention of pancreatic cancer using fat-1 mice .
they generated compound fat-1-kras transgenic mice and showed a dramatic reduction in incidence of pancreatic ductal adenocarcinoma ( 84% ; p<0.02 ) in fat-1-kras mice compared to kras mice . besides of gi cancers , the growth of hepatocellular carcinoma ( hcc ) in vivo was significantly reduced in the fat-1 transgenic mice.5153 mice expressing mmtv - neu(ndl)-yd5 and fat-1 , which were bred with mouse mammary tumor virus ( mmtv)-neu(ndl)-yd5 mice ( an aggressive breast cancer model ) , displayed significant ( p<0.05 ) reductions in tumor volume ( 30% ) and multiplicity ( 33%).54 recently , in our laboratory an in vivo study has shown a suppressive effect of fat-1 mice in gi cancer development ( unpublished data ) .
fat-1 mice were bred with the apc mouse colon cancer transgenic mice to generate compound fat-1-apc transgenic mice .
a dramatic reduction in incidence of aom - dss induced colon adenocarcinoma in fat-1-apc mice compared to apc mice was shown .
moreover , in the case of stomach cancer development , h. pylori initiated- , salt diet - promoted - gastric tumor was also reduced in fat-1 mice . in conclusion ,
several studies using fat-1 mice model indicate that balancing the tissue n-6/n-3 ratio could exert a significant effect on gi cancer development .
the fat-1 mouse model allows carefully controlled studies to be performed in the absence of restricted diets , which can create confounding factors that limit studies of this nature.55
gi cancer incidence and mortality are increasing in the eastern world and a high n-6 to n-3 pufa ratio in the western style diet may be a contributing factor .
there is much evidence to suggest that higher consumption of dietary n-3 pufa is associated with a lower risk of gi cancer in animal models and humans .
especially , recent studies suggest that endogenous n-3 pufa delay the progression of colon and stomach cancer and elevating n-3 pufa may be an important strategy to delay / prevent gastrointestinal cancer in high - risk patients via various mechanisms mediating cancer prevention by n-3 pufa ( fig .
2 ) . in addition , using n-3 pufa in combination with other agents with complementary antitumor action may improve their efficacy in gi cancer prevention . recently ,
manson et al . has started the vitamin d and omega-3 trial ( vital ) , a large randomized , double - blind , placebo - controlled , 22 factorial trial of vitamin d and n-3 pufa supplements in the primary prevention of cancer among a multi - ethnic population of 20,000 u.s .
men aged 50 and women aged 55.56 we expect that new findings in combination consumption of n-3 pufa with other nutrients will provide new approaches to public health implications with regard to prevention of gi cancer through dietary and lifestyle interventions .
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omega-3 polyunsaturated fatty acids ( n-3 pufas ) , particularly eicosapentanoic acid ( epa ) and docosahexanoic acid ( dha ) , has been acknowledged as essential very long - chain fatty acids contributing to either achieving optimal health or protection against diseases , and even longevity .
recent high impact studies dealing with epa and dha have sparked a renewed interest in using n-3 pufas for cancer prevention and cancer treatment , for which n-3 pufas may exert their anticancer actions by influencing multiple targets implicated in various stages of cancer development , including cell proliferation , cell survival , angiogenesis , inflammation , and metastasis against various cancers .
however , gastrointestinal cancers develop implicated with the close connection between inflammation and cancer and n-3 pufas especially imposed excellent actions of antiinflammation and antioxidation as well as their restorative actions . in detail , these beneficial lipids can restore or modify inflammation - associated lipid distorsion and alteration of lipid rafts . although the chemopreventive effect of n-3 pufas has been studied in various experimental models , our understanding regarding the underlying mechanisms of n-3 pufas against gi cancer is still limited . in this review article
, we described the in - detailed perspective and underlying mechanism of n-3 pufas application for gi cancers and added in vivo efficacy of n-3 pufas with fat-1 transgenic mice experience .
we suggest that future work should consider the n-6/n-3 fa ratio , combination treatment of other nutritions and alteration of lipid rafts to be a key element in experimental design and analysis .
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INTRODUCTION
THE EFFICACIES OF N-3 PUFA IN PREVENTION OF GI CANCERS
Esophageal cancer
Gastric cancer
Colorectal cancer
MECHANISMS OF N-3 PUFAS ON CHEMOPREVENTION
Useful animal model to study chemopreventive mechanism of n-3 PUFA:
CONCLUSIONS
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older people have particular spiritual needs that are distinct from those of others , particularly in times of ill health and death and dying .
it has been reported that spirituality has a positive effect on health outcomes and welfare of the elderly .
nowadays , aging phenomenon and relevant problems with its complexities are important issues that have attracted the attention of social and medical scientists to investigate the spiritual needs of the elderly .
spiritual health is a dimension of health that is portrayed as the validation of life in relation to god , fellow human beings , and the environment .
spirituality is the representative of the basic values that guide a person in searching to find answers to the crucial questions of life , such as the purpose and meaning of life , reality , love , good and bad , disease , and death . over the past three decades , spirituality , religion , and prayer
have turned into a subject of large interest for researchers in medicine , nursing , gerontology , physical health and mental health disciplines , often in conjunction with complementary and alternative medicine .
this tendency has been stimulated by research demonstrating physical and psychological benefits of spiritual and religious involvements .
in fact , approximately 96% of adults in the united states expressed their belief in god , and 72% of identified religion and spirituality as having the most important influence in their lives . saying prayers is a regular daily task for muslims which begins at the age of puberty and continues throughout the life .
it is a time getting rid of the material world and approaching the world beyond , as well as , to the inner self . in iran , elderly people are more religious than younger ones , and this is a norm of this culture .
a recent national survey of the iranians values and attitudes found that over 80% of the iranians practice prayer regularly as a part of their religion .
one might argue that this is a true reflection of individual culture where prayer and religious beliefs are a part of people 's everyday life .
however , prayer can go beyond just performing a religious duty , and based on one 's wishes , take different forms of formal and informal practices .
it has been shown that disease prevalence , incidence and mortality rates differ from one population to another .
this is partly ascribed to their disparities in lifestyle , dietary habits and other characteristics that are somehow affected by culture , especially religious traditions and experiences . during the past two decades
some studies have been carried out and the associations between religious beliefs or experiences and various types of physical illnesses , mental health , and mortality have been examined .
spirituality is viewed as personally important matter to the older people and it has been proved that it is highly related to older people 's health outcomes .
however , there has not been adequate experiential research on the relationship between religious or spiritual factors and healthy lifestyle behaviors , and their impact on the adult 's well - being .
scientists believe that probing the effects of religious and spiritual factors as well as healthy lifestyle behaviors may be decisive .
additionally , social science and medical research have concurrently reported that older adults are more inclined to take spiritual chase than younger people are .
kotrotsiou - barboutal et al.(2006 ) indicated that older adults are remaining members of religious associations without necessarily participating regularly in services , as there are numerous issues that can diminish the active participation in religious events , such as transportation intricacies , bad sound , or the illegibility of small font text in books of prayers available at mosques or religious centers . in spite of those complexities ,
many elderly manage to find out a way to come up to god through prayer .
it is stated that prayer increases the feelings of personal value of an elderly by reducing the feelings of loneliness and desertation , while television and radio provide support for spiritual life . in this regard , researchers have shown that some groups of elderly people with inadequate and difficult livelihood situations communicate their problems by putting their trust in god . moreover , more than half of the elder patients practiced spiritual needs throughout their hospitalization period , with the most prominent need of being in the search of the meaning of life .
extensive epidemiological studies have shown that higher levels of religiousness are related to lower mortality rates .
studies identified the multidimensional nature of religiousness and spirituality and investigated various religious dimensions , for instance private religious behaviors , devotion , spiritual transcendence , and religious adaptation .
for example , it was indicated that some aspects of religiousness predicted lower rates of disability and illness , alcoholism , cardiovascular disease , hypertension , and myocardial infarction .
furthermore , clinical research has suggested that spirituality can play a vital role in the recovery of patients suffering from physical and psychological diseases .
it seems that religious obligation has a crucial role in averting physical and mental diseases , recovery promotion , and disease adaptation .
numerous studies have revealed the positive involvement of spirituality and religious indexes on different aspects of health such as cardiac surgery , mortality , immunity system function and self esteem .
above all , religious involvement can be useful for psychological well - being and physical health .
additionally , supporting the effect of spirituality may become more and more prominent in later life as a result of declining health , social and financial sources .
researchers have proposed numerous psychological , social , and physiological interveners that may explain the spirituality - health association .
quantitative measurements and the evaluation of spiritual beliefs are difficult because quantitative studies neither reveal the content , components of the beliefs , and its factors , nor the contexts that might be included . therefore , implementing qualitative research that explores the personal and experiential aspects of beliefs seems crucial , and may shed light on a range of important issues which can be investigated in detail on larger samples by further quantitative studies in future .
spiritual or religious beliefs may affect the decisions of individuals that make about their health and illness .
thus , recognizing and understanding the spiritual and religious behaviors within the context of cultural influence by a qualitative research is critical for nurse and other health - care providers .
the main strengths of the present study are that it focuses on the spiritual life of healthy elderly people .
therefore , the present study aimed to explore the concept of spirituality from the perspectives of iranian healthy elderly people . the central question of the paper was what characterizes the spirituality in the iranian healthy older people ?
in this research , a qualitative approach was adopted using conventional content analysis of unstructured interviews carried out with 17 healthy elderly people in tehran in 2010 - 2011 .
it is worth noting that spirituality is underpinned by personal and cultural context in any community ; therefore , qualitative research is the best method to study cultural context - bound subjects .
initially purposeful sampling was used and continued with purposeful sampling according to the codes and categories emerged from data .
the study consisted of 17 healthy elders within the age range of 65 - 86 .
criteria for selection were having age above 65 , living with family , not having any cognitive problems , not having any physical limitation in adl , and willingness to participate in the study .
all participants were shia muslims . in order to achieve the wide range of experiences and perceptions of elderly participants , some field notes writing and 21 interviews were conducted according to emerged codes and categories from data . the first researcher communicated with each of the participants to describe the purpose of research and research questions .
based on the participants preference , the interviews were performed in a private room at the house of elderly , park , worksite , or mosque , using an individual semi - structured interview format and this was primarily the main method for data collection .
interview query consisted of open - ended questions to allow respondents thoroughly to describe their opinions , perceptions , and experiences .
the participants were asked to describe one day of their life and then to explain their own experiences and perceptions about spirituality in elderly adults .
what is your experience concerning the spirituality in later age? and what is the meaning of spirituality in your experience ?
each interview was transcribed verbatim and analyzed before the next interview was done , so that each interview supplied bearing for the next .
coding was carried out line by line , and comparative analysis of the quotations was carried out .
all interviews were conducted in a single session , at the request and preference of participants , except for two cases that took two sessions .
each interview session ranged from 30 minutes to 90 minute with an average of 55 minutes .
data were collected and analyzed over a six - month -- period in 2009 . in the first phase , subcategories and their domains in the data were identified and classified into categories .
the coding process was iterative , and categories evolved ( inserted , deleted and combined ) as re - readings were completed and analyses progressed . in the second phase , the subcategories and domains were regrouped into major categories .
credibility was recognized through prolonged engagement with the participants , field note writing , the participants revisions using member checking procedure , and peer checking .
the findings and explanations of this study were reviewed by two supervisors who were associate professors in nursing having a good background in qualitative research methodology . also , maximum variation of sampling established the conformability and credibility of the data .
this study provided sufficient descriptive data for researchers to criticize whether the findings were transferable that established applicability .
permission to conduct this study was obtained from the ethics committee of tarbiat modares university .
other ethical issues in this study included the assurance of confidentiality and anonymity of the participants and their responses .
all participants were aware of the purpose of the study , and their participation in the study was optional .
informed consent form was obtained from the participants who agreed to participate in the study according to the provisions of the declaration of helsinki .
in this research , a qualitative approach was adopted using conventional content analysis of unstructured interviews carried out with 17 healthy elderly people in tehran in 2010 - 2011 .
it is worth noting that spirituality is underpinned by personal and cultural context in any community ; therefore , qualitative research is the best method to study cultural context - bound subjects .
initially purposeful sampling was used and continued with purposeful sampling according to the codes and categories emerged from data .
the study consisted of 17 healthy elders within the age range of 65 - 86 .
criteria for selection were having age above 65 , living with family , not having any cognitive problems , not having any physical limitation in adl , and willingness to participate in the study .
in order to achieve the wide range of experiences and perceptions of elderly participants , some field notes writing and 21 interviews were conducted according to emerged codes and categories from data . the first researcher communicated with each of the participants to describe the purpose of research and research questions . the interview was scheduled according to the participant 's agreement .
based on the participants preference , the interviews were performed in a private room at the house of elderly , park , worksite , or mosque , using an individual semi - structured interview format and this was primarily the main method for data collection .
interview query consisted of open - ended questions to allow respondents thoroughly to describe their opinions , perceptions , and experiences .
the participants were asked to describe one day of their life and then to explain their own experiences and perceptions about spirituality in elderly adults .
what is your experience concerning the spirituality in later age? and what is the meaning of spirituality in your experience ?
each interview was transcribed verbatim and analyzed before the next interview was done , so that each interview supplied bearing for the next .
coding was carried out line by line , and comparative analysis of the quotations was carried out .
all interviews were conducted in a single session , at the request and preference of participants , except for two cases that took two sessions .
each interview session ranged from 30 minutes to 90 minute with an average of 55 minutes .
data were collected and analyzed over a six - month -- period in 2009 . in the first phase , subcategories and their domains in the data were identified and classified into categories .
the coding process was iterative , and categories evolved ( inserted , deleted and combined ) as re - readings were completed and analyses progressed . in the second phase , the subcategories and domains were regrouped into major categories .
credibility was recognized through prolonged engagement with the participants , field note writing , the participants revisions using member checking procedure , and peer checking . the findings and explanations of this study
were reviewed by two supervisors who were associate professors in nursing having a good background in qualitative research methodology . also , maximum variation of sampling established the conformability and credibility of the data .
this study provided sufficient descriptive data for researchers to criticize whether the findings were transferable that established applicability .
permission to conduct this study was obtained from the ethics committee of tarbiat modares university .
other ethical issues in this study included the assurance of confidentiality and anonymity of the participants and their responses .
all participants were aware of the purpose of the study , and their participation in the study was optional .
informed consent form was obtained from the participants who agreed to participate in the study according to the provisions of the declaration of helsinki .
the participants of the study were 11 males and 10 females , within the age range of 65 - 86 .
also , 15 out of 21 were married , four were widowed , and one was single .
five participants were illiterate , four were primary degree holders , eight had a diploma , two had bachelor degrees , and two had msc degree in human sciences . in terms of their occupation status , two were employees , 13 were pensioners , two was manual worker , and three were housewives .
three main categories emerged from the data and 3 - 4 distinctive subcategories within each category were identified .
these categories and their subcategories represent the main factors influencing the spirituality in iranian elderly people .
main categories and subcategories one of the main categories that emerged from data analysis was spiritual health . four subcategories ;
saying prayer as a calming factor , beneficence as a way to god , loss of psychological and spiritual support , and faith , a way to happiness , emerged from the participants experiences .
also many of the participants considered the prayer time as an opportunity for purification which made them more tolerant to the adversities and has a calming effect on them .
i want to say that waking up early in the morning , having the spirit for things like prayer , citing quran verses , thanking god for his blessings and doing exercise would make you quiet and happy until the end of the day .
( male ) most participants believed that dealing with the affairs of the elderly and giving them a helping hand in their lives have positive consequences for the servers .
they also believed that doing good things to older adults will lead them to pray for the servers which in turn cause good things in the servers lives .
they said that man would receive god 's blessings as a consequence of his / her deeds in the world .
additionally , they believed that the ultimate consequence of good deeds was good in return .
if she asks me to fix her air conditioner you can be sure that i ll do it .
this is because i think that by doing this i make her happy and she prays for me .
, the participants said that in iran the elderly people feel psychologically and spiritually alone and they do not receive any institutionally based mental and spiritual support .
when i think about my life , i feel that i do n't have any support from my children .
also in the street and road we do n't have appropriate social respect and i want to say that in our society there is limited support and backing for people like me .
( female ) the elderly participants lived experiences disclosed the subcategory of faith , a way to happiness in life .
one of participants described that having strong faith was very important over the elderly life and adopting a healthy life style depended mostly on their faith and beliefs .
also , they stressed that faith will lead them to a sense of physical and mental welfare and happiness .
the one who has strong faith and beliefs is always happy and confident . in my opinion
worshiping god , praying , fasting , reciting quran , purity and attending mosques are important elements in life , and bring happiness to your life and set your soul free .
( male ) spiritual beliefs was a main category that emerged from the participants lived experiences of spiritual concept and this category was further subcategorized into three subcategories including seeking help from god in difficulties
doing good deeds is the god 's will . regarding the subcategory of seeking help from god in difficulties
, the participants believed that wishing for and seeking help from god can help the elderly people to deal better with life difficulties , disperses and stresses .
this desire results in god 's attention to elderly and also creates sense of trust in older adults .
some statements said by participants are : i really believe in having trust in god . when i m trapped in a complicated situation
, i believe that this is god who helps me and i only trust in god . ( male ) one participant believed that spirituality was a vital component of older people lives .
also , he believed that god was the ultimate source to affect my life or death and he controls everything .
we re all waiting to see when the angel of death arrives and takes us away .
( male ) concerning the subcategory of doing good deeds is the god 's will , the participants believed that trust in god guided them to many positive activities in their lives .
they believed that calling the name of god while getting out of house , remembering him throughout the daytime and praying will lead them to prosperity .
one participant said that trust in god , submitting to god 's will and thinking about god all the time helped him to work and deal with anybody without any behavioral problems .
when i want to get out of my house for work , e first thing that comes to mind is that
my dear god : bestow upon me everything which is good for me and while i m out working , i do my best not to misbehave or do anything bad to anybody . ( male ) another most important category that emerged was religious practice with three distinctive subcategories including : saying prayers , reciting quran , and going to mosque , religious ceremonies and pilgrimage .
the majority of the participants indicated that they had good knowledge of quran , said prayers regularly , and went to mosque for religious and social activities .
for example one participant said that participating in religious settings and ceremonies led her to find new friends and got her out of loneliness .
we go to quran classes on friday mornings also.(male ) i have so much interest for pilgrimage and saying daily payers at the earliest recommended time when call for prayer is announced .
i go to the religious rites with the my friends and peers , and i want to get out of loneliness .
( female ) thus , it can be concluded that all of the participants rose early to start their religious acts of devotion , prayers , and worship in their home or mosque .
one of the main categories that emerged from data analysis was spiritual health .
four subcategories ; saying prayer as a calming factor , beneficence as a way to god , loss of psychological and spiritual support , and faith , a way to happiness , emerged from the participants experiences .
also many of the participants considered the prayer time as an opportunity for purification which made them more tolerant to the adversities and has a calming effect on them .
i want to say that waking up early in the morning , having the spirit for things like prayer , citing quran verses , thanking god for his blessings and doing exercise would make you quiet and happy until the end of the day .
( male ) most participants believed that dealing with the affairs of the elderly and giving them a helping hand in their lives have positive consequences for the servers .
they also believed that doing good things to older adults will lead them to pray for the servers which in turn cause good things in the servers lives .
they said that man would receive god 's blessings as a consequence of his / her deeds in the world .
additionally , they believed that the ultimate consequence of good deeds was good in return .
if she asks me to fix her air conditioner you can be sure that i ll do it .
this is because i think that by doing this i make her happy and she prays for me .
( female ) moreover regarding loss of psychological and spiritual support , the participants said that in iran the elderly people feel psychologically and spiritually alone and they do not receive any institutionally based mental and spiritual support .
when i think about my life , i feel that i do n't have any support from my children .
also in the street and road we do n't have appropriate social respect and i want to say that in our society there is limited support and backing for people like me .
( female ) the elderly participants lived experiences disclosed the subcategory of faith , a way to happiness in life .
one of participants described that having strong faith was very important over the elderly life and adopting a healthy life style depended mostly on their faith and beliefs .
also , they stressed that faith will lead them to a sense of physical and mental welfare and happiness .
the one who has strong faith and beliefs is always happy and confident . in my opinion
worshiping god , praying , fasting , reciting quran , purity and attending mosques are important elements in life , and bring happiness to your life and set your soul free .
spiritual beliefs was a main category that emerged from the participants lived experiences of spiritual concept and this category was further subcategorized into three subcategories including seeking help from god in difficulties , only god 's power over life and death , and
doing good deeds is the god 's will . regarding the subcategory of seeking help from god in difficulties , the participants believed
that wishing for and seeking help from god can help the elderly people to deal better with life difficulties , disperses and stresses .
this desire results in god 's attention to elderly and also creates sense of trust in older adults .
some statements said by participants are : i really believe in having trust in god . when i m trapped in a complicated situation
, i believe that this is god who helps me and i only trust in god . ( male ) one participant believed that spirituality was a vital component of older people lives .
also , he believed that god was the ultimate source to affect my life or death and he controls everything .
we re all waiting to see when the angel of death arrives and takes us away .
( male ) concerning the subcategory of doing good deeds is the god 's will , the participants believed that trust in god guided them to many positive activities in their lives .
they believed that calling the name of god while getting out of house , remembering him throughout the daytime and praying will lead them to prosperity .
one participant said that trust in god , submitting to god 's will and thinking about god all the time helped him to work and deal with anybody without any behavioral problems .
when i want to get out of my house for work , e first thing that comes to mind is that
my dear god : bestow upon me everything which is good for me and while i m out working , i do my best not to misbehave or do anything bad to anybody . ( male )
another most important category that emerged was religious practice with three distinctive subcategories including : saying prayers , reciting quran , and going to mosque , religious ceremonies and pilgrimage .
the majority of the participants indicated that they had good knowledge of quran , said prayers regularly , and went to mosque for religious and social activities .
for example one participant said that participating in religious settings and ceremonies led her to find new friends and got her out of loneliness .
we go to quran classes on friday mornings also.(male ) i have so much interest for pilgrimage and saying daily payers at the earliest recommended time when call for prayer is announced .
i go to the religious rites with the my friends and peers , and i want to get out of loneliness .
( female ) thus , it can be concluded that all of the participants rose early to start their religious acts of devotion , prayers , and worship in their home or mosque .
this qualitative study describes the concept of spirituality and its impression on iranian elderly people daily living behaviors .
the findings are significant as they reveal that the living experiences of saying prayer as a calming factor , beneficence as a way to god , loss of psychological and spiritual support , and faith , a way to happiness , are the most important domain and subcategory of spiritual health of elderly people , which can positively affect their health . on the other hand spiritual health
is an important dimension of human health and it can determine the individual integrity . also spiritual health is a power that coordinates physical , mental and social dimensions and is necessary in coping with diseases . when spiritual health will is at serious risk , individuals may be stricken by mental disorders such as loneliness , depression , and loss of meaning .
there are a number of studies that support the beneficial effects of spirituality on health .
behaviors such as reliance on god , pilgrimage and praying create hope and positive attitudes that lead to internal serenity , peacefulness and meaningful life .
most faithful people describe their relationship with god as a relationship with an intimate friend and believe that they can control the effect of irrepressible situations by having recourse to god .
all in all , religious adaptation relies on beliefs and religious activities and help people to control their stress and physical disorders . having meaning and goal in life , sense of belonging to a sublime source , hope for gods support in time of trouble and social and spiritual support are among the sources that help religious people to suffer less from hardships of life . in the iranian society context , where the majority of the population are muslim , and
a religious culture is dominant in the society , it is expected that tendency to spirituality could be affect their health .
praying is the expression of soul and a deep human instinct that arises from human soul and is uttered with profound words .
it is neither dependent upon any particular spirituality nor entailed in any time and place .
the content of praying includes confession of sins and weakness , pleading for forgiveness , bliss and closeness to god .
praying reduces mental and psychological pains , releases personal emotions , leading individual to attain eternal source of power .
weeping that often comes with praying greatly helps a person to release his or her distress and emotions .
also prayer helped participants to cope with disease and gave them calm , hope and inner strength .
in this regard , quran has put it magnificently : those who have faith in god and inundate their hearts with name of god , surely god remembrance ascertains their hearts ( ( quran , chapter 13 : sureh 13 , al - rad ) verse 28 ) .
many americans , regardless of their health status , rely on their religious and spiritual beliefs to cope with stressful life events .
the present study has demonstrated that one of the most important factors that help elderly in confronting with difficulties was seeking help from god .
several studies noted that spirituals beliefs had positive influence on welfare and personal and social life of elderly people .
effective spiritual coping strategies help individuals find meaning and purpose in their life , problems and difficulties . for instance
, one study showed that healthy older adults believed that a higher power supports them and that having a relationship with god forms a foundation for their psychological well - being .
thus , spirituality plays an important role in the lives of healthy individuals , too .
believing in support from spiritual / religious resource and connectedness with a higher power is beneficial and it can affect issues of control , quality of life , spiritual well - being , coping , depression , decision - making , and possibly health outcomes .
for instance , it has been reported that healthy older adults believe that a higher power supports them and that having a relationship with god forms a foundation for their psychological well - being .
thus , spirituality plays an important role in the lives of healthy individuals . in the result of our study
a sense of god 's power over life and death and doing good deeds is the god 's will were derived from participants experience around spiritual beliefs .
all the participants talked about the importance of god and belief to god 's power in their life .
moreover , the findings reveal that god 's power was very important in personal and private experiences . in this regard , roff et al.(2009 ) in a qualitative study through a thematic content analysis identified god 's power in the lived experiences of women who were suffering from breast cancer . also , in another qualitative study by taleghani et al .
( 2006 ) belief to god 's will was a important key in acceptance of disease .
2002 ) suggested that personal variables such as the concept of god and perception of others beliefs played important mediating roles in religious coping with stress situations .
religious practices were a subcategory that emerged from the lived experiences of elderly participants in this research . furthermore , the results of study by keefe et al .
( 2001 ) showed that individuals who reported frequent daily spiritual experiences had higher levels of positive mood , lower levels of daily negative mood , and higher levels of all of the social support domains .
another subcategory that emerged from participants experience was going to mosque and religious ceremonies . in this regard , palinkas ( 1982 ) found that religious ceremonies are supportive for individuals because liberating and curative friendships relevant to spiritual states brings about psychological health for them . attending religious ceremonies and shrines also reduces individuals anxieties and sense of loneliness .
these results are in accordance with our study findings which show that elderly women who participate in religious ceremonies , are relieved from loneliness . also koenig ( 1998 )
suggested that religious practices , attitudes and coping behaviors are prevalent among hospitalized medically ill older adults and are related to social , psychological and physical health outcomes .
morris ( 1983 ) examined the relationship between anxiety and pilgrimage and found that people with religious devotion experienced significantly lower rates of anxiety disorder compared to the non - religious group .
every muslim learns their prayers from a young age and prays five times a day .
in addition , those who seek guidance from the holy quran and believe that it has power over every aspect of their lives , expect and welcome any eventualities in their lives ( quran , chapter balad , verse 4 ) .
it seems that elderly people have more tendencies to religious and spiritual subjects than young people .
koenig et al . ( 2004 ) in his study showed spirituality and religion as an important factor in their lives more often than younger ones .
several experimental studies have been conducted that showed positive effects of prayer on the management of physical symptoms of illness .
kwilecki ( 1986 ) assessed the effect of prayer on stress and anxiety and found that 42% of participants report that saying prayer can diminish the stress and anxiety . also salehi ( 2001 ) found that individuals who pray regularly , experienced significantly lower rates of anxiety disorder and depression , psychological balance and hopefulness , compared to the non - prayer group .
azizi ( 1996 ) found that more than 90% of prayers attained psychological tranquility and relaxation after saying prayer .
several studies noted moderately positive correlations between indicators of physical health and prayer activities . also several studies have shown that prayer can play an important role in the recovery of patients who suffer from cardiac disease , hiv , arthritis rheumatoid , and cva attack , cancer cases , treatment of addiction , and physical , mental and social well - being .
furthermore , evidence suggests that patients with strong religious beliefs and high levels of religious activity experience lower levels of pain , have better immune function , lower death rates from cancer , fewer incidences of heart disease , lower blood pressure and levels of cholesterol , better health behaviors , and greater compliance with medical treatment .
the practical relevance of this study may conduct future work to further explicate and clarify the linkages between spirituality and physical and mental health and life style .
future empirical research and new methods of investigation such as concept analysis , grounded theory , and action research are needed to develop the new models of caring for management of this group . in the life style area ,
future research should focus on the effects of spirituality on elderly people lifestyle domains and on the development of valid and reliable instruments to measure these effects .
it is important to conduct such research in different samples , such as unhealthy elderly people ( frail , ill , and sick elderly people ) .
future empirical research and new methods of investigation such as concept analysis , grounded theory , and action research are needed to develop the new models of caring for management of this group . in the life style area ,
future research should focus on the effects of spirituality on elderly people lifestyle domains and on the development of valid and reliable instruments to measure these effects .
it is important to conduct such research in different samples , such as unhealthy elderly people ( frail , ill , and sick elderly people ) .
in summary , we found that elderly people describe several elements in their illustration of spirituality in healthcare settings .
spiritual health , spiritual beliefs , and religious practice intentionality were the main components of concept of spirituality in this group experiences .
also , the findings of this study made it clear that spirituality has a considerable effect on the health and life of iranian elderly people and is a major supportive resource for their physical and psychosocial health .
it can reduce mental distress and induce inner peace and hopefulness . because of the significant influence of spirituality on all domains of health of elderly people , it is critically important that health care providers understand how spirituality can considerably influence elderly people throughout their life .
these findings will assist health professionals such as nurses , physicians , and social workers to recognize the spiritual needs and value the role of spirituality in promoting health and well - being among elderly people from different religions and cultures worldwide .
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background : spirituality is recognized as a personally important matter to the elderly , and there are evidences of its impact on their health .
the aim of this study was to explore the concept of spirituality from the perspectives of iranian healthy elderly individuals.materials and methods : a conventional qualitative content analysis of carried out with 21 healthy elderly people from both male and female genders were chosen using a purposive sampling method in tehran in 2010 - 2011 .
data collection was done through semi structured interviews .
a qualitative content analysis was used to analyze the participants experiences and perceptions on spirituality , using a central question what characterizes the spirituality in the iranian healthy elderly people?results : the following categories emerged from the data analysis : ( 1 ) spiritual health , with four sub categories including saying prayer as a calming factor ; beneficence as a way to god ; loss of psychological and spiritual support ; faith as a way to happiness ; ( 2 ) spiritual beliefs , with three sub categories including seeking help from god in difficulties ; god 's power over life and death ; doing good deeds is the god 's will ; and ( 3 ) religious practice with three sub categories including saying prayer ; reading quran ; and going to mosque , religious ceremonies and pilgrimage.conclusions:in this study was found that spirituality was a fundamental element in elderly individuals lives that help them to adapt with daily living conditions .
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Design
Selection
Data collection
Analysis
Data trustworthiness
Ethical considerations
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Spiritual health
Spiritual beliefs
Religious practice
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Suggestions
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according to the national regulations regarding diagnostic criteria of brain death ( bd ) , instrumental confirmatory tests should be used in certain clinical situations , such as intoxications , infratentorial processes , extensive facial damage , children younger than 2 years of age , or any case in which clinical examinations are inadequate .
electrophysiological tests are often unavailable due to limited access to proper equipment and competent specialists . therefore ,
two of them , catheter angiography and computed tomographic angiography ( cta ) , are based on the detection of contrast enhancement of cerebral vessels . in these examinations ,
first is a non - filling phenomenon , which means a complete absence of contrast above the carotid siphons and foramen magnum .
second is a delayed , weak , and persistent opacification of proximal segments of the cerebral arteries not reaching cortical branches .
stasis filling causes a significant problem in interpretation of cta results in the diagnosis of bd . since 1998 ,
when cta was proposed as the new imaging technique in the diagnostics of bd , a consensus on its interpretation criteria has not been reached .
an analysis of stasis filling in a dynamic series of computed tomographic perfusion ( ctp ) can provide valuable information on the interpretation of cta results and relation of this phenomenon to brain perfusion .
the aim of this prospective study was to characterize stasis filling phenomenon in the diagnosis of bd . to achieve this
, we performed a dynamic evaluation of contrast enhancement of the cerebral and extracranial arteries in patients with bd and control subjects with the use of the 40-s series of ctp scans .
we also confronted stasis filling pattern with ctp findings to assess the relevance of this phenomenon to brain perfusion .
we examined 67 patients who fulfilled clinical bd criteria according to the national regulations ( coma , apnea , and no brainstem reflexes ) . the examination protocol consisted of cta , followed by ctp and catheter angiography .
thirty - seven subjects were excluded due to lack of contrast enhancement in the intracranial arteries in ctp series .
finally , 30 ( 44.8 % ) patients , in whom analysis of ctp series enabled us to identify contrast - enhanced intracranial arteries , were enrolled in the study .
the population consisted of 18 men and 12 women with a median age of 54.5 years ( range , 2284 years ) .
initial causes of coma were intracerebral hemorrhage ( n = 16 ) , subarachnoid hemorrhage ( n = 11 ) , cerebral edema ( n = 2 ) , and ischemic stroke ( n = 1 ) .
decompressive craniectomy was performed in 19 ( 63 % ) included patients . among 37 excluded patients ,
all patients were managed in the intensive care units of two university hospitals and one multi - profile provincial hospital . during all the examinations ,
the patients were normoventilated and mean arterial blood pressure ( mabp ) maintained at greater than 80 mmhg .
the elapsed time between the onset of bd symptoms and the radiological examination ranged between 6 and 48 h. delay was sometimes caused by a prolonged initial observation due to prior use of sedatives and sometimes the limited availability of an angiographic team . the majority of patients became actual organ donors .
in some cases , organ donation was not possible for medical reasons and occasionally because of a failure to obtain permission from relatives .
in such situations , families were informed about the termination of futile therapy , and ventilators were legally switched off .
the demographic and epidemiological characteristics of bd patients are presented in table 1.table 1demographic and clinical characteristics of bd patientsnumbersexage ( years)diagnosiscraniectomyexamined arterystasis filling in angiography1m56ichmca2m71sahmca+3m73ich+mca+4f49sah+aca5f29sahaca6m40isaca7f66ichmca8m84sah+mca9m53ichmca10f67ich+mca+11m63cemca12f58sah+mca13m34sah+aca14m34sah+mca+15m78ichmca+16m39ich+mca+17f38sah+mca18f74sahaca+19f75ichmca+20f40sah+mca+21f22ich+mca+22f34ich+mca23m50ich+aca+24m44sah+mca+25m51ich+pca+26m71ich+mca27f41ce+mca28m59ichmca29m62ich+mca30m56ich+mca+
is ischemic stroke , ce cerebral edema , aca anterior cerebral artery , mca middle cerebral artery , pca posterior cerebral artery demographic and clinical characteristics of bd patients
is ischemic stroke , ce cerebral edema , aca anterior cerebral artery , mca middle cerebral artery , pca posterior cerebral artery the control group consisted of 30 patients who underwent surgical clipping of an intracranial aneurysm .
the population consisted of 14 men and 16 women with a median age of 53 years ( range , 1865 years ) .
ctp was performed 810 days after neurosurgical clipping of the middle cerebral or anterior communicating artery aneurysms . during all the examinations , mabp stayed above 80 mmhg .
none of the control subjects presented any signs of bd , with normal pco2 and po2 .
all cta and aortocervical angiography examinations in bd patients were performed using the same methodology as described previously [ 4 , 5 ] .
we used siemens sensation 64 ( siemens ag , erlangen , germany ) to perform cta .
injection of 80 ml of iodinated contrast medium at a flow rate of 4 ml / s .
then cta was carried out in three phases , which were manually programmed with fixed delays of 25 , 40 , and 60 s after contrast injection .
siemens sensation 64 ( siemens ag , erlangen , germany)to perform ctp in both groups . in bd patients , the time interval between cta and ctp ranged from 1 to 15 min .
administration of 50 ml of the contrast medium at a flow rate of 5 ml / s , a series of scans were made at the level of the basal and thalamic nuclei above the basal cisterns .
detailed protocol is presented in table 2.table 2technical parameters of ctp examinationacquisition tube voltage ( kvp)80 tube current ( mas)270 rotation time ( s)1.0 collimation ( mm)24 1.2 scan range ( mm)28.8 cycle time ( s)1.0 scan time
( s)40 automatic dose modulationoffreconstruction slice width ( mm)3 9.6 fov ( mm)220 matrix512 512 recon algorithmh30scontrast injection volume ( ml)50 flow rate ( ml / s)5 technical parameters of ctp examination the dynamic series of ctp scans were analyzed in two different ways . in the first part of post - processing , two small circular 0.250.3-cm regions of interest ( rois )
the second roi covered the cerebral artery in the most distal visible segment ( see fig . 1 ) .
this artery was visible in all control examinations ( contralateral artery to the clipped aneurysm was always chosen ) and 23 examinations of bd patients . among the remaining seven examinations in six cases , the only opacified cerebral artery was aca and in one case pca ( see table 1 ) .
rois were automatically propagated over the entire series of scans . for each roi , time
calculation was performed using the osirix v.5.5.1 software ( pixmeo sarl , bernex , switzerland).fig .
1axial ctp scans in mip reconstruction from 40-s series in a bd patient ( a ) and in a control subject ( b ) .
rois are positioned in the distal segment of mca ( arrowhead ) and in the superficial temporal artery ( arrow ) axial ctp scans in mip reconstruction from 40-s series in a bd patient ( a ) and in a control subject ( b ) .
rois are positioned in the distal segment of mca ( arrowhead ) and in the superficial temporal artery ( arrow ) in bd patients , the same dynamic series of scans were used for measurements of cerebral blood flow ( cbf ) and cerebral blood volume ( cbv ) with the multimodality workplace and syngo ve40a software package ( siemens ag , erlangen , germany ) .
this software is based on a maximum slope method , which assumes that there is no venous outflow from the tissue volume under consideration during the time of observation .
therefore , the lack of venous outflow in bd patients was not an obstacle , and calculation of cbf and cbv was feasible in all cases .
we chose the same cerebral arteries , which were used in the first part of post - processing for calculation of tdcs .
cbf and cbv values were measured in circular 2.53.5-cm rois placed in the cortical regions of the frontal , temporal , occipital lobes , and basal nuclei of both hemispheres with exclusion of major blood vessels .
angiography was performed with a delay of 15 min to 3 h after ctp .
we used two angiographic systems : fluorospot top and axiom artis ( siemens ag , germany ) .
after positioning a pigtail 45-f catheter in the ascending aorta , 30 ml of contrast was injected at a flow rate of 15 ml / s .
we registered 3050 s series with a frequency of 2 f / s visualizing head and neck area with the use of a digital subtraction technique . according to the national guidelines ,
cerebral circulatory arrest was diagnosed in two situations : non - filling of intracranial vessels with preserved flow in the external carotid arteriesstasis filling delayed , weak , and persistent opacification of the proximal cerebral arterial segments , without opacification of the cortical branches or venous outflow non - filling of intracranial vessels with preserved flow in the external carotid arteries stasis filling delayed , weak , and persistent opacification of the proximal cerebral arterial segments , without opacification of the cortical branches or venous outflow demographic data recorded for bd patients and control subjects were age and sex .
in addition , for bd patients , we registered initial cause of coma and the presence of craniectomy . for each extracranial and cerebral roi in the ctp series
, the following parameters were measured : baseline density : ct density on the first scanenhancement at 20 , 30 , and 40 s : difference between density at the time point of 20 , 30 , and 40 s and baseline densitypeak enhancement : difference between the highest density and baseline densityc / e peak ratio : ratio of peak enhancement in a cerebral artery to peak enhancement in the superficial temporal arterytime to peak : period of time in seconds from the time when the first image was acquired to the time when the highest density was reacheddelay of cerebral peak : period of time in seconds from peak enhancement in the superficial temporal artery to the peak enhancement in the cerebral artery baseline density : ct density on the first scan enhancement at 20 , 30 , and 40 s : difference between density at the time point of 20 , 30 , and 40 s and baseline density peak enhancement : difference between the highest density and baseline density c / e peak ratio : ratio of peak enhancement in a cerebral artery to peak enhancement in the superficial temporal artery time to peak : period of time in seconds from the time when the first image was acquired to the time when the highest density was reached delay of cerebral peak : period of time in seconds from peak enhancement in the superficial temporal artery to the peak enhancement in the cerebral artery in bd patients , the additional recorded data were values of cbf , cbv , and results of catheter angiography classified as non - filling or stasis filling .
whitney test was used for the analysis of differences between the groups because the distributions of most of the quantitative variables were significantly different from the normal distribution ( p < 0.05 , shapiro
all cta and aortocervical angiography examinations in bd patients were performed using the same methodology as described previously [ 4 , 5 ] .
we used siemens sensation 64 ( siemens ag , erlangen , germany ) to perform cta .
injection of 80 ml of iodinated contrast medium at a flow rate of 4 ml / s .
then cta was carried out in three phases , which were manually programmed with fixed delays of 25 , 40 , and 60 s after contrast injection .
we used the same scanner siemens sensation 64 ( siemens ag , erlangen , germany)to perform ctp in both groups . in bd patients , the time interval between cta and ctp ranged from 1 to 15 min .
administration of 50 ml of the contrast medium at a flow rate of 5 ml / s , a series of scans were made at the level of the basal and thalamic nuclei above the basal cisterns .
detailed protocol is presented in table 2.table 2technical parameters of ctp examinationacquisition tube voltage ( kvp)80 tube current ( mas)270 rotation time ( s)1.0 collimation ( mm)24 1.2 scan range ( mm)28.8 cycle time ( s)1.0 scan time ( s)40 automatic dose modulationoffreconstruction slice width ( mm)3 9.6 fov ( mm)220 matrix512 512 recon algorithmh30scontrast injection volume ( ml)50 flow rate ( ml / s)5 technical parameters of ctp examination the dynamic series of ctp scans were analyzed in two different ways . in the first part of post - processing ,
two small circular 0.250.3-cm regions of interest ( rois ) were positioned in each ctp series of scans .
the second roi covered the cerebral artery in the most distal visible segment ( see fig . 1 ) .
this artery was visible in all control examinations ( contralateral artery to the clipped aneurysm was always chosen ) and 23 examinations of bd patients . among the remaining seven examinations in six cases , the only opacified cerebral artery was aca and in one case pca ( see table 1 ) .
rois were automatically propagated over the entire series of scans . for each roi , time density curve ( tdc ) was plotted .
calculation was performed using the osirix v.5.5.1 software ( pixmeo sarl , bernex , switzerland).fig .
1axial ctp scans in mip reconstruction from 40-s series in a bd patient ( a ) and in a control subject ( b ) .
rois are positioned in the distal segment of mca ( arrowhead ) and in the superficial temporal artery ( arrow ) axial ctp scans in mip reconstruction from 40-s series in a bd patient ( a ) and in a control subject ( b ) .
rois are positioned in the distal segment of mca ( arrowhead ) and in the superficial temporal artery ( arrow ) in bd patients , the same dynamic series of scans were used for measurements of cerebral blood flow ( cbf ) and cerebral blood volume ( cbv ) with the multimodality workplace and syngo ve40a software package ( siemens ag , erlangen , germany ) .
this software is based on a maximum slope method , which assumes that there is no venous outflow from the tissue volume under consideration during the time of observation .
therefore , the lack of venous outflow in bd patients was not an obstacle , and calculation of cbf and cbv was feasible in all cases .
we chose the same cerebral arteries , which were used in the first part of post - processing for calculation of tdcs .
cbf and cbv values were measured in circular 2.53.5-cm rois placed in the cortical regions of the frontal , temporal , occipital lobes , and basal nuclei of both hemispheres with exclusion of major blood vessels .
angiography was performed with a delay of 15 min to 3 h after ctp .
we used two angiographic systems : fluorospot top and axiom artis ( siemens ag , germany ) .
a typical femoral approach was used in all cases . after positioning a pigtail 45-f catheter in the ascending aorta ,
30 ml of contrast was injected at a flow rate of 15 ml / s .
we registered 3050 s series with a frequency of 2 f / s visualizing head and neck area with the use of a digital subtraction technique . according to the national guidelines ,
cerebral circulatory arrest was diagnosed in two situations : non - filling of intracranial vessels with preserved flow in the external carotid arteriesstasis filling delayed , weak , and persistent opacification of the proximal cerebral arterial segments , without opacification of the cortical branches or venous outflow non - filling of intracranial vessels with preserved flow in the external carotid arteries stasis filling delayed , weak , and persistent opacification of the proximal cerebral arterial segments , without opacification of the cortical branches or venous outflow
in addition , for bd patients , we registered initial cause of coma and the presence of craniectomy . for each extracranial and cerebral roi in the ctp series
, the following parameters were measured : baseline density : ct density on the first scanenhancement at 20 , 30 , and 40 s : difference between density at the time point of 20 , 30 , and 40 s and baseline densitypeak enhancement : difference between the highest density and baseline densityc / e peak ratio : ratio of peak enhancement in a cerebral artery to peak enhancement in the superficial temporal arterytime to peak : period of time in seconds from the time when the first image was acquired to the time when the highest density was reacheddelay of cerebral peak : period of time in seconds from peak enhancement in the superficial temporal artery to the peak enhancement in the cerebral artery baseline density : ct density on the first scan enhancement at 20 , 30 , and 40 s : difference between density at the time point of 20 , 30 , and 40 s and baseline density peak enhancement : difference between the highest density and baseline density c / e peak ratio : ratio of peak enhancement in a cerebral artery to peak enhancement in the superficial temporal artery time to peak : period of time in seconds from the time when the first image was acquired to the time when the highest density was reached delay of cerebral peak : period of time in seconds from peak enhancement in the superficial temporal artery to the peak enhancement in the cerebral artery in bd patients , the additional recorded data were values of cbf , cbv , and results of catheter angiography classified as non - filling or stasis filling .
whitney test was used for the analysis of differences between the groups because the distributions of most of the quantitative variables were significantly different from the normal distribution ( p < 0.05 , shapiro
for cerebral arteries , tdcs in bd patients represented flat curves in contrast to tdcs in the control group , which formed steep and narrow gaussian curves ( see fig . 2 ) .
baseline density for cerebral and extracranial arteries was significantly higher in bd patients in comparison to the control group ( 60.5 vs. 41.5 hu ( hounsfield unit ) ;
we found significantly longer time to peak enhancement in cerebral arteries in bd patients than in controls ( median , 32 vs. 21 s ; p <
in bd patients , peak enhancement in cerebral arteries occurred with a median delay of 14.5 s to peak in extracranial arteries while practically no delay was noted in controls ( it occurred 1 s earlier , which is represented as median 1 s value ; p <
cerebral arteries in bd patients showed significantly lower peak enhancement in comparison to the control group ( 34.5 vs. 81.5 hu ; p < 0.0001 ) ( see fig .
density curves in a bd patient ( a ) and in a control subject ( b ) .
3distribution of baseline density values in bd patients and controls in cerebral and extracranial arteriesfig .
4distribution of time to peak in cerebral and extracranial arteries in bd patients and controlsfig .
6distribution of peak enhancement in cerebral and extracranial arteries in bd patients and controls time
density curves in a bd patient ( a ) and in a control subject ( b ) .
time to peak ( ttp ) enhancement distribution of baseline density values in bd patients and controls in cerebral and extracranial arteries distribution of time to peak in cerebral and extracranial arteries in bd patients and controls distribution of delay of cerebral peak values in bd patients and controls distribution of peak enhancement in cerebral and extracranial arteries in bd patients and controls for extracranial arteries , tdcs represented a similar shape in both groups .
however , we observed higher enhancement in bd patients at 20 , 30 , and 40 s ( see table 3 ) .
peak enhancement was significantly higher and earlier in bd patients compared to controls ( p = 0.0087 and p < 0.0001 , respectively ) ( see figs . 4 and 6 ) .
the intensity of peak enhancement in cerebral arteries in relation to extracranial arteries expressed as c / e peak ratio appeared to be significantly lower in bd patients in comparison to the control group ( 0.31 vs. 0.87 ; p < 0.0001 ) ( see fig .
7).table 3variables calculated from dynamic ctp series in bd patients vs. controlsparameterbd ( n = 30)controls ( n = 30 )
p valueenhancement at 20 s ( hu ) cerebral artery7.5 ( 24)41 ( 25)<0.0001 extracranial artery53.5 ( 40)41.5 ( 30)nsenhancement at 30 s ( hu ) cerebral artery18.5 ( 14)25 ( 19)ns extracranial artery36 ( 23)26 ( 20)0.0225enhancement at 40 s ( hu ) cerebral artery20 ( 15)16.5 ( 8)ns extracranial artery36.5 ( 18)17 ( 10)<0.0001values expressed as median ( interquartile range )
ns not significantfig
. 7distribution of cerebral / extracranial peak ratio values in bd patients and controls variables calculated from dynamic ctp series in bd patients vs. controls values expressed as median ( interquartile range ) distribution of cerebral / extracranial peak ratio values in bd patients and controls analyzing the influence of demographic and clinical features on dynamic ct parameters , we revealed significant differences in time to peak and delay of peak enhancement in cerebral arteries between subgroups of bd patients with and without craniectomy . in the subgroup with craniectomy , time to peak and delay of cerebral peak were shorter in comparison to the subgroup without craniectomy ( see table 4 ) .
a statistically insignificant trend was found in the analysis of tdcs in bd patients with subarachnoid hemorrhage ( sah ) and intracerebral hemorrhage ( ich ) .
we revealed a tendency to longer time to peak and longer delay of cerebral peak in the group with sah compared to the group with ich ( see table 5 ) .
we did not observe any significant differences between ct parameters in relation to sex or examined artery.table 4variables calculated from dynamic ctp series in bd patients without vs. with craniectomyparameterbd patients without craniectomy ( n = 11)bd patients with craniectomy ( n = 19 )
p valuetime to peak in cerebral artery ( s)34 ( 6)26 ( 9)0.007delay of cerebral peak ( s)18 ( 8)13 ( 9)0.04values expressed as median ( interquartile range)table 5variables calculated from dynamic ctp series in bd patients with intracerebral vs. subarachnoid hemorrhageparameterbd patients with ich ( n = 16)bd patients with sah ( n = 11 )
p valuetime to peak in cerebral artery ( s)27.5 ( 10)33 ( 9)0.34 ( ns)delay of cerebral peak ( s)13.5 ( 8)18 ( 13)0.16 ( ns)values expressed as median ( interquartile range )
ns not significant variables calculated from dynamic ctp series in bd patients without vs. with craniectomy values expressed as median ( interquartile range ) variables calculated from dynamic ctp series in bd patients with intracerebral vs. subarachnoid hemorrhage values expressed as median ( interquartile range ) in all 30 bd patients , ctp revealed zero values of cbf and cbv in all rois .
stasis filling pattern was observed in 14 ( 46.7 % ) and non - filling in 16 ( 53.3 % ) cases . in 37
excluded bd patients , angiography revealed a non - filling pattern in all cases ; no stasis filling was noted .
the term stasis filling for describing a delayed , weak , and persistent intracranial opacification in patients with bd was used for the first time by kricheff et al . in 1978 .
munari et al . reported stasis filling in 5 % ( one out of 20 ) , braun et al . in 11 % ( 15/140 ) , bradac et al . in 12.5 % ( two out of 16 ) , kricheff et al . in 15 % ( three out of 20 ) , and savard et al . in 28 % ( nine out of 32 ) of cases [ 2 , 69 ] .
noted stasis filling in 43 % ( six out of 14 ) and welschehold et al . in 59 % ( 37/63 ) of cases [ 3 , 10 ] .
stasis filling , as specific angiographic pattern of cerebral circulatory arrest , is a consequence of two major factors : raised intracranial pressure ( icp ) and high cerebrovascular resistance ( cvr ) .
they both cause reduction of cbf , according to poiseuille s law : cbf = cpp / cvr = ( mabp icp)/cvr , where cpp stands for cerebral perfusion pressure . increased cvr in extreme cerebral hemodynamic disturbances
was revealed in studies using a transcranial doppler ( tcd ) [ 11 , 12 ] .
this is caused mainly by altered cerebral autoregulation mechanisms , which are sufficient to preserve almost constant cbf when cpp is above 50 mmhg .
cessation of capillary circulation is consistent with cerebral circulatory arrest while proximal arterial segments are still patent . at this stage
, tcd frequently shows to and fro or narrow systolic spike patterns , which reflect blood movement [ 11 , 12 ] . in such circumstances ,
heartbeat driven slow propagation of contrast column in proximal cerebral arteries is possible . in the present study ,
recently revealed a potential efficacy of ctp for assessing brain death . in this material ,
ctp detected complete absence of brain perfusion reflected by zero values of cbf and cbv in all cases .
these findings clearly show that stasis filling is not an indicator of any residual brain perfusion but only ineffective propagation of contrast in cerebral vessels ; thus , it does not preclude diagnosis of brain death .
ctp results are complementary to the recent report of selcuk et al . , which revealed global reduction of adc consistent with necrosis of neurons using diffusion - weighted mri technique .
the findings showed two features of stasis filling phenomenon : delay and weakness of intracranial opacification .
tdcs for cerebral arteries in bd patients were characterized by significantly longer times to peak ( median , 32 s ) compared to controls ( 21 s ) . moreover
, intracranial peak enhancement occurred with a median delay of 14.5 s to extracranial peak .
this is consistent with observations in cta studies , in which intracranial filling in bd patients is usually detected in the late phase ( performed 60 s after contrast injection ) and very rarely in the early phase of scanning [ 3 , 10 , 17 ] .
correlation with ctp results shows that such delayed vascular opacification does not provide any brain perfusion , thus can not preclude diagnosis of brain death .
typical cerebral tdc in bd patients was a flat curve with a low peak ( median value of 34.5 hu ) ; about three times lower compared to the peak in the extracranial artery .
in contrast , extra- and intracranial tdcs in the control group represented steep and narrow gaussian curves , and both peaks were of similar height .
such low peak values could be the reason of lower detectability of stasis filling by catheter angiography compared to ct ( 14/67 = 21 % vs. 30/67 = 45 % cases ) observed in the present study .
these findings are consistent with results of others as was presented in the first paragraph of discussion .
also observed underestimation of intracranial filling in catheter angiography compared to cta in four out of seven cases .
although , a larger amount of contrast was used in dynamic ct than in catheter angiography ( 50 vs. 30 ml ) , but in angiography , it was injected intra - arterially , which minimizes the effect of dilution .
this discrepancy could be a result of advanced reconstruction algorithms , systems of noise reduction , and signal amplification used in ct scanners , which reduce noise , improve spatial resolution , and low contrast detectability
. the other explanation could be the time sequence in performing ctp and catheter angiography .
angiography was carried out with a delay from 15 min to 3 h after ctp . during this period
, slow rising icp could stop the propagation of contrast at the level of skull base . analyzing the influence of demographic and clinical features on tdc s parameters
, we found a statistically significant trend towards shorter time to peak and shorter delay of peak in the cerebral arteries of bd patients with craniectomy compared to those without it .
moreover , the incidence of craniectomy was much lower in bd patients excluded from the study due to lack of intracranial opacification ( four out of 37 = 11 % ) compared to included patients who presented intracranial filling ( 19/30 = 63 % ) .
several authors previously reported a relationship between skull defect and preserved intracranial filling in bd patients [ 7 , 18 , 19 ] .
this can be explained by a decreased icp enabling propagation of contrast in the cerebral vessels .
a statistically insignificant trend was found in the analysis of tdcs in bd patients with sah and intracerebral hemorrhage .
we revealed a tendency to longer time to peak and longer delay of cerebral peak in the group with sah compared to the group with ich .
this difference was probably caused by vasospasm after sah , which independently increases cvr and additionally contributes to slowing down the propagation of contrast .
the major drawback of this study is a consequence of performing ctp shortly after cta in bd patients .
the influence of residual contrast injected for cta was reflected by higher baseline density in the cerebral and extracranial arteries , higher and earlier peak enhancement , and shorter time to peak in the extracranial vessels in bd patients compared to controls .
however , this contrast contamination should not significantly change values of delay of cerebral peak or c / e peak ratio as they were calculated on the basis of both cerebral and extracranial tcds .
in this study , we assessed the characteristic features of intracranial filling in bd patients delay and weakness of cerebrovascular opacification .
it led to the conclusion that delayed and weak opacification of cerebral arteries do not necessarily mean the presence of cerebral perfusion , thus does not preclude diagnosis of bd .
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introductionstasis filling , defined as delayed , weak , and persistent opacification of proximal segments of the cerebral arteries , is frequently found in brain dead patients .
this phenomenon causes a major problem in the development of reliable computed tomographic angiography ( cta ) protocol in the diagnosis of brain death ( bd ) .
the aim of our study was to characterize stasis filling in the diagnosis of bd . to achieve this
, we performed a dynamic evaluation of contrast enhancement of the cerebral and extracranial arteries in patients with bd and controls.methodsstudy population included 30 bd patients , who showed stasis filling in computed tomographic perfusion ( ctp ) series .
thirty patients , after clipping of an intracranial aneurysm , constituted the control group .
the study protocol consisted of cta , ctp , and angiography .
time
density curves ( tdcs ) of cerebral and extracranial arteries were generated using 40-s series of ctp.resultscerebral tdcs in bd patients represented flat curves in contrast to tdcs in controls , which formed steep and narrow gaussian curves .
we found longer time to peak enhancement in bd patients than in controls ( 32 vs. 21 s ; p < 0.0001 ) . in bd patients , peak enhancement in the cerebral arteries occurred with a median delay of 14.5 s to peak in extracranial arteries , while no delay was noted in controls ( p < 0.0001 ) .
cerebral arteries in bd patients showed lower peak enhancement than controls ( 34.5 vs. 81.5 hu ; p < 0.0001 ) . in all bd patients
, ctp revealed zero values of cerebral blood flow and volume .
angiography showed stasis filling in 14 ( 46.7 % ) and non - filling in 16 ( 53.3 % ) cases.conclusiona confrontation of stasis filling with ctp results showed that stasis filling is not consistent with preserved cerebral perfusion , thus does not preclude diagnosis of bd .
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Background
Material and methods
CTA
CTP
Catheter angiography
Data collection
Statistical analysis
Results
Discussion
Limitations
Conclusions
Conflict of interest
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the skin is the largest organ of the body and protects the organism against external physical , chemical , and biological insults such as wounding , uvb radiation , and microorganisms .
this major barrier resides in the upper layers of the epidermis ( for review see segre , 2006 ) . the epidermis is the upper part of the skin that is continuously renewed .
the basal layer , or stratum basale , of the epidermis contains proliferating keratinocytes ( fig .
1 ) . upon withdrawal from the cell cycle , these basal keratinocytes detach from the basement membrane and undergo a terminal differentiation program to become corneocytes in the outer layers of the epidermis .
the cells reinforce their cytoskeletal keratin filament network , and adjacent cells interact via many desmosomes , a specialized type of cell junction , to resist physical trauma . in the stratum granulosum ,
the keratinocytes become more flattened and express certain proteins such as profilaggrin and loricrin , which aggregate to form the typical keratohyalin granules of the stratum granulosum . in addition , lipids are produced and stored in lamellar bodies . at the final stage of differentiation , the keratinocytes lose their organelles , including the nucleus , and become the dead , flattened corneocytes of the stratum corneum . during cornification , proteins are cross - linked at the inner side of the cytoplasmic membrane to form a cornified envelope ( for review see candi et al . , 2005 ) . in the transitional layer between the stratum granulosum and the stratum corneum , lipids are extruded to form a water - repelling envelope around the cornified envelope , thereby assuring an adequate permeability barrier function of the mammalian epidermis .
improper formation of these envelopes results in an impaired epidermal barrier that can not protect against dehydration , uvb , and infection .
the signaling cascades involved in epidermal barrier formation are largely unknown , but the many proteases that seem to be involved are currently being intensively studied .
see introduction for details . since the cloning of caspase-14 in the late nineties , it has become clear that this protease is a unique member of the caspase family .
unlike apoptotic caspases , which evolved in common ancestors such as hydra , echinodermata , insects , nematodes , and chordates , caspase-14 has so far been found only in terrestrial mammals ( lamkanfi et al . , 2002 ) .
in contrast to the ubiquitously expressed other members of the caspase family , caspase-14 is expressed and activated mainly in the epidermis and is absent from most other adult tissues ( eckhart et al .
recently , caspase-14 was found to be involved in epidermal barrier formation ( denecker et al . , 2007 ) .
in this review , we discuss current knowledge of the expression , regulation , and function of caspase-14 .
the expression pattern of caspase-14 is unique among the caspases , as it is present mainly in cornifying epithelia , such as the epidermis , the hassall 's bodies of the thymus , and the forestomach of rodents ( lippens et al . , 2000 , 2005 ;
in skin , caspase-14 is expressed only in the differentiating and cornifying layers of the epidermis and the hair follicle ( lippens et al . , 2000 ;
this is consistent with the observation that , in vitro , caspase-14 is only expressed in differentiating but not in proliferating keratinocytes ( lippens et al .
remarkably , nail matrix keratinocytes that differentiate into specialized nail corneocytes , the building blocks of the nail plate , do not express caspase-14 ( jager et al .
in addition , caspase-14 is not expressed in the noncornifying keratinocytes of the sweat gland or the mouth epithelium ( lippens et al . , 2000 ;
ultrastructural analysis demonstrated that spatial distribution of caspase-14 in the epidermis and hair follicles is strongly conserved among several mammalian species ( alibardi et al .
caspase-14 was found to be associated with the nucleus , the keratohyalin granules , and the desmosomes , whereas in corneocytes , caspase-14 was found in the cytoplasm and was associated with corneodesmosomes ( a modified version of desmosomes ) and nuclear remnants .
these observations suggested a role for caspase-14 in nuclear degradation during cornification , but nuclear degradation was not affected in caspase-14deficient mice ( denecker et al . , 2007 ) .
the expression of caspase-14 in the hassall 's bodies of the thymus and in the forestomach of rodents is somewhat expected , as they are cornifying structures and express the typical late differentiation markers , such as profilaggrin and loricrin , which are also found in the epidermis ( laster and haynes , 1986 ; favre , 1989 ; jarnik et al . , 1996 ) .
protein expression of caspase-14 has also been reported in several noncornifying tissues ( lippens et al . , 2003 ; krajewska et al . , 2004 , 2005 ; kam et al . , 2005
however , these observations should be interpreted carefully , as we have recently shown that the reported expression of caspase-14 in such tissues can be the result of aspecific staining ( denecker et al . , 2007 ) .
remarkably , so far caspase-14 has been found only in terrestrial mammals but not in birds or reptiles .
whereas birds and reptiles have a stiff , dry , scaly epidermis , mammals have a soft stratum corneum because of the larger amounts of histidine - rich late differentiation markers ( e.g. , profilaggrin ; alibardi , 2003 ) .
interestingly , profilaggrin is a direct substrate of caspase-14 ( denecker et al . , 2007 ) .
this could indicate that the occurrence of a soft stratum corneum and the caspase-14 gene are associated during evolution .
although the expression of caspase-14 is very restricted , little is known about the transcriptional regulation of its gene . in vitro
, caspase-14 is only expressed when keratinocytes are forced to differentiate by growing them postconfluently or in suspension or by adding vitamin d3 ( eckhart et al .
in contrast , adding ca at high concentrations to the medium , a method frequently used to induce differentiation , did not induce caspase-14 expression ( eckhart et al .
retinoids , which suppress keratinocyte differentiation , down - regulate caspase-14 expression ( rendl et al . ,
these results indicate that transcription factors that are specifically active during terminal differentiation are required to regulate caspase-14 expression . whether caspase-14 expression levels can be regulated at the posttranscriptional level is not known .
down - regulation of several differentiation - associated genes by retinoids has been shown to be mediated by the transrepression of activator protein 1 ( ap-1)mediated gene activation ( fisher and voorhees , 1996 ) .
indeed , the caspase-14 promoter contains at least two potential ap-1binding sites ( unpublished data ) .
the green tea phenol ( )-epigallocatechin-3-gallate ( egcg ) is a potent activator of ap-1 and has been shown to up - regulate caspase-14 in a p38- and jnk - dependent way ( hsu et al . , 2005 , 2007 ) .
ap-1 alone is probably not sufficient to drive caspase-14 expression because tnf and 12-o - tetradecanoyl - phorbol 13-acetate , two potent activators of ap-1 in keratinocytes ( arnott et al . , 2002 )
, did not induce caspase-14 expression in keratinocytes ( lippens et al . , 2004 ) .
differentiation - dependent expression of caspase-14 in keratinocytes could also result from a strong transcriptional repression in proliferating keratinocytes .
this possibility is supported by the observation that mice deficient in nuclear receptor corepressor hairless ( hr ) had 510-fold higher levels of caspase-14 and profilaggrin mrna , starting from postnatal day 6 and progressing during development ( zarach et al . , 2004 ) .
these alterations in gene expression were detected mainly in the keratinocytes of the utricle , an abnormal pouch - like structure at the upper part of the hair follicle . increased
gene expression occurred before the morphologically distinct utricle could be identified , indicating that the up - regulation was probably a cause rather than a consequence of utricle formation
. it would be interesting to analyze both caspase-14 activation and filaggrin processing in these mice .
multiple mutant hr alleles in mice and in humans show phenotypic variations that include congenital hair loss , skin wrinkling , and papular rash ( cichon et al . , 1998 ;
1998 ) . whether caspase-14 overexpression is important for the observed phenotypes could be addressed by generating epidermis - specific caspase-14 transgenic mice or by crossing the hr mice with caspase-14deficient mice .
procaspases consist of a prodomain , a large subunit ( p20 ) , and a small subunit ( p10 ) .
activation of caspases is induced by dimerization , ( auto-)proteolytic cleavage at asp residues , and/or conformational changes ( lamkanfi et al . , 2003 ) .
so far , maturation of caspase-14 by proteolytic cleavage into p20 and p10 subunits has been consistently observed only in cornifying epithelia such as the epidermis and the rodent forestomach ( lippens et al . , 2000 ;
although some investigators suggested that caspase-8 and -10 can activate caspase-14 in vitro ( ahmad et al .
, 1998 ; van de craen et al . , 1998 ) , this could not be confirmed by others ( lippens et al .
furthermore , caspase-14 is probably not proteolytically activated by a caspase in vivo , as other caspases are not activated during epidermal differentiation ( eckhart et al .
2000 ; raymond et al . , 2007 ) , and caspase-14 is not processed at an aspartate residue like other caspases but is processed at ile in man and presumably at leu in the mouse ( chien et al . , 2002 ) .
alignment of the protease - sensitive loop between the p20 and p10 subunits of the known mammalian procaspase-14 amino acid sequences ( fig .
2 ) reveals a conserved hydrophobic patch that is n terminal of the caspase-14 cleavage site .
this patch contains p1-preferred amino acids of elastase - like serine proteases , such as val , ala , leu , and ile ( mallory and travis , 1975 ; vered et al . , 1985 ; takahashi et al . ,
1989 ) , suggesting that a serine protease with elastase - like properties could be involved in caspase-14 activation ( unpublished data ) . all together ,
these data indicate that during skin homeostasis , the caspase-14activating protease is not a caspase , separating caspase-14 activation from the apoptotic and inflammatory caspase cascades that could be detrimental to epidermal integrity .
the precise epidermal layer in which caspase-14 is processed and activated is unknown because antiserum specifically recognizing activated caspase-14 is not yet available .
however , the following findings indicate that activation of caspase-14 occurs at the interface between the granular and cornified layers of the epidermis or early during cornification .
first , both the proform and activated form of caspase-14 can be found in total epidermal extracts , whereas in the cornified layer only activated caspase-14 is found ( fischer et al . , 2004 ) .
second , caspase-14 activation coincides with stratum corneum formation both during embryonic development and in organotypic skin cultures ( eckhart et al .
this implies that the main biological function of caspase-14 is exercised in the stratum corneum , as proven by the phenotype of the caspase-14deficient mice ( see the next section ) .
sequence analysis indicates that a hydrophobic patch in the protease - sensitive loop is conserved .
only part of the alignment is shown here , including the c - terminal part of the p20 subunit , the protease - sensitive loop , and the n - terminal part of the p10 subunit .
the darker the yellow , the more the amino acids are conserved between species . the catalytic qacrg box is delineated with a green box .
the conserved hydrophobic patch is delineated with a red box , and the cleavage site in human caspase-14 is indicated with a red arrow .
the alignment was performed using clustalw ( mega version 3.1 ; kumar et al . , 2004 ) and
identification of caspase-14 substrates has been hampered for a long time by the unavailability of enzymatically active caspase-14 .
however , it was recently shown that proteolytically processed caspase-14 requires high concentrations of kosmotropic salt to be active in vitro , such as sodium citrate , in addition to proteolytic cleavage between the p20 and p10 subunit ( mikolajczyk et al . , 2004 ) .
these salts induce both dimerization and ordering of active site loops by partial desolvation of the protein to a more compact , catalytically active protease .
the cellular environment of the stratum corneum of the epidermis probably favors caspase-14 activity in a similar way .
indeed , the water content decreases from 45% at the transitional layer to 1525% at the skin surface ( warner et al . , 1988 ; caspers et al . , 2001 ) .
human caspase-14 preferentially accommodates tryptophan or tyrosine in the s4 subsite , whereas mouse caspase-14 is more tolerant , with almost equal preferences for -branched and aromatic amino acids ( mikolajczyk et al . , 2004 ) .
for example , both human and mouse caspase-14 efficiently cleave the fluorescent peptide substrate wehd - amc , but only mouse caspase-14 cleaves ietd - amc as efficiently ( fischer et al . , 2004 ; mikolajczyk et al . ,
these substrate preferences would classify human caspase-14 as an inflammatory caspase and mouse caspase-14 as an inflammatory and apoptotic initiator caspase ( thornberry et al .
, 1997 ; thornberry , 1998 ) . however , human caspase-14 can not proteolytically activate the inflammatory cytokines pro
2004 ) , and there are no data supporting a direct role for caspase-14 in apoptosis ( lippens et al . , 2000 ; denecker et al . , 2007 ) .
whether the substrate preferences of human and murine caspase-14 observed in vitro on peptide substrates also occur in vivo is not clear .
importantly , profilaggrin , a major structural protein in the differentiating epidermis , has been shown to be a physiological substrate of caspase-14 ( denecker et al . , 2007 ) .
identification of additional substrates and determination of the cleavage sites will provide more insight into the preferred recognition sequence of caspase-14 in the context of a protein .
keratinocytes can die by two different processes : apoptotic cell death induced by damaging agents such as uvb , chemicals , and cytotoxic cytokines or by a continuous process of differentiation leading to the formation of corneocytes .
these processes are clearly distinct pathways executed by different players ( for review see lippens et al . , 2005 ) , but the role of caspases in these two cell death programs has long been controversial .
although almost all procaspases are constitutively expressed in the epidermis , only caspase-14 has consistently been shown to be activated during epidermal cornification ( eckhart et al .
, 2000b ; lippens et al . , 2000 ; raymond et al . , 2007 ) .
in addition , knockouts for apoptotic caspases were not reported to have a phenotypic skin anomaly except for caspase-3deficient mice , in which keratinocyte differentiation is delayed in the embryo but normalized at birth ( okuyama et al . , 2004 ) .
however , others could not confirm the activation of caspase-3 during embryonic epidermal development ( fischer et al .
although they are not activated during cornification , apoptotic caspases , in contrast to caspase-14 , become activated during uvb- , staurosporine- , tnf- , and tnf - related apoptosis - inducing ligand induced apoptosis of keratinocytes and , thereby , play a role in the apoptotic cell death of keratinocytes ( for review see lippens et al . , 2005 ) .
thus , we conclude that apoptotic caspases are not involved in the physiological keratinocyte cell death program leading to cornification .
furthermore , caspase-14deficient epidermal cells can undergo classical apoptosis , which genetically demonstrates that caspase-14 is dispensable for the apoptosis of keratinocytes . during development , caspase-14 protein expression is detectable from embryonic day ( e ) 15.5 on , and its processing is observed from e17.5 ( hu et al . , 1998 ; van de craen et al . , 1998 ; fischer et al . , 2005 ) , which coincides with stratum corneum formation and establishment of the epidermal barrier .
however , no differences in outside - in barrier formation of the skin of caspase-14deficient mice during embryogenesis were observed ( denecker et al . , 2007 ) .
furthermore , caspase-14deficient mice were born at the expected mendelian ratios , were fertile , and had long survival rates .
detailed analysis of caspase-14deficient mice indicated that caspase-14 has an important role in cornification , hydration , and uvb protection .
the skin of caspase-14deficient mice was shinier , characterized by deeper skin lines , and had larger scales ( denecker et al . , 2007 ) even though the shape and size of the cornified envelopes themselves were not altered .
biochemical analysis indicated that caspase-14 was responsible for the correct processing and degradation of ( pro)filaggrin , as epidermis lacking caspase-14 was characterized by an altered profilaggrin processing and staining pattern ( fig .
3 ) and by the presence of aberrant keratohyalin granules , the profilaggrin storage granules . profilaggrin is a large , insoluble protein consisting of a calcium - binding a domain , a b domain , and several tandem repeats of filaggrin units . in the transitional layer
4 ) , which aid in the bundling of keratin intermediate filaments and formation of the cornified envelope ( for review see candi et al . , 2005 ) .
subsequently , filaggrin is deiminated ( conversion of arginine to citrulline by elimination of the imino group of arginine by peptidylarginine deiminases ) , causing its release from keratin and allowing its degradation into free hygroscopic amino acids that act as natural moisturizing factors of the stratum corneum ( scott and harding , 1986 ; rawlings and matts , 2005 ) .
therefore , filaggrin plays an important role in skin hydration . although the filaggrin unit was detected in caspase-14deficient epidermis by western blot analysis , lower molecular weight filaggrin fragments were also present ( denecker et al . , 2007 ) .
immunofluorescence analysis showed that in these mice , filaggrin immunoreactive fragments accumulated in the upper layers of the stratum corneum ( fig .
this indicates that the correct degradation of filaggrin into free amino acids was affected in caspase-14deficient skin .
interestingly , caspase-14 was found to be associated with keratohyalin granules in the stratum granulosum and to remain cytoplasmic in the stratum corneum ( alibardi et al . , 2004 ) , which could correlate with its possible involvement in the generation of free amino acids . expression of caspase-14 and ( pro)filaggrin in wild - type and caspase-14deficient skin .
immunofluorescence staining for caspase-14 ( red ) and ( pro)filaggrin ( green ) on paraffin sections of 5.5-d - old skin of both wild - type ( + /+ ) and caspase-14deficient ( / ) mice ( denecker et al . , 2007 ) .
fluorescence microscopy was performed on a cellm system ( olympus ) with an upright microscope ( bx61 ; olympus ) .
a specific dapi emission band - pass filter ( 450470 nm ) and a gfp emission band - pass filter ( 510550 nm ) were used .
image acquisition and processing were performed with the cellm software using a cooled ccd camera with a 1,344 1,024 pixel resolution .
image intensity scaling and color conversion were completed in imagej ( national institutes of health ) .
caspase-14 is expressed mainly in the spinous , granular , and cornified layers of wild - type mice and is absent in caspase-14deficient mice .
( pro)filaggrin is expressed in the granular layer and in the lower cornified layer in wild - type skin . in caspase-14deficient skin , additional filaggrin
caspase-14 protects the skin against uvb photo damage and water loss and is involved in the processing of ( pro)filaggrin .
caspase-14 expression starts in the spinous layer ( indicated in shades of red ) , and cleavage into its p20 and p10 subunits occurs at the transition of the granular to the cornified layer .
caspase-14 is active in the dehydrating environment of the cornified layer , where it has an important function in formation of the epidermal barrier leading to protection against uvb and water loss ( denecker et al . , 2007 ) .
profilaggrin is a large structural molecule consisting of an n - terminal a domain and a b domain followed by multiple filaggrin repeats and a unique c - terminal sequence ( for review see candi et al . , 2005 ) .
profilaggrin undergoes many posttranslational modifications , eventually leading to release from the keratin intermediate filaments ( see the section on the function of caspase-14 for details ) . in the lower stratum corneum
these amino acids compose 40% of the natural moisturizing factors present in the stratum corneum and are important for maintaining epidermal hydration ( rawlings and matts , 2005 ) . in caspase-14deficient skin ,
accumulating filaggrin fragments are present ( denecker et al . , 2007 ) , indicating that an unidentified protease ( asterisk ) cleaves the filaggrin monomer into these fragments and that caspase-14 is responsible for the further processing and degradation of these fragments into free amino acids .
as it is very unlikely that caspase-14 is directly responsible for degradation of the filaggrin fragments into free amino acids , we propose two possible mechanisms : ( 1 ) caspase-14 could first cleave these filaggrin fragments , leading to further degradation into free amino acids by another endo- and/or exopeptidase ; or ( 2 ) caspase-14 could directly or indirectly ( by inactivating an inhibitor ) activate an endo- and/or exopeptidase that further processes the smaller filaggrin fragments .
kg , keratohyalin granule ; kif , keratin intermediate filament ; nmf , natural moisturizing factors ; sb , stratum basale ; sc , stratum corneum ; sg , stratum granulosum ; ss , stratum spinosum ; tg , transglutaminase .
these results , together with the finding that caspase-14 can directly cleave ( pro)filaggrin in vitro , demonstrate that caspase-14 has a critical role in the correct processing of ( pro)filaggrin during cornification . whether the degradation of other differentiation - associated proteins is also affected in caspase-14deficient mice
first , caspase-14 may cleave the filaggrin fragments and , thereby , expose cleavage sites that can be recognized by other endo- and/or exopeptidases for further degradation .
second , caspase-14 may be the activator of an endo- and/or exopeptidase that cleaves and degrades filaggrin .
direct degradation of filaggrin fragments into free amino acids by caspase-14 can be ruled out , as caspases only cleave after aspartate residues .
the lack of filaggrin processing into free hygroscopic amino acids in caspase-14deficient mice may lead to the reduced epidermal hydration and increased trans - epidermal water loss observed in these mice ( denecker et al . , 2007 ) .
these results point to an important function of caspase-14 in the maintenance of epidermal hydration .
although a profilaggrin - deficient mouse has not been generated , it has been demonstrated that flaky tail ( ft / ft ) mice , which have an autosomal recessive mutation in the flaky tail gene ( probably the profilaggrin gene ) , lack a functional filaggrin monomer ( presland et al . , 2000 ) .
these mice have been proposed as a model for the filaggrin - deficient skin disease ichthyosis vulgaris because they have dry , flaky skin and irregular scales of variable size .
the importance of filaggrin has been underscored recently by human genetic studies demonstrating that loss - of - function mutations in the profilaggrin gene are the primary cause of the skin disease ichthyosis vulgaris ( smith et al . , 2006 ) , which is characterized by silvery scales on the abdomen and palmar hyperlinearity .
2006 ) , possibly as a result of a defect in epidermal barrier function that allows the increased entry of allergens and infectious agents .
two of the important functions of the skin are prevention of water loss and protection against environmental stress , such as protection against uvb radiation , which are essential for terrestrial life .
the development of caspase-14deficient mice revealed that the absence of caspase-14 enhances sensitivity toward uvb - induced photo damage and apoptosis of the skin ( denecker et al . , 2007 ) .
importantly , this is not caused by cell - autonomous differences in dna damage sensitivity and apoptosis between wild - type and caspase-14deficient keratinocytes .
instead , the uvb - filtering capacity of the stratum corneum is severely reduced in caspase-14deficient skin , as higher levels of cyclobutane pyrimidine dimers are detected immediately after uvb irradiation .
this indicates that caspase-14 has an indispensable role in the photoprotective function of the stratum corneum .
interestingly , topical application of egcg , an inducer of caspase-14 expression , has been shown to be photoprotective ( elmets et al . , 2001 ) . how caspase-14 alters the structural and biochemical properties of the stratum corneum is currently under investigation .
caspase-14 was shown to be expressed at the protein level in several cancer cell lines ( pistritto et al . , 2002 ; koenig et al . , 2005 ; krajewska et al . , 2005 ) .
in addition , caspase-14 mrna , together with keratin 1 and profilaggrin mrna , was decreased in murine uvb - induced squamous cell carcinoma , possibly reflecting reduced differentiation in the tumor ( rundhaug et al . , 2005 ) .
furthermore , in some cases , caspase-14 protein expression was associated with highly differentiated cornified areas of lung squamous cell carcinoma and cervix carcinoma ( koenig et al . , 2005 ) . however
, caspase-14 activation in tumors has not been shown , and so it might not be responsible for the tumor phenotype .
presumably , the ectopic caspase-14 expression is caused by the changed transcriptional activity in these epithelial tumors .
mutations in the caspase-14 gene have not been found in human carcinomas except very rarely in colorectal tumors , which most probably were not the cause of altered caspase-14 expression ( koenig et al . , 2005 ;
we as well as other investigators demonstrated that caspase-14 expression is substantially down - regulated in psoriatic lesions but is unaffected in the nonlesional epidermis ( lippens et al . , 2000 , 2004 ;
psoriasis is an autoimmune disease characterized by the uncontrolled proliferation of keratinocytes and impaired cornification , which results in the aberrant presence of nuclei in the cornified layer , also called parakeratosis .
although caspase-14 is absent in these parakeratotic regions , this is probably not the cause of the development of parakeratotic plaques , as caspase-14deficient mice did not show spontaneous parakeratosis ( denecker et al . , 2007 ) .
more likely , caspase-14 down - regulation results from the impairment of terminal differentiation or up - regulation of transcriptional repressors .
the absence of caspase-14 in psoriatic plaques may lead to the formation of a defective barrier and , therefore , to the aggravation of psoriatic lesions . treating the parakeratotic plaques of patients with a vitamin d3 analogue results in the up - regulation of caspase-14 and
likewise , in the flaky skin ( fsn / fsn ) mouse model of psoriasis , topical egcg treatment causes the up - regulation of caspase-14 and the amelioration of psoriasis ( hsu et al . , 2007 ) .
interestingly , the expression of junb is strongly down - regulated in psoriatic lesions , and inducible epidermal deletion of both junb and c - jun in mice results in a psoriatic phenotype ( zenz et al . , 2005 ) . because caspase-14 might be regulated by these transcription factors
, it would be interesting to elucidate whether caspase-14 is down - regulated in junb / c - jun deficient mice .
recent evidence sheds light on the crucial role of caspase-14 in the skin , but several major questions remain .
activation of caspase-14 occurs most probably at the interface between the granular and cornified layer , implicating a role for caspase-14 in the stratum corneum .
it is now clear that the caspase-14activating protease is not a caspase but probably an epidermis - specific serine protease with elastase - like properties .
this is not surprising , as it has been known for a long time that serine proteases are of major importance in epidermal homeostasis .
determination of the in vitro conditions for caspase-14 activity that mimic stratum corneum conditions , together with the generation of caspase-14deficient mice , led to the identification of ( pro)filaggrin as the first known physiological caspase-14 substrate .
importantly , caspase-14 seems to be involved in the correct processing of filaggrin preceding its degradation into free hygroscopic amino acids , which might explain its role in the prevention of water loss from the epidermis .
proteomic approaches could lead to the identification of additional caspase-14 substrates , which would contribute to understanding the role of caspase-14 in the skin .
caspase-14 also protects against uvb - induced damage , which means that it is involved in the establishment of the biochemical or structural properties of the stratum corneum as a uvb filter . how caspase-14 establishes the uvb - filtering capacity of the corneum is not completely understood .
an extensive biochemical analysis of caspase-14deficient epidermis could reveal these mechanisms . whether caspase-14 , its activating protease , or its substrates could be used as therapeutic agents or as targets to improve formation of the epidermal barrier is a challenging research goal .
|
caspase-14 is a unique member of the evolutionarily conserved family of cysteinyl aspartate
specific proteinases , which are mainly involved in inflammation and apoptosis .
however , recent evidence also implicates these proteases in proliferation and differentiation .
although most caspases are ubiquitously expressed , caspase-14 expression is confined mainly to cornifying epithelia , such as the skin . moreover ,
caspase-14 activation correlates with cornification , indicating that it plays a role in terminal keratinocyte differentiation .
the determination of in vitro conditions for caspase-14 activity paved the way to identifying its substrates .
the recent development of caspase-14deficient mice underscored its importance in the correct degradation of ( pro)filaggrin and in the formation of the epidermal barrier that protects against dehydration and uvb radiation .
here , we review the current knowledge on caspase-14 in skin homeostasis and disease .
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Introduction
Caspase-14 expression and regulation
Activation of caspase-14
Function: caspase-14 is involved in cornification, hydration, and protection against UVB
Caspase-14 and disease
Conclusions
|
nine male competitive cyclists participated in this study . their mean sd age , height , and body mass were 33.3 7.5 yr , 184 4 cm , and 77.3 7.0 kg , respectively .
they had a maximal oxygen consumption ( vo2max ) of 4.8 0.2 lmin ( oxycon pro ; viasys healthcare , germany ) with a peak power output of 418 16 w ( protocol , 25-w increase every minute from 100 w until exhaustion ) , corresponding to a performance level of 4 ( i.e. , well - trained cyclist ( 7 ) ) .
all cyclists were experienced with performing tt in cold - to - temperate conditions ( < 25c ) and provided their written informed consent to participate in this study .
the protocol conformed to the recommendations of the declaration of helsinki and was approved by an independent ethics committee .
the participants were northern european residents having had no exposure to environmental temperatures above 10c for the last 4 months before the study ( i.e. , november to march ) .
they trained 14 h 40 min 4 h 40 min per week before the acclimatization and 13 h 1 min 1 h 6 min during heat acclimatization .
they were encouraged to maintain a constant sleep routine throughout the protocol and remain hydrated ( table 1 ) .
the cyclists performed 3 tt in hot ambient conditions ( tth , see following section ) .
the first tt in hot conditions ( tth-1 ) was not preceded by any outdoor riding in hot conditions ; tth-2 was preceded by 5 d in hot ambient conditions , and tth-3 was preceded by 13 d in the heat .
the participants spent a minimum of 4 hd outside ( average temperature , 34c 3c ; relative humidity , 18% 5% ) but slept , rested , and ate indoors in an air - conditioned facility .
two additional tt were performed in a cool condition ( ttc , see following section ) before and after the heat intervention and were averaged to represent ttc ( no difference in power output between them ) .
the outdoor ttc and tth were respectively performed in denmark and qatar as multilap looping circuits at sea level on flat terrain ( maximal elevation difference , 10 m ) .
cyclists had 36 h ( i.e. , two nights ) of rest after arrival in qatar before performing tth-1 , allowing for recovery from travel fatigue .
the time difference between denmark and qatar is only 2 h , which should not induce significant jet lag ( 34 ) . on the day of the tt
, the riders performed a self - paced warm - up ( approximately two laps on the tt course ) .
the cyclists were allowed to drink water and energy drinks ad libitum before and during the tt . during tt ,
the riders had access to hr , speed , distance , and power output data .
wind speed was 6.0 ms during ttc , and 6.8 , 5.8 , and 4.4 ms during tth-1 , -2 , and -3 , respectively .
environmental temperature was 8.2c 3.5c during ttc and 36.0c 0.4c , 37.4c 0.8c , and 36.2c 1.6c during tth-1 , -2 , and -3 , respectively .
relative humidity was 30% 8% during ttc and 13% 1% , 16% 2% , and 12% 3% during tth-1 , -2 , and -3 , respectively . on the basis of the ideal gas law ( pv = nrt ) adapted to a mixture of ideal gases ( dry air and humid air )
, the air density ( number of mol ( n)/volume ( v ) ) is inversely proportional to temperature and was calculated to be 1.249 kgm during ttc and 1.135 , 1.127 , and 1.131 kgm during tth-1 , -2 , and -3 , respectively .
power output and speed during the tt were measured with powertap wheel sets ( powertap , madison , wi ) logged continuously on garmin devices ( garmin 705 edge ) and afterwards extracted with the software trainingpeaks and exported in 1-hz resolution for subsequent average by 10% of the tt .
all powertap wheel sets were measured within 10 w from a power2max power meter ( power2max , berlin , germany ) , and each rider used the same equipment during the tt .
hr was continuously recorded during all tt via a chest strap ( polar team system 2 ; polar electro , kempele , finland ) .
rectal temperature was measured at the end of each tt by a clinical thermometer ( precision , 0.1c ; depth , approximately 2 cm ) .
in addition , rectal temperature was continuously recorded during tth-1 and tth-3 via a telemetric sensor ( precision , 0.01c ; vitalsense ; mini mitter , respironics , herrsching , germany ) inserted the length of a gloved index finger beyond the anal sphincter .
body mass losses were estimated from the changes in body weight from before to after tth ( seca 769 ; seca , hamburg , germany ) ( precision , 0.1 kg ) .
continuously recorded data ( power , hr , and temperature ) were coded in 10% increments of the tt and analyzed via two - way repeated - measures anova ( four conditions 10 times ) . in addition , total time was analyzed via one - way repeated - measures anova .
anova assumptions were verified preceding all statistical analyses ; logarithmic transformations and greenhouse geisser corrections were applied where appropriate . in case of post hoc comparisons ,
reported p values were adjusted for multiple comparisons using a bonferroni correction ( i.e. , correction for six potential comparisons between conditions ) .
effect sizes are described in terms of partial eta - squared ( ; with 0.06 representing moderate difference and 0.14 , large difference ) .
data are presented as mean sd along with the mean differences ( 95% confidence interval ) .
nine male competitive cyclists participated in this study . their mean sd age , height , and body mass were 33.3 7.5 yr , 184 4 cm , and 77.3 7.0 kg , respectively .
they had a maximal oxygen consumption ( vo2max ) of 4.8 0.2 lmin ( oxycon pro ; viasys healthcare , germany ) with a peak power output of 418 16 w ( protocol , 25-w increase every minute from 100 w until exhaustion ) , corresponding to a performance level of 4 ( i.e. , well - trained cyclist ( 7 ) ) .
all cyclists were experienced with performing tt in cold - to - temperate conditions ( < 25c ) and provided their written informed consent to participate in this study .
the protocol conformed to the recommendations of the declaration of helsinki and was approved by an independent ethics committee .
the participants were northern european residents having had no exposure to environmental temperatures above 10c for the last 4 months before the study ( i.e. , november to march ) .
they trained 14 h 40 min 4 h 40 min per week before the acclimatization and 13 h 1 min 1 h 6 min during heat acclimatization .
they were encouraged to maintain a constant sleep routine throughout the protocol and remain hydrated ( table 1 ) .
the cyclists performed 3 tt in hot ambient conditions ( tth , see following section ) .
the first tt in hot conditions ( tth-1 ) was not preceded by any outdoor riding in hot conditions ; tth-2 was preceded by 5 d in hot ambient conditions , and tth-3 was preceded by 13 d in the heat .
the participants spent a minimum of 4 hd outside ( average temperature , 34c 3c ; relative humidity , 18% 5% ) but slept , rested , and ate indoors in an air - conditioned facility .
two additional tt were performed in a cool condition ( ttc , see following section ) before and after the heat intervention and were averaged to represent ttc ( no difference in power output between them ) .
the outdoor ttc and tth were respectively performed in denmark and qatar as multilap looping circuits at sea level on flat terrain ( maximal elevation difference , 10 m ) .
cyclists had 36 h ( i.e. , two nights ) of rest after arrival in qatar before performing tth-1 , allowing for recovery from travel fatigue .
the time difference between denmark and qatar is only 2 h , which should not induce significant jet lag ( 34 ) . on the day of the tt
, the riders performed a self - paced warm - up ( approximately two laps on the tt course ) .
the cyclists were allowed to drink water and energy drinks ad libitum before and during the tt . during tt ,
the riders had access to hr , speed , distance , and power output data .
wind speed was 6.0 ms during ttc , and 6.8 , 5.8 , and 4.4 ms during tth-1 , -2 , and -3 , respectively .
environmental temperature was 8.2c 3.5c during ttc and 36.0c 0.4c , 37.4c 0.8c , and 36.2c 1.6c during tth-1 , -2 , and -3 , respectively .
relative humidity was 30% 8% during ttc and 13% 1% , 16% 2% , and 12% 3% during tth-1 , -2 , and -3 , respectively . on the basis of the ideal gas law ( pv = nrt ) adapted to a mixture of ideal gases ( dry air and humid air )
, the air density ( number of mol ( n)/volume ( v ) ) is inversely proportional to temperature and was calculated to be 1.249 kgm during ttc and 1.135 , 1.127 , and 1.131 kgm during tth-1 , -2 , and -3 , respectively .
power output and speed during the tt were measured with powertap wheel sets ( powertap , madison , wi ) logged continuously on garmin devices ( garmin 705 edge ) and afterwards extracted with the software trainingpeaks and exported in 1-hz resolution for subsequent average by 10% of the tt .
all powertap wheel sets were measured within 10 w from a power2max power meter ( power2max , berlin , germany ) , and each rider used the same equipment during the tt .
hr was continuously recorded during all tt via a chest strap ( polar team system 2 ; polar electro , kempele , finland ) .
rectal temperature was measured at the end of each tt by a clinical thermometer ( precision , 0.1c ; depth , approximately 2 cm ) .
in addition , rectal temperature was continuously recorded during tth-1 and tth-3 via a telemetric sensor ( precision , 0.01c ; vitalsense ; mini mitter , respironics , herrsching , germany ) inserted the length of a gloved index finger beyond the anal sphincter .
body mass losses were estimated from the changes in body weight from before to after tth ( seca 769 ; seca , hamburg , germany ) ( precision , 0.1 kg ) .
continuously recorded data ( power , hr , and temperature ) were coded in 10% increments of the tt and analyzed via two - way repeated - measures anova ( four conditions 10 times ) . in addition , total time was analyzed via one - way repeated - measures anova .
anova assumptions were verified preceding all statistical analyses ; logarithmic transformations and greenhouse geisser corrections were applied where appropriate . in case of post hoc comparisons ,
reported p values were adjusted for multiple comparisons using a bonferroni correction ( i.e. , correction for six potential comparisons between conditions ) .
effect sizes are described in terms of partial eta - squared ( ; with 0.06 representing moderate difference and 0.14 , large difference ) .
data are presented as mean sd along with the mean differences ( 95% confidence interval ) .
nine male competitive cyclists participated in this study . their mean sd age , height , and body mass were 33.3 7.5 yr , 184 4 cm , and 77.3 7.0 kg , respectively .
they had a maximal oxygen consumption ( vo2max ) of 4.8 0.2 lmin ( oxycon pro ; viasys healthcare , germany ) with a peak power output of 418 16 w ( protocol , 25-w increase every minute from 100 w until exhaustion ) , corresponding to a performance level of 4 ( i.e. , well - trained cyclist ( 7 ) ) .
all cyclists were experienced with performing tt in cold - to - temperate conditions ( < 25c ) and provided their written informed consent to participate in this study .
the protocol conformed to the recommendations of the declaration of helsinki and was approved by an independent ethics committee .
the participants were northern european residents having had no exposure to environmental temperatures above 10c for the last 4 months before the study ( i.e. , november to march ) .
they trained 14 h 40 min 4 h 40 min per week before the acclimatization and 13 h 1 min 1 h 6 min during heat acclimatization .
they were encouraged to maintain a constant sleep routine throughout the protocol and remain hydrated ( table 1 ) .
the cyclists performed 3 tt in hot ambient conditions ( tth , see following section ) .
the first tt in hot conditions ( tth-1 ) was not preceded by any outdoor riding in hot conditions ; tth-2 was preceded by 5 d in hot ambient conditions , and tth-3 was preceded by 13 d in the heat .
the participants spent a minimum of 4 hd outside ( average temperature , 34c 3c ; relative humidity , 18% 5% ) but slept , rested , and ate indoors in an air - conditioned facility .
two additional tt were performed in a cool condition ( ttc , see following section ) before and after the heat intervention and were averaged to represent ttc ( no difference in power output between them ) .
the outdoor ttc and tth were respectively performed in denmark and qatar as multilap looping circuits at sea level on flat terrain ( maximal elevation difference , 10 m ) .
cyclists had 36 h ( i.e. , two nights ) of rest after arrival in qatar before performing tth-1 , allowing for recovery from travel fatigue .
the time difference between denmark and qatar is only 2 h , which should not induce significant jet lag ( 34 ) . on the day of the tt
, the riders performed a self - paced warm - up ( approximately two laps on the tt course ) .
the cyclists were allowed to drink water and energy drinks ad libitum before and during the tt . during tt ,
the riders had access to hr , speed , distance , and power output data .
wind speed was 6.0 ms during ttc , and 6.8 , 5.8 , and 4.4 ms during tth-1 , -2 , and -3 , respectively .
environmental temperature was 8.2c 3.5c during ttc and 36.0c 0.4c , 37.4c 0.8c , and 36.2c 1.6c during tth-1 , -2 , and -3 , respectively .
relative humidity was 30% 8% during ttc and 13% 1% , 16% 2% , and 12% 3% during tth-1 , -2 , and -3 , respectively . on the basis of the ideal gas law ( pv = nrt ) adapted to a mixture of ideal gases ( dry air and humid air )
, the air density ( number of mol ( n)/volume ( v ) ) is inversely proportional to temperature and was calculated to be 1.249 kgm during ttc and 1.135 , 1.127 , and 1.131 kgm during tth-1 , -2 , and -3 , respectively .
power output and speed during the tt were measured with powertap wheel sets ( powertap , madison , wi ) logged continuously on garmin devices ( garmin 705 edge ) and afterwards extracted with the software trainingpeaks and exported in 1-hz resolution for subsequent average by 10% of the tt .
all powertap wheel sets were measured within 10 w from a power2max power meter ( power2max , berlin , germany ) , and each rider used the same equipment during the tt .
hr was continuously recorded during all tt via a chest strap ( polar team system 2 ; polar electro , kempele , finland ) .
rectal temperature was measured at the end of each tt by a clinical thermometer ( precision , 0.1c ; depth , approximately 2 cm ) .
in addition , rectal temperature was continuously recorded during tth-1 and tth-3 via a telemetric sensor ( precision , 0.01c ; vitalsense ; mini mitter , respironics , herrsching , germany ) inserted the length of a gloved index finger beyond the anal sphincter .
body mass losses were estimated from the changes in body weight from before to after tth ( seca 769 ; seca , hamburg , germany ) ( precision , 0.1 kg ) .
continuously recorded data ( power , hr , and temperature ) were coded in 10% increments of the tt and analyzed via two - way repeated - measures anova ( four conditions 10 times ) . in addition , total time was analyzed via one - way repeated - measures anova .
anova assumptions were verified preceding all statistical analyses ; logarithmic transformations and greenhouse geisser corrections were applied where appropriate . in case of post hoc comparisons , reported p values were adjusted for multiple comparisons using a bonferroni correction ( i.e. , correction for six potential comparisons between conditions ) .
effect sizes are described in terms of partial eta - squared ( ; with 0.06 representing moderate difference and 0.14 , large difference ) .
data are presented as mean sd along with the mean differences ( 95% confidence interval ) .
there was a large ( = 0.88 ) and significant condition effect on the time to complete the tt ( table 1 ) ( p < 0.001 ) . the time to complete ttc ( 66 min 13 s 3 min 26 s ) and tth-3 ( 65 min 37 s 3 min 44 s ) was not significantly different ( 0.6 ( 2.5 to + 1.4 ) min , p > 0.999 ) but was significantly shorter than tth-1 ( 77 min 17 s 6 min 26 s ) and tth-2 ( 69 min 25 s 4 min 37 s ) ( all p < 0.01 ) .
speed ( table 1 ) followed a similar pattern of evolution with significantly lower speeds during tth-1 than those during ttc , followed by increases from tth-1 to tth-2 and from tth-2 to tth-3 ( all p < 0.001 ) .
speed during tth-3 was similar to that during ttc ( + 0.4 ( 0.5 to + 1.7 ) kmh , p = 0.797 ) .
average power output was significantly lower in tth-1 than that in ttc ( 48 ( 67 to 30 ) w , p < 0.001 ) .
this decrement was partly restored after 1 wk of acclimatization ( tth-2 vs ttc , 24 ( 40 to 9 ) w , p = 0.003 ) and further restored after the second week ( tth-3 vs ttc , 11 ( 21 to 0 ) w , p = 0.042 ) ( table 1 ) .
1 ) ( = 0.90 , p < 0.001 ) and showed a large ( = 0.51 ) and significant ( p < 0.001 ) time condition interaction .
the post hoc analysis revealed that there was no effect of condition during that first 20% of the tt ( fig .
1 ) ( all p > 0.05 ) . however , power output during tth-1 became and remained lower than both those during ttc and tth-3 from 30% of the distance covered onward ( p < 0.01 ) and lower than that during tth-2 from 80% onward ( fig .
power output during tth-2 became lower than that during ttc from 50% of the distance covered onward ( fig .
power output during tth-3 was lower than that during ttc in one segment of the tt only ( i.e. , 70% , fig .
power output during a 43.4-km cycling tt in ttc ( plain line ) and in tth-1 ( long dashed line ) , tth-2 ( short dashed line ) , and tth-3 ( dotted line ) .
ttc was significantly ( p < 0.05 ) higher than tth-1 , tth-2 , and tth-3 , respectively .
2 , hr significantly increased during the tt ( = 0.67 , p < 0.001 ) relative to testing conditions ( = 0.24 , p < 0.001 ) .
the post hoc analysis showed that hr was significantly elevated during tth-1 as compared with that during both ttc and tth-3 during the first 20% of the tt ( all p < 0.05 ) .
consequently , hr was different between conditions ( = 0.41 , p = 0.024 ) , without pairwise differences reaching significance ( e.g. , tth-1 vs ttc , + 7 ( 2 to + 15 ) bpm , p = 0.127 ; tth-2 vs ttc , + 4 ( 4 to + 12 ) bpm , p = 0.616 ; tth-3 vs ttc , + 5 ( 2 to + 13 ) bpm , p = 0.254 ) .
hr ( upper panel ) and rectal temperature ( lower panel ) during a 43.4-km cycling tt in ttc ( plain line ) and in tth-1 ( long dashed line ) , tth-2 ( short dashed line ) , and tth-3 ( dotted line ) . *
rectal temperature continuously recorded during tth-1 and tth-3 showed an increase during the tt ( = 0.93 , p < 0.001 ) and was higher during the first 80% of tth-1 than that during tth-3 ( fig .
2 ) , leading to an overall higher temperature during tth-3 than that during tth-1 ( + 0.3c ( 0.1c0.5c ) , = 0.63 , p = 0.019 ) . however , final rectal temperature ( table 1 ) was significantly higher after the tth than that after ttc ( all p < 0.001 ) ( fig .
2 ) but without differences between tth ( all p > 0.999 ) . as displayed in table 1 ,
the differences in body mass loss ( p = 0.071 , = 0.28 ) and fluid consumption ( p = 0.095 , = 0.30 ) between the tth did not reach significance .
there was a large ( = 0.88 ) and significant condition effect on the time to complete the tt ( table 1 ) ( p < 0.001 ) . the time to complete ttc ( 66 min 13 s 3 min 26 s ) and tth-3 ( 65 min 37 s 3 min 44 s ) was not significantly different ( 0.6 ( 2.5 to + 1.4 ) min , p > 0.999 ) but was significantly shorter than tth-1 ( 77 min 17 s 6 min 26 s ) and tth-2 ( 69 min 25 s 4 min 37 s ) ( all p < 0.01 ) .
speed ( table 1 ) followed a similar pattern of evolution with significantly lower speeds during tth-1 than those during ttc , followed by increases from tth-1 to tth-2 and from tth-2 to tth-3 ( all p < 0.001 ) .
speed during tth-3 was similar to that during ttc ( + 0.4 ( 0.5 to + 1.7 ) kmh , p = 0.797 ) .
average power output was significantly lower in tth-1 than that in ttc ( 48 ( 67 to 30 ) w , p < 0.001 ) .
this decrement was partly restored after 1 wk of acclimatization ( tth-2 vs ttc , 24 ( 40 to 9 ) w , p = 0.003 ) and further restored after the second week ( tth-3 vs ttc , 11 ( 21 to 0 ) w , p = 0.042 ) ( table 1 ) .
1 ) ( = 0.90 , p < 0.001 ) and showed a large ( = 0.51 ) and significant ( p < 0.001 ) time condition interaction .
the post hoc analysis revealed that there was no effect of condition during that first 20% of the tt ( fig .
1 ) ( all p > 0.05 ) . however , power output during tth-1 became and remained lower than both those during ttc and tth-3 from 30% of the distance covered onward ( p < 0.01 ) and lower than that during tth-2 from 80% onward ( fig .
power output during tth-2 became lower than that during ttc from 50% of the distance covered onward ( fig .
power output during tth-3 was lower than that during ttc in one segment of the tt only ( i.e. , 70% , fig .
power output during a 43.4-km cycling tt in ttc ( plain line ) and in tth-1 ( long dashed line ) , tth-2 ( short dashed line ) , and tth-3 ( dotted line ) .
ttc was significantly ( p < 0.05 ) higher than tth-1 , tth-2 , and tth-3 , respectively .
2 , hr significantly increased during the tt ( = 0.67 , p < 0.001 ) relative to testing conditions ( = 0.24 , p < 0.001 ) .
the post hoc analysis showed that hr was significantly elevated during tth-1 as compared with that during both ttc and tth-3 during the first 20% of the tt ( all p < 0.05 ) .
consequently , hr was different between conditions ( = 0.41 , p = 0.024 ) , without pairwise differences reaching significance ( e.g. , tth-1 vs ttc , + 7 ( 2 to + 15 ) bpm , p = 0.127 ; tth-2 vs ttc , + 4 ( 4 to + 12 ) bpm , p = 0.616 ; tth-3 vs ttc , + 5 ( 2 to + 13 ) bpm , p = 0.254 ) .
hr ( upper panel ) and rectal temperature ( lower panel ) during a 43.4-km cycling tt in ttc ( plain line ) and in tth-1 ( long dashed line ) , tth-2 ( short dashed line ) , and tth-3 ( dotted line ) . *
rectal temperature continuously recorded during tth-1 and tth-3 showed an increase during the tt ( = 0.93 , p < 0.001 ) and was higher during the first 80% of tth-1 than that during tth-3 ( fig .
2 ) , leading to an overall higher temperature during tth-3 than that during tth-1 ( + 0.3c ( 0.1c0.5c ) , = 0.63 , p = 0.019 ) . however , final rectal temperature ( table 1 ) was significantly higher after the tth than that after ttc ( all p < 0.001 ) ( fig .
2 ) but without differences between tth ( all p > 0.999 ) . as displayed in table 1 ,
the differences in body mass loss ( p = 0.071 , = 0.28 ) and fluid consumption ( p = 0.095 , = 0.30 ) between the tth did not reach significance .
there was a large ( = 0.88 ) and significant condition effect on the time to complete the tt ( table 1 ) ( p < 0.001 ) . the time to complete ttc ( 66 min 13 s 3 min 26 s ) and tth-3 ( 65 min 37 s 3 min 44 s ) was not significantly different ( 0.6 ( 2.5 to + 1.4 ) min , p > 0.999 ) but was significantly shorter than tth-1 ( 77 min 17 s 6 min 26 s ) and tth-2 ( 69 min 25 s 4 min 37 s ) ( all p < 0.01 ) .
speed ( table 1 ) followed a similar pattern of evolution with significantly lower speeds during tth-1 than those during ttc , followed by increases from tth-1 to tth-2 and from tth-2 to tth-3 ( all p < 0.001 ) .
speed during tth-3 was similar to that during ttc ( + 0.4 ( 0.5 to + 1.7 ) kmh , p = 0.797 ) .
average power output was significantly lower in tth-1 than that in ttc ( 48 ( 67 to 30 ) w , p < 0.001 ) .
this decrement was partly restored after 1 wk of acclimatization ( tth-2 vs ttc , 24 ( 40 to 9 ) w , p = 0.003 ) and further restored after the second week ( tth-3 vs ttc , 11 ( 21 to 0 ) w , p = 0.042 ) ( table 1 ) .
1 ) ( = 0.90 , p < 0.001 ) and showed a large ( = 0.51 ) and significant ( p < 0.001 ) time condition interaction .
the post hoc analysis revealed that there was no effect of condition during that first 20% of the tt ( fig .
1 ) ( all p > 0.05 ) . however , power output during tth-1 became and remained lower than both those during ttc and tth-3 from 30% of the distance covered onward ( p < 0.01 ) and lower than that during tth-2 from 80% onward ( fig .
power output during tth-2 became lower than that during ttc from 50% of the distance covered onward ( fig .
power output during tth-3 was lower than that during ttc in one segment of the tt only ( i.e. , 70% , fig .
power output during a 43.4-km cycling tt in ttc ( plain line ) and in tth-1 ( long dashed line ) , tth-2 ( short dashed line ) , and tth-3 ( dotted line ) .
ttc was significantly ( p < 0.05 ) higher than tth-1 , tth-2 , and tth-3 , respectively .
2 , hr significantly increased during the tt ( = 0.67 , p < 0.001 ) relative to testing conditions ( = 0.24 , p < 0.001 ) .
the post hoc analysis showed that hr was significantly elevated during tth-1 as compared with that during both ttc and tth-3 during the first 20% of the tt ( all p < 0.05 ) .
consequently , hr was different between conditions ( = 0.41 , p = 0.024 ) , without pairwise differences reaching significance ( e.g. , tth-1 vs ttc , + 7 ( 2 to + 15 ) bpm , p = 0.127 ; tth-2 vs ttc , + 4 ( 4 to + 12 ) bpm , p = 0.616 ; tth-3 vs ttc , + 5 ( 2 to + 13 ) bpm , p = 0.254 ) .
hr ( upper panel ) and rectal temperature ( lower panel ) during a 43.4-km cycling tt in ttc ( plain line ) and in tth-1 ( long dashed line ) , tth-2 ( short dashed line ) , and tth-3 ( dotted line ) . *
rectal temperature continuously recorded during tth-1 and tth-3 showed an increase during the tt ( = 0.93 , p < 0.001 ) and was higher during the first 80% of tth-1 than that during tth-3 ( fig .
2 ) , leading to an overall higher temperature during tth-3 than that during tth-1 ( + 0.3c ( 0.1c0.5c ) , = 0.63 , p = 0.019 ) . however , final rectal temperature ( table 1 ) was significantly higher after the tth than that after ttc ( all p < 0.001 ) ( fig .
2 ) but without differences between tth ( all p > 0.999 ) . as displayed in table 1 ,
the differences in body mass loss ( p = 0.071 , = 0.28 ) and fluid consumption ( p = 0.095 , = 0.30 ) between the tth did not reach significance .
the current study is the first to determine the effects of acute heat exposure and heat acclimatization on performance and pacing during outdoor cycling tt in experienced cyclists .
the cyclists initiated all tt in the heat with a similar power output as maintained during the first 20% of ttc .
however , while maintaining similar hr , they subsequently experienced a marked decrease in power output and speed in the heat .
these decrements were progressively restored with heat acclimatization , despite core temperature in all tt in the heat increasing significantly more than that in cool conditions .
our data showed that mean power output during tth-1 decreased by 16% 5% in unacclimatized cyclists .
this decrement in power output for an increase in air temperature of approximately 28c between ttc and tth-1 represents an average decrement in performance of 0.5% per 1c increase . even if this decrement is not linear , as the effect of an absolute increase in air temperature is more important in warm than in cold environments ( 13 ) , this rate is comparable with the decrements reported during laboratory tt ( 0.3% to 0.9% per 1c ( 12,23,24,32 ) .
in addition , the current study quantified the effects of heat stress on performance at different acclimatization stages . to date , most studies investigating the effects of heat on performance have examined unacclimatized participants .
these have shown that artificial heat acclimatization increases the ability to cycle in a hot laboratory ( 18,21 ) and that natural heat acclimatization increases physical performance during sporting activities in hot environments ( 25,26,33 ) .
however , a comparison of the magnitude of improvement in performance after heat acclimatization , relative to the initial decrement in performance associated with the first exposure to heat stress , has yet to be examined .
our data showed that the decrement in cycling performance was progressively restored as the cyclists acclimatized . from an average power decrement of 16% 5% on the first day of heat exposure ( tth-1 )
relative to ttc , the decrement was reduced to 8% 4% after 1 wk of training in the heat ( tth-2 ) and to 3% 4% after 2 wk ( tth-3 ) . despite the cooling effect of air movement
, our data showed that the average final temperature of the riders was above 40c during tth , irrespective of heat acclimatization ( table 1 ) .
one rider complained of nausea after tth-1 but did not require medical attention and participated in the following training sessions and tests without any sequelae . despite an average final temperature of 40.2c ( range , 39.6c41.0c ) ,
this confirms that well - prepared athletes reach high core temperatures while exercising in the heat , asymptomatic of heat illness ( 4 ) , and that there is no absolute critical temperature threshold set at 40c ( 11 ) . in the current study , despite the decrease in power output ( fig .
1 ) and the possibility to drink ad libitum on the bike , participants lost more than 2% body mass and core temperature reached final values above 40c in the hot conditions ( fig .
our data showed a decrement in absolute intensity ( i.e. , power output ) during the tt but the likely maintenance of a similar relative intensity .
indeed , it has been shown that the rise in cardiovascular strain in hot conditions during both constant rate ( 1,36,37 ) and self - paced ( 24 ) exercise mediates a decrease in maximal aerobic capacity , resulting in an increase in relative intensity for a given absolute work rate .
although power output decreases during prolonged self - paced exercise in the heat , it is proposed that a similar relative intensity to that of cool conditions is maintained and is reflected by a similar or slightly elevated hr ( 24 ) .
it therefore seems that despite a decrease in power output , hr remained elevated and stable ( fig .
moreover , the pacing pattern was not dependent on the environmental conditions or the acclimatization level , which is in line with recent reports that pacing strategies are not affected by environmental temperature ( 23 ) , thermal perception ( 2 ) , or the presence of previous muscle fatigue ( 6 ) .
rather , it seems that pacing during a cycling tt in hot or temperate conditions relates to the maintenance of a physiological threshold or relative intensity ( manifested by hr ) .
given the progressive reduction in vo2max as hyperthermia develops , sustainable power output is reduced , owing to a reduction in work rate for a given relative exercise intensity ( 24 ) . furthermore , it is remarkable that experienced cyclists started their tt at the same absolute intensity regardless of the environmental conditions or their level of heat acclimatization .
notwithstanding , it has previously been reported that tt are initiated at the same power output in hot and cool conditions ( 11,24,31 ) .
this similar work rate adopted seems to correspond to a critical power ( 16 ) .
given that vo2max does not typically decrease in the first approximately 15 min of exercise in the heat ( 27,29,35 ) , athletes seem to adopt a relative intensity associated with this critical power output . as vo2max progressively decreases with the development of thermal and cardiovascular strain in the heat , the maintenance of a similar relative intensity requires reduction in power output ( 24 ) .
however , given the role of previous experiences on pacing ( 30 ) , one would expect that the large decrease in power output experienced by the athletes during tth-1 would have led to a conservative start during tth-2 .
initial power output was similar between trials and decreased thereafter in relation to acclimatization state .
indeed , early studies suggest that heat acclimatization attenuates the circulatory strain associated with thermoregulation ( 19 ) , allowing normalization of the relation between physiological responses and work intensity ( 10 ) .
consequently , the cyclists seem to have finished all tt at a similar relative intensity , as suggested by hr and the similar core temperatures recorded upon completion but at a higher power output as acclimatization progressed . of note , the slightly higher hr in tth may be attributable to higher skin blood flow , as suggested by the higher core temperature , although the cyclists were wearing thermal clothing in ttc , which would also have lead to increase in skin temperature and blood flow .
the current study is the first to investigate cycling performance during outdoor tt in a hot environment .
. however , the evaporative capacity of the environment is improved with higher air velocities , reducing heat stress and dehydration during outdoor cycling compared with indoor laboratory - based experiments ( 28 ) . in the current study ,
the cyclists wore thermal clothing in ttc including long tights , long sleeves , and gloves , which limited the evaporative and convective power of the environment , whereas they wore short tights and a jersey with short sleeves in the heat ( except one cyclist who was consistently using white long sleeves ) .
therefore , it can not be ruled out that overall performance may be optimized in a slightly warmer temperature than in the ttc conditions of the current study .
interestingly , the detrimental effect of hot ambient conditions on speed was not as important as that on power output ( table 1 ) .
the different relations between the environment and speed and power output could be partly related to a temperature effect on air density , as the aerodynamic drag of a cyclist is related to air density and temperature ( 3,9,17 ) . for example , at an air temperature of 11c , the temperature reported to optimize laboratory cycling capacity ( 14 ) , air density is approximately 1.245 kgm at sea level .
in contrast , air density drops to approximately 1.165 kgm at a temperature of 30c , reducing the drag force by approximately 6% .
consequently , the decrease in air density noted in hot ambient conditions is likely to partially attenuate the performance decrement associated with development of hyperthermia during outdoor cycling . in the current study ,
air density was 9.4% lower during tth-3 ( 1.131 kgm ) than that during ttc ( 1.249 kgm ) , representing a power economy of almost 8% ( 22 ) .
thus , despite the slightly lower power output in tth-3 than that in ttc ( 3% ) , the average speeds were not significantly different .
our data showed that mean power output during tth-1 decreased by 16% 5% in unacclimatized cyclists .
this decrement in power output for an increase in air temperature of approximately 28c between ttc and tth-1 represents an average decrement in performance of 0.5% per 1c increase . even if this decrement is not linear , as the effect of an absolute increase in air temperature is more important in warm than in cold environments ( 13 ) , this rate is comparable with the decrements reported during laboratory tt ( 0.3% to 0.9% per 1c ( 12,23,24,32 ) .
in addition , the current study quantified the effects of heat stress on performance at different acclimatization stages .
to date , most studies investigating the effects of heat on performance have examined unacclimatized participants .
these have shown that artificial heat acclimatization increases the ability to cycle in a hot laboratory ( 18,21 ) and that natural heat acclimatization increases physical performance during sporting activities in hot environments ( 25,26,33 ) .
however , a comparison of the magnitude of improvement in performance after heat acclimatization , relative to the initial decrement in performance associated with the first exposure to heat stress , has yet to be examined .
our data showed that the decrement in cycling performance was progressively restored as the cyclists acclimatized . from an average power decrement of 16% 5% on the first day of heat exposure ( tth-1 )
relative to ttc , the decrement was reduced to 8% 4% after 1 wk of training in the heat ( tth-2 ) and to 3% 4% after 2 wk ( tth-3 ) . despite the cooling effect of air movement
, our data showed that the average final temperature of the riders was above 40c during tth , irrespective of heat acclimatization ( table 1 ) .
one rider complained of nausea after tth-1 but did not require medical attention and participated in the following training sessions and tests without any sequelae . despite an average final temperature of 40.2c ( range , 39.6c41.0c ) ,
this confirms that well - prepared athletes reach high core temperatures while exercising in the heat , asymptomatic of heat illness ( 4 ) , and that there is no absolute critical temperature threshold set at 40c ( 11 ) . in the current study , despite the decrease in power output ( fig .
1 ) and the possibility to drink ad libitum on the bike , participants lost more than 2% body mass and core temperature reached final values above 40c in the hot conditions ( fig .
our data showed a decrement in absolute intensity ( i.e. , power output ) during the tt but the likely maintenance of a similar relative intensity .
indeed , it has been shown that the rise in cardiovascular strain in hot conditions during both constant rate ( 1,36,37 ) and self - paced ( 24 ) exercise mediates a decrease in maximal aerobic capacity , resulting in an increase in relative intensity for a given absolute work rate .
although power output decreases during prolonged self - paced exercise in the heat , it is proposed that a similar relative intensity to that of cool conditions is maintained and is reflected by a similar or slightly elevated hr ( 24 ) .
it therefore seems that despite a decrease in power output , hr remained elevated and stable ( fig .
moreover , the pacing pattern was not dependent on the environmental conditions or the acclimatization level , which is in line with recent reports that pacing strategies are not affected by environmental temperature ( 23 ) , thermal perception ( 2 ) , or the presence of previous muscle fatigue ( 6 ) .
rather , it seems that pacing during a cycling tt in hot or temperate conditions relates to the maintenance of a physiological threshold or relative intensity ( manifested by hr ) .
given the progressive reduction in vo2max as hyperthermia develops , sustainable power output is reduced , owing to a reduction in work rate for a given relative exercise intensity ( 24 ) . furthermore , it is remarkable that experienced cyclists started their tt at the same absolute intensity regardless of the environmental conditions or their level of heat acclimatization .
notwithstanding , it has previously been reported that tt are initiated at the same power output in hot and cool conditions ( 11,24,31 ) .
this similar work rate adopted seems to correspond to a critical power ( 16 ) .
given that vo2max does not typically decrease in the first approximately 15 min of exercise in the heat ( 27,29,35 ) , athletes seem to adopt a relative intensity associated with this critical power output . as vo2max progressively decreases with the development of thermal and cardiovascular strain in the heat , the maintenance of a similar relative intensity requires reduction in power output ( 24 ) .
however , given the role of previous experiences on pacing ( 30 ) , one would expect that the large decrease in power output experienced by the athletes during tth-1 would have led to a conservative start during tth-2 .
initial power output was similar between trials and decreased thereafter in relation to acclimatization state .
indeed , early studies suggest that heat acclimatization attenuates the circulatory strain associated with thermoregulation ( 19 ) , allowing normalization of the relation between physiological responses and work intensity ( 10 ) .
consequently , the cyclists seem to have finished all tt at a similar relative intensity , as suggested by hr and the similar core temperatures recorded upon completion but at a higher power output as acclimatization progressed . of note , the slightly higher hr in tth may be attributable to higher skin blood flow , as suggested by the higher core temperature , although the cyclists were wearing thermal clothing in ttc , which would also have lead to increase in skin temperature and blood flow .
the current study is the first to investigate cycling performance during outdoor tt in a hot environment .
however , the evaporative capacity of the environment is improved with higher air velocities , reducing heat stress and dehydration during outdoor cycling compared with indoor laboratory - based experiments ( 28 ) . in the current study ,
the cyclists wore thermal clothing in ttc including long tights , long sleeves , and gloves , which limited the evaporative and convective power of the environment , whereas they wore short tights and a jersey with short sleeves in the heat ( except one cyclist who was consistently using white long sleeves ) . therefore ,
it can not be ruled out that overall performance may be optimized in a slightly warmer temperature than in the ttc conditions of the current study .
interestingly , the detrimental effect of hot ambient conditions on speed was not as important as that on power output ( table 1 ) .
the different relations between the environment and speed and power output could be partly related to a temperature effect on air density , as the aerodynamic drag of a cyclist is related to air density and temperature ( 3,9,17 ) . for example , at an air temperature of 11c , the temperature reported to optimize laboratory cycling capacity ( 14 ) , air density is approximately 1.245 kgm at sea level . in contrast
, air density drops to approximately 1.165 kgm at a temperature of 30c , reducing the drag force by approximately 6% .
consequently , the decrease in air density noted in hot ambient conditions is likely to partially attenuate the performance decrement associated with development of hyperthermia during outdoor cycling . in the current study ,
air density was 9.4% lower during tth-3 ( 1.131 kgm ) than that during ttc ( 1.249 kgm ) , representing a power economy of almost 8% ( 22 ) .
thus , despite the slightly lower power output in tth-3 than that in ttc ( 3% ) , the average speeds were not significantly different .
our data showed that mean power output during tth-1 decreased by 16% 5% in unacclimatized cyclists .
this decrement in power output for an increase in air temperature of approximately 28c between ttc and tth-1 represents an average decrement in performance of 0.5% per 1c increase . even if this decrement is not linear , as the effect of an absolute increase in air temperature is more important in warm than in cold environments ( 13 ) , this rate is comparable with the decrements reported during laboratory tt ( 0.3% to 0.9% per 1c ( 12,23,24,32 ) .
in addition , the current study quantified the effects of heat stress on performance at different acclimatization stages .
to date , most studies investigating the effects of heat on performance have examined unacclimatized participants .
these have shown that artificial heat acclimatization increases the ability to cycle in a hot laboratory ( 18,21 ) and that natural heat acclimatization increases physical performance during sporting activities in hot environments ( 25,26,33 ) .
however , a comparison of the magnitude of improvement in performance after heat acclimatization , relative to the initial decrement in performance associated with the first exposure to heat stress , has yet to be examined .
our data showed that the decrement in cycling performance was progressively restored as the cyclists acclimatized . from an average power decrement of 16% 5% on the first day of heat exposure ( tth-1 )
relative to ttc , the decrement was reduced to 8% 4% after 1 wk of training in the heat ( tth-2 ) and to 3% 4% after 2 wk ( tth-3 ) . despite the cooling effect of air movement
, our data showed that the average final temperature of the riders was above 40c during tth , irrespective of heat acclimatization ( table 1 ) .
one rider complained of nausea after tth-1 but did not require medical attention and participated in the following training sessions and tests without any sequelae . despite an average final temperature of 40.2c ( range , 39.6c41.0c ) ,
this confirms that well - prepared athletes reach high core temperatures while exercising in the heat , asymptomatic of heat illness ( 4 ) , and that there is no absolute critical temperature threshold set at 40c ( 11 ) . in the current study , despite the decrease in power output ( fig .
1 ) and the possibility to drink ad libitum on the bike , participants lost more than 2% body mass and core temperature reached final values above 40c in the hot conditions ( fig .
our data showed a decrement in absolute intensity ( i.e. , power output ) during the tt but the likely maintenance of a similar relative intensity .
indeed , it has been shown that the rise in cardiovascular strain in hot conditions during both constant rate ( 1,36,37 ) and self - paced ( 24 ) exercise mediates a decrease in maximal aerobic capacity , resulting in an increase in relative intensity for a given absolute work rate .
although power output decreases during prolonged self - paced exercise in the heat , it is proposed that a similar relative intensity to that of cool conditions is maintained and is reflected by a similar or slightly elevated hr ( 24 ) .
it therefore seems that despite a decrease in power output , hr remained elevated and stable ( fig .
moreover , the pacing pattern was not dependent on the environmental conditions or the acclimatization level , which is in line with recent reports that pacing strategies are not affected by environmental temperature ( 23 ) , thermal perception ( 2 ) , or the presence of previous muscle fatigue ( 6 ) .
rather , it seems that pacing during a cycling tt in hot or temperate conditions relates to the maintenance of a physiological threshold or relative intensity ( manifested by hr ) .
given the progressive reduction in vo2max as hyperthermia develops , sustainable power output is reduced , owing to a reduction in work rate for a given relative exercise intensity ( 24 ) . furthermore , it is remarkable that experienced cyclists started their tt at the same absolute intensity regardless of the environmental conditions or their level of heat acclimatization .
notwithstanding , it has previously been reported that tt are initiated at the same power output in hot and cool conditions ( 11,24,31 ) .
this similar work rate adopted seems to correspond to a critical power ( 16 ) .
given that vo2max does not typically decrease in the first approximately 15 min of exercise in the heat ( 27,29,35 ) , athletes seem to adopt a relative intensity associated with this critical power output . as vo2max progressively decreases with the development of thermal and cardiovascular strain in the heat , the maintenance of a similar relative intensity requires reduction in power output ( 24 ) .
however , given the role of previous experiences on pacing ( 30 ) , one would expect that the large decrease in power output experienced by the athletes during tth-1 would have led to a conservative start during tth-2 .
initial power output was similar between trials and decreased thereafter in relation to acclimatization state .
indeed , early studies suggest that heat acclimatization attenuates the circulatory strain associated with thermoregulation ( 19 ) , allowing normalization of the relation between physiological responses and work intensity ( 10 ) .
consequently , the cyclists seem to have finished all tt at a similar relative intensity , as suggested by hr and the similar core temperatures recorded upon completion but at a higher power output as acclimatization progressed . of note , the slightly higher hr in tth may be attributable to higher skin blood flow , as suggested by the higher core temperature , although the cyclists were wearing thermal clothing in ttc , which would also have lead to increase in skin temperature and blood flow .
the current study is the first to investigate cycling performance during outdoor tt in a hot environment .
however , the evaporative capacity of the environment is improved with higher air velocities , reducing heat stress and dehydration during outdoor cycling compared with indoor laboratory - based experiments ( 28 ) . in the current study ,
the cyclists wore thermal clothing in ttc including long tights , long sleeves , and gloves , which limited the evaporative and convective power of the environment , whereas they wore short tights and a jersey with short sleeves in the heat ( except one cyclist who was consistently using white long sleeves ) .
therefore , it can not be ruled out that overall performance may be optimized in a slightly warmer temperature than in the ttc conditions of the current study .
interestingly , the detrimental effect of hot ambient conditions on speed was not as important as that on power output ( table 1 ) .
the different relations between the environment and speed and power output could be partly related to a temperature effect on air density , as the aerodynamic drag of a cyclist is related to air density and temperature ( 3,9,17 ) . for example , at an air temperature of 11c , the temperature reported to optimize laboratory cycling capacity ( 14 ) , air density is approximately 1.245 kgm at sea level .
in contrast , air density drops to approximately 1.165 kgm at a temperature of 30c , reducing the drag force by approximately 6% .
consequently , the decrease in air density noted in hot ambient conditions is likely to partially attenuate the performance decrement associated with development of hyperthermia during outdoor cycling . in the current study ,
air density was 9.4% lower during tth-3 ( 1.131 kgm ) than that during ttc ( 1.249 kgm ) , representing a power economy of almost 8% ( 22 ) .
thus , despite the slightly lower power output in tth-3 than that in ttc ( 3% ) , the average speeds were not significantly different .
the novel findings of this investigation are that competitive cyclists performing an outdoor tt undertake their effort at the same power output , irrespective of the environmental conditions or previous experience .
consequently , non heat - acclimatized cyclists are incapable of sustaining this absolute effort .
however , the decrement in power output is partly recovered after 1 wk of heat acclimatization and almost fully restored after 2 wk .
furthermore , our data seem to confirm that sustainable power output is related to a given relative intensity ( 24 ) , which is partly reflected in the maintenance of hr within a certain range . finally , because of the reduction in air density associated with cycling in hot ambient conditions , speed was not different between tth-3 and ttc .
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abstractpurposethis study aimed to determine the effects of heat acclimatization on performance and pacing during outdoor cycling time trials ( tt , 43.4 km ) in the heat.methodsnine cyclists performed three tt in hot ambient conditions ( tth , approximately 37c ) on the first ( tth-1 ) , sixth ( tth-2 ) , and 14th ( tth-3 ) days of training in the heat . data were compared with the average of two tt in cool condition ( approximately 8c ) performed before and after heat acclimatization ( ttc).resultstth-1 ( 77 6 min ) was slower ( p = 0.001 ) than tth-2 ( 69 5 min ) , and both were slower ( p < 0.01 ) than ttc and tth-3 ( 66 3 and 66 4 min , respectively ) , without differences between ttc and tth-3 ( p > 0.05 ) .
the cyclists initiated the first 20% of all tt at a similar power output , irrespective of climate and acclimatization status ; however , during tth-1 , they subsequently had a marked decrease in power output , which was partly attenuated after 6 d of acclimatization and was further reduced after 14 d. hr was higher during the first 20% of tth-1 than that in the other tt ( p < 0.05 ) , but there were no differences between conditions from 30% onward .
final rectal temperature was similar in all tth ( 40.2c 0.4c , p = 1.000 ) and higher than that in ttc ( 38.5c 0.6c , p < 0.001).conclusionsafter 2 wk of acclimatization , trained cyclists are capable of completing a prolonged tt in a similar time in the heat compared with cool conditions , whereas in the unacclimatized state , they experienced a marked decrease in power output during the tth .
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METHODS
None
Participants
General procedure
Measures
Statistical analyses
RESULTS
None
Performance
Pacing of power output
HR and temperature responses
DISCUSSION
None
Power output and pacing
Effect of air density and air movement while cycling in the heat
CONCLUSIONS
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this retrospective observational study was conducted using electronic medical records from the south central veterans affairs health care network ( visn 16 ) , one of the largest of the 23 visns in the veterans health administration ( vha ) .
the vha is a national integrated health care system providing a set of comprehensive services to veterans . as of 2010
the visn 16 data warehouse is an integrated , de - identified , individual - level database representing 7.8% of u.s .
veterans and covers a geographic region of 170,000 square miles , including the states of arkansas , louisiana , mississippi , and oklahoma , and parts of alabama , florida , missouri , and texas .
it includes records for > 445,000 veterans from 10 medical centers and 40 outpatient clinics , with information regarding demographics , vital signs , laboratory results , diagnoses , procedures , inpatient and outpatient services ( e.g. , admission date , length of stay , and emergency room visits ) , drug prescriptions , and database enrollment history .
as in the national vha population , patients in visn 16 are predominantly male ( 90.1% ) .
the study protocol was approved by the institutional review board and research and development committee of the southeast louisiana veterans health care systems .
adult patients ( 18 years of age ) were included in the study if they had two or more diagnoses of t2 dm between 1 january 2004 and 30 june 2010 .
patients had at least one measurement of hba1c and ldl - c within 30 days of each other ( paired measurements ) after the first diabetes diagnosis ; the earlier date of the hba1c or ldl - c measurement was defined as the index date .
all patients were further required to have at least one more measurement of hba1c and ldl - c within 1 year after the index date , irrespective of the gap between the measurements .
the final sample included patients who were enrolled in the database for at least 12 months after the index date .
average hba1c and ldl - c levels were estimated for each cycle using the area under the curve method ( 24,25 ) . for each cycle , these estimated averages were used to stratify patients into one of four goal achievement categories : dual goal ( average hba1c < 7% [ 53 mmol / mol ] and average ldl - c < 100 mg / dl ) , hba1c only ( average hba1c < 7% [ 53 mmol / mol ] and average ldl - c 100 mg / dl ) , ldl - c only ( average ldl - c < 100 mg / dl and average hba1c 7% [ 53 mmol / mol ] ) , or no goal ( average hba1c 7% [ 53 mmol / mol ] and average ldl - c 100 mg / dl ) .
patient characteristics as of the first cycle were summarized for the overall population , as well as stratified according to goal achievement status .
demographic information included age on index date , sex , race , bmi , and year of index date .
the history of diabetes - related complications ( microvascular , macrovascular , and other ) , comorbidities , and surgical procedures was identified as of the first cycle using icd-9 , clinical modification ( icd-9-cm ) codes .
medication use during the first cycle was categorized by drug therapeutic class ; health care resource utilization during the first cycle was categorized by hospitalization days and outpatient visits .
characteristics were compared across the four groups according to goal achievement status using the anova method for continuous variables and tests for categorical variables .
clinical outcomes were selected a priori and comprised 1 ) a composite cardiovascular - related end point ( cerebrovascular disease [ stroke ] , acute myocardial infarction , or cardiovascular death [ defined by a diagnosis of coronary artery disease or cerebrovascular disease on the day of death ] ) , 2 ) a composite end point for microvascular complications ( diabetic retinopathy , nephropathy , or neuropathy ) , 3 ) acute coronary syndromes ( acs ; acute myocardial infarction or unstable angina ) , and 4 ) cardiovascular procedures ( percutaneous coronary intervention [ pci ] or coronary artery bypass graft [ cabg ] ) .
for each clinical outcome , goal achievement and patient characteristics were measured in a given cycle and outcomes were assessed for the following cycle .
the time to the first clinical event was evaluated using a cox proportional hazards model , with goal achievement status as a time - dependent variable , controlling for patient demographics and other potential confounding factors such as cumulative comorbidity history , resource utilization , and medication use .
all clinical outcomes were measured from the start of the second cycle until the first event , death , or end of data ; for the analyses of specific clinical outcomes , patients were excluded from the analysis if any clinical event defining the particular outcome occurred before the end of the first cycle .
the numbers of diabetes - related hospitalization days and outpatient visits were estimated for each 6-month cycle .
diabetes - related medical service costs were measured in each cycle using the average cost method ( 26 ) .
dollars according to the medical care services component of the consumer price index ( 27,28 ) . utilization and costs were considered diabetes related if they were associated with diagnoses of any of the following : diabetes , macrovascular complications , or microvascular complications .
the associations between goal achievement status in a given study cycle and utilization in the following cycle were assessed using generalized linear regression models ( glms ) with a poisson distribution ; results are reported as adjusted incidence rate ratios with 95% cis .
the associations between goal achievement status in a given cycle and costs in the following cycle were assessed using glms with a distribution , and adjusted results are reported as annualized incremental cost differences .
all longitudinal glms accounted for within - patient correlation using a generalized estimating equation approach and controlled for demographics and time - dependent variables such as cumulative comorbidity history , resource utilization , and medication use .
sas software version 9.2 was used to conduct statistical analyses , and a two - tailed level of 0.05 was used to determine statistical significance .
this retrospective observational study was conducted using electronic medical records from the south central veterans affairs health care network ( visn 16 ) , one of the largest of the 23 visns in the veterans health administration ( vha ) .
the vha is a national integrated health care system providing a set of comprehensive services to veterans . as of 2010
the visn 16 data warehouse is an integrated , de - identified , individual - level database representing 7.8% of u.s .
veterans and covers a geographic region of 170,000 square miles , including the states of arkansas , louisiana , mississippi , and oklahoma , and parts of alabama , florida , missouri , and texas .
it includes records for > 445,000 veterans from 10 medical centers and 40 outpatient clinics , with information regarding demographics , vital signs , laboratory results , diagnoses , procedures , inpatient and outpatient services ( e.g. , admission date , length of stay , and emergency room visits ) , drug prescriptions , and database enrollment history .
as in the national vha population , patients in visn 16 are predominantly male ( 90.1% ) .
the study protocol was approved by the institutional review board and research and development committee of the southeast louisiana veterans health care systems .
adult patients ( 18 years of age ) were included in the study if they had two or more diagnoses of t2 dm between 1 january 2004 and 30 june 2010 .
patients had at least one measurement of hba1c and ldl - c within 30 days of each other ( paired measurements ) after the first diabetes diagnosis ; the earlier date of the hba1c or ldl - c measurement was defined as the index date .
all patients were further required to have at least one more measurement of hba1c and ldl - c within 1 year after the index date , irrespective of the gap between the measurements .
the final sample included patients who were enrolled in the database for at least 12 months after the index date .
average hba1c and ldl - c levels were estimated for each cycle using the area under the curve method ( 24,25 ) . for each cycle , these estimated averages were used to stratify patients into one of four goal achievement categories : dual goal ( average hba1c < 7% [ 53 mmol / mol ] and average ldl - c < 100 mg / dl ) , hba1c only ( average hba1c < 7% [ 53 mmol / mol ] and average ldl - c 100 mg / dl ) , ldl - c only ( average ldl - c < 100 mg / dl and average hba1c 7% [ 53 mmol / mol ] ) , or no goal ( average hba1c 7% [ 53 mmol / mol ] and average ldl - c 100 mg / dl ) .
patient characteristics as of the first cycle were summarized for the overall population , as well as stratified according to goal achievement status .
demographic information included age on index date , sex , race , bmi , and year of index date .
the history of diabetes - related complications ( microvascular , macrovascular , and other ) , comorbidities , and surgical procedures was identified as of the first cycle using icd-9 , clinical modification ( icd-9-cm ) codes .
medication use during the first cycle was categorized by drug therapeutic class ; health care resource utilization during the first cycle was categorized by hospitalization days and outpatient visits .
characteristics were compared across the four groups according to goal achievement status using the anova method for continuous variables and tests for categorical variables .
clinical outcomes were selected a priori and comprised 1 ) a composite cardiovascular - related end point ( cerebrovascular disease [ stroke ] , acute myocardial infarction , or cardiovascular death [ defined by a diagnosis of coronary artery disease or cerebrovascular disease on the day of death ] ) , 2 ) a composite end point for microvascular complications ( diabetic retinopathy , nephropathy , or neuropathy ) , 3 ) acute coronary syndromes ( acs ; acute myocardial infarction or unstable angina ) , and 4 ) cardiovascular procedures ( percutaneous coronary intervention [ pci ] or coronary artery bypass graft [ cabg ] ) . for each clinical outcome , goal achievement and patient characteristics were measured in a given cycle and outcomes were assessed for the following cycle .
the time to the first clinical event was evaluated using a cox proportional hazards model , with goal achievement status as a time - dependent variable , controlling for patient demographics and other potential confounding factors such as cumulative comorbidity history , resource utilization , and medication use .
all clinical outcomes were measured from the start of the second cycle until the first event , death , or end of data ; for the analyses of specific clinical outcomes , patients were excluded from the analysis if any clinical event defining the particular outcome occurred before the end of the first cycle .
the numbers of diabetes - related hospitalization days and outpatient visits were estimated for each 6-month cycle .
diabetes - related medical service costs were measured in each cycle using the average cost method ( 26 ) .
dollars according to the medical care services component of the consumer price index ( 27,28 ) . utilization and costs were considered diabetes related if they were associated with diagnoses of any of the following : diabetes , macrovascular complications , or microvascular complications .
the associations between goal achievement status in a given study cycle and utilization in the following cycle were assessed using generalized linear regression models ( glms ) with a poisson distribution ; results are reported as adjusted incidence rate ratios with 95% cis .
the associations between goal achievement status in a given cycle and costs in the following cycle were assessed using glms with a distribution , and adjusted results are reported as annualized incremental cost differences .
all longitudinal glms accounted for within - patient correlation using a generalized estimating equation approach and controlled for demographics and time - dependent variables such as cumulative comorbidity history , resource utilization , and medication use .
sas software version 9.2 was used to conduct statistical analyses , and a two - tailed level of 0.05 was used to determine statistical significance .
of the 149,613 patients with at least two recorded diagnoses of t2 dm between 1 january 2004 and 30 june 2010 , a total of 75,646 patients met the selection criteria and were included in the analysis .
as shown in table 1 , as of the index date , most patients were older than 55 years ( 84.1% ; mean age 64.7 years ) and had an average bmi of 31.6 kg / m .
almost all patients were men ( 97.4% ) , and approximately two in three were white ( 67.4% ) . during the first cycle , 35.1% of patients achieved both goals ( dual - goal achievers ) , whereas 21.6% achieved only the ldl - c goal ( ldl - c achievers ) , 24.6% achieved only the hba1c goal ( hba1c achievers ) , and 18.6% did not achieve either goal ( no - goal achievers ) ( table 1 ) . compared with all other groups , dual - goal achievers were older ( 67.1 vs. 61.464.4 years ) .
rates of microvascular complications were lower for dual - goal ( 24.1% ) and hba1c achievers ( 22.4% ) than for ldl - c ( 33.0% ) and no - goal achievers ( 30.2% ) ; similar differences were observed for the usage of insulin ( 10.4 and 7.5% vs. 34.6 and 30.1% , respectively ) and oral antidiabetic drugs ( 67.4 and 64.0% vs. 82.2 and 82.7% , respectively ) . a higher rate of macrovascular complications was observed among dual - goal ( 47.4% ) and ldl - c achievers ( 45.6% ) than among hba1c ( 33.8% ) and no - goal achievers ( 33.9% ) ( table 1 ) .
patient baseline characteristics , demographics , comorbidities , complications , medications , and resource use the median duration of follow - up time was 4.5 years from the index date . after adjusting for demographics , diabetes - related complications , comorbidities , surgical procedures , diabetic medication use , and health care utilization , dual - goal achievement , when compared with achievement of ldl - c goal alone ,
was associated with a significantly reduced risk of microvascular complications ( adjusted hazard ratio 0.79 [ 95% ci 0.760.82 ] ) , acs ( 0.88 [ 0.810.96 ] ) , pci ( 0.78 [ 0.670.90 ] ) , and cabg ( 0.74 [ 0.600.92 ] ) , but not of the composite cardiovascular - related end point ( 1.00 [ 0.941.06 ] ) ( fig .
dual - goal achievement was associated with a lower risk of cabg ( 0.62 [ 0.490.79 ] ) and the composite cardiovascular end point ( 0.87 [ 0.810.93 ] ) .
dual , patients achieving both ldl - c and hba1c goals ; hba1c , patients achieving only the hba1c goal ; ldl - c , patients achieving only the ldl - c goal ; none , patients achieving neither goal ( see text for details ) . compared with no - goal achievers , dual - goal achievers had a significant 1930% lower hazard of the cardiovascular end point or diabetes - related microvascular or acs - related events , and a 4549% lower hazard of undergoing pci or cabg .
in addition , relative to no - goal achievers , both groups of single - goal achievers had significant reductions in hazard for all categories of complications and surgical procedures , except microvascular complications among ldl - c achievers . after controlling for demographics , diabetes - related complications , comorbidities , surgical procedures , diabetic medication use , and health care utilization ,
dual - goal achievers generally used less health care resources compared with single- or no - goal achievers ( fig .
2 ) . in particular , compared with ldl - c achievers , dual - goal achievers had significantly fewer hospitalization days ( adjusted incidence rate ratio 0.93 [ 95% ci 0.871.00 ] ) and outpatient visits ( 0.88 [ 0.870.89 ] ) . compared with hba1c achievers , dual - goal achievers had significantly fewer outpatient visits ( 0.98 [ 0.971.00 ] ) , but there was no statistical difference in the number of hospitalization days ( 0.98 [ 0.891.07 ] ) . compared with no - goal achievers , dual - goal achievers had significantly fewer hospitalization days ( 0.81 [ 0.720.90 ] ) and outpatient visits ( 0.86 [ 0.850.87 ] ) . similarly , both groups of single - goal achievers also had significantly fewer hospitalization days ( ldl - c achievers , 0.87 [ 0.780.96 ] ; hba1c achievers , 0.83 [ 0.730.94 ] ) and outpatient visits ( ldl - c achievers , 0.98 [ 0.960.99 ] ; hba1c achievers , 0.87 [ 0.860.89 ] ) than patients who did not achieve either goal .
dual , patients achieving both ldl - c and hba1c goals ; hba1c , patients achieving only the hba1c goal ; ldl - c , patients achieving only the ldl - c goal ; none , patients achieving neither goal . *
p < 0.05 ; * * p < 0.01 ; * * * p < 0.001 . after adjusting for demographics , diabetes - related complications , comorbidities , surgical procedures , diabetic medication use , and health care utilization , dual - goal achievers incurred significantly lower annualized diabetes - related medical service costs compared with those who achieved only the ldl - c goal ( $130.89 ; p = 0.016 ) , but no statistically significant difference was observed between dual - goal achievers and hba1c goal achievers ( $56.17 ; p = 0.404 ) ( fig .
diabetes - related medical service costs were significantly lower for dual - goal ( $376.50 ; p < 0.001 ) , ldl - c ( $245.61 ; p < 0.001 ) , and hba1c achievers ( $320.32 ; p < 0.001 ) compared with those who did not achieve either goal .
dual , patients achieving both ldl - c and hba1c goals ; hba1c , patients achieving only the hba1c goal ; ldl - c , patients achieving only the ldl - c goal ; none , patients achieving neither goal .
of the 149,613 patients with at least two recorded diagnoses of t2 dm between 1 january 2004 and 30 june 2010 , a total of 75,646 patients met the selection criteria and were included in the analysis .
as shown in table 1 , as of the index date , most patients were older than 55 years ( 84.1% ; mean age 64.7 years ) and had an average bmi of 31.6 kg / m .
almost all patients were men ( 97.4% ) , and approximately two in three were white ( 67.4% ) . during the first cycle , 35.1% of patients achieved both goals ( dual - goal achievers ) , whereas 21.6% achieved only the ldl - c goal ( ldl - c achievers ) , 24.6% achieved only the hba1c goal ( hba1c achievers ) , and 18.6% did not achieve either goal ( no - goal achievers ) ( table 1 ) . compared with all other groups , dual - goal achievers were older ( 67.1 vs. 61.464.4 years ) .
rates of microvascular complications were lower for dual - goal ( 24.1% ) and hba1c achievers ( 22.4% ) than for ldl - c ( 33.0% ) and no - goal achievers ( 30.2% ) ; similar differences were observed for the usage of insulin ( 10.4 and 7.5% vs. 34.6 and 30.1% , respectively ) and oral antidiabetic drugs ( 67.4 and 64.0% vs. 82.2 and 82.7% , respectively ) . a higher rate of macrovascular complications was observed among dual - goal ( 47.4% ) and ldl - c achievers ( 45.6% ) than among hba1c ( 33.8% ) and no - goal achievers ( 33.9% ) ( table 1 ) .
the median duration of follow - up time was 4.5 years from the index date . after adjusting for demographics , diabetes - related complications , comorbidities , surgical procedures , diabetic medication use , and health care utilization , dual - goal achievement , when compared with achievement of ldl - c goal alone ,
was associated with a significantly reduced risk of microvascular complications ( adjusted hazard ratio 0.79 [ 95% ci 0.760.82 ] ) , acs ( 0.88 [ 0.810.96 ] ) , pci ( 0.78 [ 0.670.90 ] ) , and cabg ( 0.74 [ 0.600.92 ] ) , but not of the composite cardiovascular - related end point ( 1.00 [ 0.941.06 ] ) ( fig .
dual - goal achievement was associated with a lower risk of cabg ( 0.62 [ 0.490.79 ] ) and the composite cardiovascular end point ( 0.87 [ 0.810.93 ] ) .
dual , patients achieving both ldl - c and hba1c goals ; hba1c , patients achieving only the hba1c goal ; ldl - c , patients achieving only the ldl - c goal ; none , patients achieving neither goal ( see text for details ) . compared with no - goal achievers , dual - goal achievers had a significant 1930% lower hazard of the cardiovascular end point or diabetes - related microvascular or acs - related events , and a 4549% lower hazard of undergoing pci or cabg .
in addition , relative to no - goal achievers , both groups of single - goal achievers had significant reductions in hazard for all categories of complications and surgical procedures , except microvascular complications among ldl - c achievers .
after controlling for demographics , diabetes - related complications , comorbidities , surgical procedures , diabetic medication use , and health care utilization , dual - goal achievers generally used less health care resources compared with single- or no - goal achievers ( fig .
2 ) . in particular , compared with ldl - c achievers , dual - goal achievers had significantly fewer hospitalization days ( adjusted incidence rate ratio 0.93 [ 95% ci 0.871.00 ] ) and outpatient visits ( 0.88 [ 0.870.89 ] ) . compared with hba1c achievers , dual - goal achievers had significantly fewer outpatient visits ( 0.98 [ 0.971.00 ] ) , but there was no statistical difference in the number of hospitalization days ( 0.98 [ 0.891.07 ] ) . compared with no - goal achievers , dual - goal achievers had significantly fewer hospitalization days ( 0.81 [ 0.720.90 ] ) and outpatient visits ( 0.86 [ 0.850.87 ] ) .
similarly , both groups of single - goal achievers also had significantly fewer hospitalization days ( ldl - c achievers , 0.87 [ 0.780.96 ] ; hba1c achievers , 0.83 [ 0.730.94 ] ) and outpatient visits ( ldl - c achievers , 0.98 [ 0.960.99 ] ; hba1c achievers , 0.87 [ 0.860.89 ] ) than patients who did not achieve either goal .
dual , patients achieving both ldl - c and hba1c goals ; hba1c , patients achieving only the hba1c goal ; ldl - c , patients achieving only the ldl - c goal ; none , patients achieving neither goal .
* p < 0.05 ; * * p < 0.01 ; * * * p < 0.001 .
after adjusting for demographics , diabetes - related complications , comorbidities , surgical procedures , diabetic medication use , and health care utilization , dual - goal achievers incurred significantly lower annualized diabetes - related medical service costs compared with those who achieved only the ldl - c goal ( $130.89 ; p = 0.016 ) , but no statistically significant difference was observed between dual - goal achievers and hba1c goal achievers ( $56.17 ; p = 0.404 ) ( fig .
diabetes - related medical service costs were significantly lower for dual - goal ( $376.50 ; p < 0.001 ) , ldl - c ( $245.61 ; p < 0.001 ) , and hba1c achievers ( $320.32 ; p < 0.001 ) compared with those who did not achieve either goal .
dual , patients achieving both ldl - c and hba1c goals ; hba1c , patients achieving only the hba1c goal ; ldl - c , patients achieving only the ldl - c goal ; none , patients achieving neither goal . *
the study results showed that , compared with the achievement of only the ldl - c goal , achievement of both hba1c and ldl - c goals is associated with a lower risk of microvascular complications , acs , and cardiovascular surgeries ( pci or cabg ) , lower utilization of health care resources , and lower costs of care , but no additional effect was observed for the composite cardiovascular - related end point .
in addition , dual - goal achievement relative to hba1c goal achievement is associated with a lower risk of the composite cardiovascular - related end point , cabg , and outpatient visits . to our knowledge , this is the first study that was designed to quantify the differences in clinical and economic outcomes between dual - goal and single - goal achievement in patients with t2 dm . the results from our study are generally consistent with findings from previous studies designed to assess the benefits of treatment paradigms aimed at achieving more than one clinical goal in diabetes . in steno-2 , a danish open - label ,
randomized , parallel - group study of patients with established t2 dm , patients assigned to receive an intensive treatment targeting tighter goals for blood pressure ( systolic < 130140 mmhg ; diastolic < 8085 mmhg ) , hba1c ( < 6.5% [ 48 mmol / mol ] ) , total cholesterol ( < 175190 mg / dl ) , and triglycerides ( < 150 mg / dl ) had a significantly lower risk of cardiovascular disease and microvascular complications over an average follow - up of 7.8 years , compared with patients assigned to antidiabetic treatment in accordance with national guidelines ( 21 ) .
a 5.5-year extension of that same study demonstrated that the multifactorial therapy was associated with sustained lower risk of cardiovascular events or death ( 20 ) . in the steno-2 study ,
lipid - lowering treatments were suggested to have had the greatest contribution to cardiovascular risk reduction , whereas antiglycemic and antihypertensive treatments were considered to have accounted for the greatest reduction in microvascular complications ( 29 ) .
these results are in alignment with our findings that suggest that there are additional cardiovascular benefits associated with the achievement of both ldl - c and hba1c goals when compared with only hba1c goal achievement , while there are additional microvascular benefits associated with the achievement of both hba1c and ldl - c goals when compared with only ldl - c goal achievement .
in contrast to the steno-2 study ( 21 ) , we did not assess the impact of multiple interventions or goal achievements other than hba1c and ldl - c .
the blood pressure level cutoff recommended by major national and international guidelines for patients with diabetes ( < 130/80 mmhg ) may be difficult to achieve , and the benefits of achieving this blood pressure goal are unclear ( 30 ) .
we decided to await final consensus on the optimal blood pressure goal for patients with diabetes before creating appropriate models to account for blood pressure goal achievements .
current lipid and glycemic goals , however , are relatively easy to achieve , as can be seen from our study , and their effects are therefore easier to take into consideration .
our results are also consistent with findings from large randomized trials that evaluated situations analogous to the achievement of single metabolic goals .
the 10-year uk prospective diabetes study found that intensive glycemic control reduces the risk of microvascular complications but does not affect the risk of macrovascular disease ( 31 ) .
this lack of association between intensive glycemic control and macrovascular benefits has been observed in other large trials , such as action in diabetes and vascular disease : preterax and diamicron modified release controlled evaluation ( advance ) ( 4 ) , action to control cardiovascular risk in diabetes ( accord ) ( 2,3 ) , and veterans affairs diabetes trial ( vadt ) ( 32 ) , except in the case of patients with newly diagnosed diabetes ( 5,33 ) .
our study showed a 6% lower hazard of cardiovascular - related end point for hba1c achievers compared with no - goal achievers .
our findings are in agreement with the results from several large randomized trials that found that the treatments aimed at lowering ldl - c were associated with a reduced risk of cardiovascular events in patients with diabetes ( 610 ) . in our study ,
achievement of the hba1c goal alone and the ldl - c goal alone were each associated with a lower risk of cardiovascular surgeries , which is consistent with other research in patients with diabetes which has shown that patients who undergo cabg ( 3436 ) and pci ( 37,38 ) often have elevated hba1c levels , and that lowering ldl - c levels is also associated with lower risks of coronary events ( 6,7,39 ) .
this is the first study to examine the differences between dual- and single - goal achievement in terms of health care resource utilization and costs in patients with diabetes .
our results suggest that dual - goal achievement provides additional economic benefits . over ldl - c goal alone , but not over hba1c goal alone .
our results are consistent with previous studies that show that control of either ldl - c ( 40 ) or hba1c ( 15 ) is associated with cost savings . for example , a 2003 study designed to assess cost of statin therapy for the primary prevention of major coronary events in u.s .
patients with diabetes and ldl - c levels > 100 mg / dl found that among individuals with ldl - c levels of 100129 mg / dl and 130 mg / dl , the annual cost difference between patients with major coronary events with statin treatment versus without statin treatment was $ 480950 and $ 5901,920 , respectively ( 16 ) .
a 2005 analysis conducted to predict costs and outcomes for patients with uncontrolled t1 dm and t2 dm , compared with patients who remained at hba1c levels of 7% ( 53 mmol / mol ) or 6.5% ( 48 mmol / mol ) , found that efficient targeting of financial resources toward achievement of hba1c goals in the u.s . would result in $ 3572 billion savings over the subsequent 10 years ( 15 ) .
first , the data used for this study include laboratory measurements for patients over time .
second , this study used a longitudinal design that was able to capture the time - varying nature of laboratory measurements .
this allowed for better estimation of the association between goal achievement and risk of complications over time , compared with a simple cross - sectional design , which would use baseline laboratory values in regression models .
first , due to the retrospective observational design , the analysis may have been affected by unobserved factors that were not taken into account in the model .
second , the electronic medical records did not include information on disease severity , disease duration , lifestyle modifications , or other interventions .
third , although patients enrolled in the vha do not typically use services outside of the system , any health care services that were administered by a provider outside of the vha were not included in the electronic records .
fourth , because we used vha data , the patients in our study were predominantly male .
although our sample was representative of the vha population , gender imbalance may limit the generalization of our findings . as the vha population may have characteristics that are distinct from those in the general population , similar studies in the general population should be performed .
finally , studies on the clinical and economic benefits associated with triple goal achievement of hba1c , ldl - c , and blood pressure goals may shed additional light on the appropriate management of patients with t2 dm . in conclusion ,
veterans suggests that the achievement of both ldl - c and hba1c goals is associated with additional clinical and economic benefits , compared with the achievement of either goal alone
. these findings may facilitate decision making when considering the health and pharmacoeconomic benefits of various treatment strategies to target multiple treatment goals for individuals with diabetes .
to our knowledge , this is the first study that was designed to quantify the differences in clinical and economic outcomes between dual - goal and single - goal achievement in patients with t2 dm . the results from our study are generally consistent with findings from previous studies designed to assess the benefits of treatment paradigms aimed at achieving more than one clinical goal in diabetes . in steno-2 , a danish open - label ,
randomized , parallel - group study of patients with established t2 dm , patients assigned to receive an intensive treatment targeting tighter goals for blood pressure ( systolic < 130140 mmhg ; diastolic < 8085 mmhg ) , hba1c ( < 6.5% [ 48 mmol / mol ] ) , total cholesterol ( < 175190 mg / dl ) , and triglycerides ( < 150 mg / dl ) had a significantly lower risk of cardiovascular disease and microvascular complications over an average follow - up of 7.8 years , compared with patients assigned to antidiabetic treatment in accordance with national guidelines ( 21 ) .
a 5.5-year extension of that same study demonstrated that the multifactorial therapy was associated with sustained lower risk of cardiovascular events or death ( 20 ) . in the steno-2 study ,
lipid - lowering treatments were suggested to have had the greatest contribution to cardiovascular risk reduction , whereas antiglycemic and antihypertensive treatments were considered to have accounted for the greatest reduction in microvascular complications ( 29 ) .
these results are in alignment with our findings that suggest that there are additional cardiovascular benefits associated with the achievement of both ldl - c and hba1c goals when compared with only hba1c goal achievement , while there are additional microvascular benefits associated with the achievement of both hba1c and ldl - c goals when compared with only ldl - c goal achievement .
in contrast to the steno-2 study ( 21 ) , we did not assess the impact of multiple interventions or goal achievements other than hba1c and ldl - c .
the blood pressure level cutoff recommended by major national and international guidelines for patients with diabetes ( < 130/80 mmhg ) may be difficult to achieve , and the benefits of achieving this blood pressure goal are unclear ( 30 ) .
we decided to await final consensus on the optimal blood pressure goal for patients with diabetes before creating appropriate models to account for blood pressure goal achievements .
current lipid and glycemic goals , however , are relatively easy to achieve , as can be seen from our study , and their effects are therefore easier to take into consideration .
our results are also consistent with findings from large randomized trials that evaluated situations analogous to the achievement of single metabolic goals .
the 10-year uk prospective diabetes study found that intensive glycemic control reduces the risk of microvascular complications but does not affect the risk of macrovascular disease ( 31 ) .
this lack of association between intensive glycemic control and macrovascular benefits has been observed in other large trials , such as action in diabetes and vascular disease : preterax and diamicron modified release controlled evaluation ( advance ) ( 4 ) , action to control cardiovascular risk in diabetes ( accord ) ( 2,3 ) , and veterans affairs diabetes trial ( vadt ) ( 32 ) , except in the case of patients with newly diagnosed diabetes ( 5,33 ) .
our study showed a 6% lower hazard of cardiovascular - related end point for hba1c achievers compared with no - goal achievers .
our findings are in agreement with the results from several large randomized trials that found that the treatments aimed at lowering ldl - c were associated with a reduced risk of cardiovascular events in patients with diabetes ( 610 ) . in our study ,
achievement of the hba1c goal alone and the ldl - c goal alone were each associated with a lower risk of cardiovascular surgeries , which is consistent with other research in patients with diabetes which has shown that patients who undergo cabg ( 3436 ) and pci ( 37,38 ) often have elevated hba1c levels , and that lowering ldl - c levels is also associated with lower risks of coronary events ( 6,7,39 ) .
this is the first study to examine the differences between dual- and single - goal achievement in terms of health care resource utilization and costs in patients with diabetes .
our results suggest that dual - goal achievement provides additional economic benefits . over ldl - c goal alone , but not over hba1c goal alone .
our results are consistent with previous studies that show that control of either ldl - c ( 40 ) or hba1c ( 15 ) is associated with cost savings .
for example , a 2003 study designed to assess cost of statin therapy for the primary prevention of major coronary events in u.s .
patients with diabetes and ldl - c levels > 100 mg / dl found that among individuals with ldl - c levels of 100129 mg / dl and 130 mg / dl , the annual cost difference between patients with major coronary events with statin treatment versus without statin treatment was $ 480950 and $ 5901,920 , respectively ( 16 ) . a 2005 analysis conducted to predict costs and outcomes for patients with uncontrolled t1 dm and t2 dm ,
compared with patients who remained at hba1c levels of 7% ( 53 mmol / mol ) or 6.5% ( 48 mmol / mol ) , found that efficient targeting of financial resources toward achievement of hba1c goals in the u.s . would result in $ 3572 billion savings over the subsequent 10 years ( 15 ) .
first , the data used for this study include laboratory measurements for patients over time .
second , this study used a longitudinal design that was able to capture the time - varying nature of laboratory measurements .
this allowed for better estimation of the association between goal achievement and risk of complications over time , compared with a simple cross - sectional design , which would use baseline laboratory values in regression models .
first , due to the retrospective observational design , the analysis may have been affected by unobserved factors that were not taken into account in the model .
second , the electronic medical records did not include information on disease severity , disease duration , lifestyle modifications , or other interventions .
third , although patients enrolled in the vha do not typically use services outside of the system , any health care services that were administered by a provider outside of the vha were not included in the electronic records .
fourth , because we used vha data , the patients in our study were predominantly male .
although our sample was representative of the vha population , gender imbalance may limit the generalization of our findings . as the vha population may have characteristics that are distinct from those in the general population , similar studies in the general population should be performed . finally , studies on the clinical and economic benefits associated with triple goal achievement of hba1c
, ldl - c , and blood pressure goals may shed additional light on the appropriate management of patients with t2 dm . in conclusion , this retrospective study among u.s .
veterans suggests that the achievement of both ldl - c and hba1c goals is associated with additional clinical and economic benefits , compared with the achievement of either goal alone .
these findings may facilitate decision making when considering the health and pharmacoeconomic benefits of various treatment strategies to target multiple treatment goals for individuals with diabetes .
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objectivethis study compared the clinical and economic benefits associated with dual - goal achievement , glycated hemoglobin ( hba1c ) < 7% ( 53 mmol / mol ) and ldl cholesterol ( ldl - c ) < 100 mg / dl , with achievement of only the ldl - c goal or only the hba1c goal in veterans with type 2 diabetes mellitus ( t2dm).research design and methodsthis retrospective cohort analysis evaluated electronic medical records ( veterans integrated service network 16 ) in adult t2 dm patients with two or more measurements of ldl - c and hba1c between 1 january 2004 and 30 june 2010 ( n = 75,646 ) .
cox proportional hazards models were used to compare microvascular and cardiovascular outcomes by goal achievement status ; generalized linear regression models were used to assess diabetes - related resource utilization ( hospitalization days and number of outpatient visits ) and medical service costs.resultsrelative to achievement of only the ldl - c goal , dual - goal achievement was associated with lower risk of microvascular complications ( adjusted hazard ratio [ ahr ] 0.79 ) , acute coronary syndrome ( 0.88 ) , percutaneous coronary intervention ( 0.78 ) , and coronary artery bypass graft ( cabg ) ( 0.74 ) ; it was also associated with fewer hospitalization days ( adjusted incidence rate ratio [ airr ] 0.93 ) and outpatient visits ( 0.88 ) , as well as lower diabetes - related annual medical costs ( $130.89 ) .
compared with achievement of only the hba1c goal , dual - goal achievement was associated with lower risk of the composite cardiovascular - related end point ( ahr 0.87 ) and cabg ( ahr 0.62 ) , as well as fewer outpatient visits ( airr 0.98).conclusionsachieving both hba1c and ldl - c goals in diabetes care is associated with additional clinical and economic benefits , as compared with the achievement of either goal alone .
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RESEARCH DESIGN AND METHODS
Data source
Sample selection
Data preparation
Patient characteristics
Clinical outcomes
Economic outcomes
RESULTS
Patient characteristics
Clinical outcomes
Resource utilization
Medical service costs
CONCLUSIONS
Clinical outcomes: dual- vs. single-goal achievement
Clinical outcomes: single- vs. no-goal achievement
Economic outcomes
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cancer places a huge burden on society and has been identified as the leading cause of death in both more and less economically developed countries .
projections based on the globocan 2012 estimates predict a substantive increase to 19.3 million new cancer cases per year by 2025 , due to growth and ageing of the global population .
south africa , like other developing countries , is also experiencing an increase in the overall burden of disease attributable to cancer , with the number of new cancer cases predicted to increase by 46% by 2030 .
this is likely to result in an increase in the use of cancer chemotherapy agents , which assist in preventing the proliferation , invasion , and metastases of the cancer cells .
the basis for chemotherapy is anticancer drugs containing platinum , that is , cisplatin ( cis - diamminedichloroplatinum ii ) and carboplatin ( cis - diammine 1,1-cyclobutane dicarboxylatoplatinum ii ) .
other chemotherapy drugs include nitrogen mustard , amino - nicotinamide , dichloromethotrexate , bleomycin , and 5-fluorouracil [ 5 , 6 ] .
the first of these drugs , that is , cisplatin , consists of a divalent pt ( ii ) central atom and four ligands of cis - positioned pairs of chlorine atoms or amine groups .
since its discovery in the 1970s , cisplatin continues to be hailed as one of the most potent cancer chemotherapeutics in children and adults , as it is unique and unmatched in its effectiveness against many cancers , namely , osteogenic sarcoma , medulloblastoma , testicular , cervical , and ovarian cancers .
similarly , its toxicity profile is expansive , involving the gastrointestinal , hematologic , renal , and auditory systems .
while the use of saline hydration and mannitol diuresis may prevent nephrotoxicity , neurotoxicity is still not curable or preventable .
ototoxicity refers to the hearing disorder that results from the temporary or permanent inner ear dysfunction after treatment with an ototoxic drug .
other drug classes known to have ototoxic properties include aminoglycosides , loop diuretics , quinine , nonsteroidal anti - inflammatory drugs , and antiretroviral therapy ( art ) .
this is of concern in south africa , as it is estimated that 12.2% of the population ( 6.4 million persons ) were hiv positive in 2012 , which is 1.2 million more people living with hiv than in 2008 ( 10.6% , or 5.2 million ) .
resultantly , art exposure had almost doubled from 16.6% in 2008 to 31.2% in 2012 .
not only will many infected people be at risk for ototoxicity due to arts , but a large number will also be susceptible to hiv - related cancers , such as kaposi 's sarcoma , non - hodgkin 's lymphoma , and cervical cancer , as well as infectious diseases such as tuberculosis , conditions that often require pharmacological therapy with the adverse side effect of ototoxicity .
it is possible that their treatments could consist of simultaneous use of more than one ototoxic drug , increasing the likelihood of ototoxicity .
all health care professionals managing patients with cancer should therefore be knowledgeable about the ototoxic properties of cisplatin .
however , malhotra indicated that most oncologists in india do not make referrals for audiological evaluations of patients receiving cisplatin , while a study in south africa revealed that the effects of ototoxicity , the role of audiologists , and need for their expertise were not fully realized by the oncologists sampled .
this is further supported by evidence from the south african study of khoza - shangase and jina which indicated that most general practitioners sampled also do not appear to carry out ototoxicity monitoring strategies , despite being aware of their own role within an ototoxicity monitoring programme .
this review therefore aims to serve as resource for health professionals to enhance their understanding of ototoxicity as well as their roles within an ototoxicity monitoring programme by providing an overview and description of this condition in patients diagnosed with cancer and receiving cisplatin chemotherapy .
the review identified peer - reviewed articles available from january 1975 to july 2015 on the topic of cisplatin - associated ototoxicity and ototoxicity monitoring and included english articles only .
the same researcher conducted the literature search and reviewed the abstracts and articles for inclusion in the study .
studies were identified using keyword and mesh term searches of electronic databases depicted in table 1 . a manual search of relevant authors and journals
the references cited by each publication , review , or book chapter were reviewed in order to locate additional potential publications . in order to be selected , the article had to present data on either cisplatin ototoxicity and/or ototoxicity monitoring in human participants , and no research designs were excluded . running these searches yielded a total of 2106 records , of which 1581 were excluded based on the title and/or abstract as well as duplication .
eighty - five relevant articles , comprising six national and 79 international articles , were selected .
a perusal of narrative reviews of other auditory pathologies was conducted in an attempt to determine areas of significance for an overview of cisplatin ototoxicity .
this resulted in the following eight areas being included : the mechanisms of cisplatin ototoxicity , clinical presentation , risk factors , incidence rates in adults and children , the effect on quality of life , ototoxicity monitoring , otoprotective strategies , and management of an ototoxic hearing loss .
one such mechanism , the antioxidant model , involves the formation of reactive oxygen species ( ros ) within the cochlea and consequent reduction in antioxidant enzymes following exposure to cisplatin chemotherapy [ 1620 ] .
another mechanism of cisplatin ototoxicity involves the significant contribution of nicotinamide adenine dinucleotide phosphate oxidase 3 isoform ( nox3 ) to the generation of reactive oxygen species within the cochlea , when activated by cisplatin [ 17 , 21 ] , while a third mechanism relates to the activation of transient receptor potential vanilloid 1 channel ( trpv1 ) [ 2224 ] .
the molecular mechanisms of cisplatin ototoxicity therefore include the following:creation of reactive oxygen species , depletion of antioxidant glutathione and its regenerating enzymes , increased rate of lipid peroxidation , oxidative modifications of proteins , nucleic acids damage by caspase system activation ands - nitrosylation of cochlear proteins .with the cellular mechanisms of cisplatin - associated ototoxicity including damage to the outer hair cells , supporting cells , marginal cells of the stria vascularis , spiral ligament , and the spiral ganglion cells , it is evident that the structures of the inner ear are most susceptible to damage by cisplatin chemotherapy , with apoptotic degeneration of the hair cell in the organ of corti being most prominent .
the outer hair cells in the basal turn of the cochlea are most affected [ 27 , 28 ] .
this leads to an initial elevation of high frequency audiometric thresholds , followed by a progressive loss into the lower frequencies with continued therapy [ 27 , 28 ] .
knowledge of the different mechanisms of cisplatin ototoxicity is important for health care professionals as it will create an awareness of its complexity and the resulting clinical presentation .
creation of reactive oxygen species , depletion of antioxidant glutathione and its regenerating enzymes , increased rate of lipid peroxidation , oxidative modifications of proteins , nucleic acids damage by caspase system activation and s - nitrosylation of cochlear proteins .
cisplatin - associated ototoxicity usually manifests as irreversible , progressive , bilateral , high frequency sensorineural hearing loss with tinnitus .
the latter may occur with or without a hearing loss and may be permanent or transient , sometimes disappearing a few hours after treatment or alternatively persisting a week after treatment . while most of the hearing loss is permanent , there is sometimes sporadic and partial recovery . in addition ,
rare cases of unilateral hearing loss have been reported , which are usually explained by tumour location and surgical or therapeutic intervention on the affected side . moreover
, the hearing loss is not always symmetrical [ 33 , 34 ] , with jenkins et al . finding that 75% of women on cisplatin chemotherapy displayed an asymmetry of hearing thresholds of at least 10 db between ears posttreatment .
schmidt et al . , in their investigation of 55 children on cisplatin chemotherapy , found that the high frequency hearing thresholds were slightly elevated in the left ear and that males had a greater degree of hearing loss than the females .
the degree of hearing loss is often variable and is related to the dose ; that is , the higher the cumulative dose , the greater the ototoxic effect [ 35 , 36 ] .
the duration , number of cycles administered , and method of administration also influence cisplatin - associated ototoxicity .
additional factors that may increase ototoxicity include exposure to concomitant noise , chemicals , and other ototoxic medications .
furthermore , evidence also shows that melanin content is related to an increased risk of cisplatin - associated ototoxicity .
individuals with dark eyes and therefore a higher melanin content in the cochlear are at greater risk of ototoxic damage , as the melanin causes retention of the platinum within the cochlear and subsequently increases the risk of damage [ 41 , 42 ] .
individuals presenting with renal insufficiency , that is , high levels of serum creatinine , are at a greater risk for cisplatin - associated ototoxicity . genetic risk factors , such as megalin and glutathione s - transferases gene polymorphism , have also been reported to influence cisplatin ototoxicity , as do physiological factors such as age , with younger children and older adults ( older than 46 years ) presenting with a greater severity of hearing damage .
awareness of these risk factors may assist health care professionals with informational counselling of the patient receiving cisplatin chemotherapy .
the incidence of cisplatin ototoxicity is variable in adults ( table 2 ) and children ( table 3 ) .
the variations may be due to a number of factors , such as differences in the dose , both within a cycle and the total amount administered over multiple cycles , time interval between courses , method of administration , and treatment duration , as well as differences in patient population .
ototoxicity poses a major problem to the cancer patient , as the quality of life after receiving cisplatin chemotherapy may be negatively affected due to hearing loss , resulting in social , emotional , and vocational difficulties , as effective communication is often hindered .
tasks that normal hearing persons take for granted may become challenging and frustrating . in addition , an individual 's safety may be compromised due to the hearing loss , as appropriate response to alarms and warning signals may be delayed .
furthermore , a hearing loss may also result in psychosocial and physical health problems , as well as depression and social isolation .
hence , hearing loss , often referred to as the invisible condition , has serious visible ramifications on the quality of life of a hearing impaired individual .
this is particularly relevant if the individual has already experienced the hearing world , as the hearing function is never restored to normal , even though patients may benefit from the use of assistive listening devices , such as hearing aids and cochlear implants .
the impact of an ototoxic hearing loss may be more profound for infants and young children who are at a critical stage of their speech and language development .
furthermore , the high frequency nature of an ototoxic hearing loss may result in speech recognition and comprehension being compromised , resulting in possible neurocognitive and psychosocial delays .
there is also an elevated risk for academic learning problems and psychosocial difficulties in school - aged children and adolescents .
literature indicated that childhood survivors of neuroblastoma had twice the rate of difficulties , as indicated by parent reports , with reading and math skills , and/or attention and a higher risk of a general learning disability than those without a hearing loss .
there was also poorer self - reported quality of life scores in these children with regard to school functioning .
hence , cisplatin - associated ototoxicity further complicates the morbidity of cancer patients , as it would also isolate them from family members and significant others at a time when they require the greatest support .
advancements in medical knowledge and technology , such as screening and early detection of several cancers , have resulted in notable improvements in relative five - year survival rates for cancer [ 64 , 65 ] .
therefore , improving the quality of life after cisplatin - based chemotherapy becomes increasingly important , and resulting comorbidities such as ototoxicity can be managed appropriately and immediately if adequate monitoring is in place .
the nature of ototoxicity is such that it often goes undetected until speech intelligibility is affected and is usually detected when a communication problem becomes evident .
communication problems , such as constantly asking for repetition or not responding when spoken to , signify that the hearing loss has progressed to the frequencies important for understanding speech . in this case ,
an audiological monitoring programme can avert , to a large extent , the reduced quality of life as a result of hearing loss , as patients on cisplatin chemotherapy can be identified early , counselled , monitored , and managed appropriately through interventions in a logical , systematic , and coherent manner .
audiological monitoring should aim to identify the hearing loss early and reduce its impact on the individual 's life by means of proper medical and hearing intervention .
prospective audiological evaluations remain the only reliable method for detecting ototoxicity before it becomes symptomatic .
an ototoxicity monitoring programme should involve a health care team comprising of an oncology nurse , oncologists , audiologist , and pharmacist to ensure effective sustainability of such a programme , if implemented , with the patient being the central focus .
the audiologist is involved in identifying an ototoxic hearing loss , informing the oncologist of such a development , counselling the patient and their family , and prescribing amplification devices , such as hearing aids and cochlear implants .
early identification of an ototoxic hearing loss provides oncologists with an opportunity to adjust the chemotherapy regimen in order to reduce or prevent further deterioration of hearing . the oncologist and nurses should also counsel patients on the side effects of cisplatin , including ototoxicity , in an attempt to prepare them for treatment outcomes and help them set realistic expectations .
pharmacists who have access to a patient 's medication list may also alert the oncologists and audiologists to those who are on other ototoxic medication and therefore at a greater risk for cisplatin - induced ototoxicity .
effective management of such patients using evidence - based practices may improve management of those with cancer , ensuring that they and their families are counselled and appropriate interventions are timeously implemented .
the principles of early identification and early intervention are a part of ototoxicity monitoring , and the audiologist can manage such a programme . in countries without ototoxicity management guidelines , the guidelines for the audiological management of individuals receiving cochleotoxic drug therapy developed by the american association of speech - language - hearing association may , consequently , guide the audiologist in the implementation of an ototoxicity monitoring programme within a local , regional , or national setting . for widespread acceptance and use ,
ototoxicity monitoring programmes need to incorporate efficient and cost - effective ototoxicity identification techniques , while considering the health care system and demographics of the patient population being managed .
for any population receiving ototoxic medication , the following should be considered : ( 1 ) the patient 's level of alertness or ability to respond reliably ; ( 2 ) the most appropriate times during the treatment protocol for test administration , and ; ( 3 ) the test should comprise the baseline , monitoring and post - treatment evaluations .
appropriate time intervals for audiological assessments may differ depending on the type of cancer as well as the frequency and dose of cisplatin ( figure 2 ) .
the audiological assessments should incorporate a detailed case history , otoscopic examination , immittance audiometry , speech audiometry , dpoaes , and conventional and extended high frequency audiometry ( i.e. , up to 20 000 hz ) ( hfa ) [ 69 , 73 ] . these procedures are all conducted for the baseline assessment and the six - month follow - up evaluation [ 69 , 73 ] . while auditory brainstem response may be used , it is not considered a standard procedure for monitoring ototoxicity . monitoring audiological evaluations during treatment and the one- and three - month follow - up evaluations include case interview , otoscopy , and immittance audiometry as well as air conduction pure tone and objective testing . however , full - frequency threshold testing is impractical for many patients on cisplatin chemotherapy , as these individuals are often extremely ill and easily fatigued . the use of abbreviated threshold monitoring procedures that are clinically practical for these patients is therefore recommended .
one such method involves the use of the sensitive range for ototoxicity ( sro ) .
this is the highest frequency with a threshold at or below 100 db spl followed by the next six lower adjacent frequencies in 1/6-octave steps or the one octave range near the highest audible frequency .
sro is usually determined during the baseline evaluation and is dependent on each patient 's hearing threshold configuration . during monitoring evaluations
however , full - frequency testing should be conducted within the same session if an asha significant hearing change is noted within the sro .
if a patient on cisplatin chemotherapy is still responsive and alert , the protocol presented above would be suitable .
however , a patient who has limited responsiveness may be required to undergo the same audiological evaluations , except speech audiometry .
patients who are responsive as well as those who have limited responses can undergo both behavioural and objective testing . however , those patients who are too ill or too young to respond should undergo only objective testing , such as otoscopy , tympanometry , acoustic reflexes , and dpoaes or abrs . while pure tone audiometry in the conventional frequency range is suitable for evaluating hearing in the range responsible for speech understanding , as well as for differential diagnosis , it is less sensitive to detecting early ototoxic change [ 11 , 70 ] .
the two tests identified as being the most important for the early detection of cisplatin ototoxicity are hfas and oaes , each also having limitations ( see table 4 )
. therefore , using each test in isolation may not be as effective as utilizing a test battery approach , as it increases the chances of obtaining reliable audiologic monitoring data over time .
in addition , these two tests could be used to complement one another in every cycle of chemotherapy to ensure the earliest detection of ototoxicity . the ototoxicity monitoring protocol proposed by
asha represents an aggressive and ideal approach for monitoring ototoxicity and is dependent on a country 's infrastructure and resource constraints .
the asha guidelines may therefore not be generalized to a country without considering the contextual factors that may influence its applicability to that country .
however , it does provide guidance towards creating a roadmap that countries , such as south africa , may aspire towards in implementing an ototoxicity monitoring programme .
similar to india , no programmes have been formally implemented to identify and monitor ototoxicity in patients on cancer chemotherapy in south africa . as a result , there is no contextually relevant research to steer the implementation of an accountable and effective ototoxicity monitoring program in the country .
this is probably one of the main reasons for ototoxicity monitoring programmes not being commonplace in local hospitals and clinics .
in addition , the health of south africans is characterized by a quadruple burden of disease , encompassing the occurrence of infectious diseases , the rise of noncommunicable diseases , and perinatal and maternal disorders , as well as injuries and violence , which may result in cancer receiving low priority for health care services . however
, the creation of an audiological monitoring programme allows for better control of cancer related comorbidities . over the years
, a number of studies have investigated the use of otoprotectants with cisplatin , their purpose being to protect the inner ear from any injury while not interfering with the antitumor effects of cisplatin .
otoprotective strategies include reducing the formation of free radicals by maintaining glutathione levels and antioxidant activity .
three mechanisms may provide protection against cisplatin , these being endogenous molecules , exogenous agents , or a combination of exogenous agents that trigger endogenous protective mechanisms .
however , endogenous agents are not effective against cisplatin when the dose exceeds a certain threshold [ 17 , 81 ] .
nearly all of the otoprotective agents are sulfur- or sulfhydryl - containing compounds ( thio compounds ) , known as antioxidants , and potent heavy metal chelators .
the numerous otoprotective agents utilized in clinical and animal studies include amifostine , d - or l - methionine , methylthiobenzoic acid , lipoic acid , tiopronin , glutathione ester , sodium thiosulfate , melatonin , vitamin e , n - acetylcysteine , dexamethasone , and resveratrol . however
, none of these agents have been found to be unequivocally beneficial in preventing cisplatin ototoxicity and no agent is currently recommended for routine use . further research is therefore needed to find new methods and optimize old ones to prevent and/or treat hearing loss during cisplatin therapy .
in addition , intratympanic administration of medication together with gene therapy needs to be further explored .
intratympanic administration involves the diffusion of the otoprotective agent across the round window into the inner ear , where its therapeutic effect is exerted .
an advantage of this method of administration is that there are higher concentrations of the otoprotective agent in the inner ear , this being in comparison to the use of oral or parenteral administration , without potentially reducing the efficacy of the cisplatin treatment [ 89 , 90 ] .
the disadvantage of this procedure , however , is that each ear would have to be treated with a moderately invasive procedure . alternatively
, gene therapy may prove to be beneficial in protecting an individual against cisplatin - induced hearing loss as several genes , namely , megalin , glutathione - s - transferases , thiopurine s - methyltransferase , and catechol - o - methyl transferase , may be responsible for susceptibility to hearing loss .
if a cisplatin - associated hearing loss results in communication difficulties , it is the audiologist 's ethical responsibility to begin or recommend aural rehabilitation .
however , this intervention should not only occur once hearing loss has been detected but before the patient begins the cisplatin chemotherapy .
aural rehabilitation techniques such as speech reading and counselling on compensatory communication strategies should be conducted .
the counselling should include spouses and significant other , as hearing loss may not only impact the person with cancer but also frequent communication partners .
patients with sensorineural hearing loss due to the use of cisplatin may also benefit from the use of assistive listening devices such as hearing aids or cochlear implants .
children with ototoxic hearing loss may also require the use of personal frequency modulated systems in the classroom .
furthermore , with the recent developments in hearing aid technology , a patient with an ototoxic hearing loss is more likely to receive the desired amplification benefit .
these hearing aids are able to amplify sounds at and above 8000 hz . however , there is limited data indicating significant improvements in speech recognition with the use of this technology .hearing aids with frequency lowering technology achieved by linear frequency transposition , nonlinear frequency compression , or spectral envelope warping .
frequency lowering is used to overcome the limits of either the bandwidth of the device or the functional bandwidth of the ear , by lowering high frequency energy to a region that is more likely to provide and/or benefit from audible sound .
while there are no published studies suggesting one approach to be superior to another , frequency lowering technology has been found to improve audibility and speech understanding of high frequency sounds .
commercially available types of frequency lowering signal processing include frequency transposition ( widex ) , nonlinear frequency compression ( phonak ) , and frequency translation ( starkey ) .
these processors are commercially labelled as audibility extender , soundrecover , and spectral iq , respectively .
these hearing aids are able to amplify sounds at and above 8000 hz . however , there is limited data indicating significant improvements in speech recognition with the use of this technology .
hearing aids with frequency lowering technology achieved by linear frequency transposition , nonlinear frequency compression , or spectral envelope warping .
frequency lowering is used to overcome the limits of either the bandwidth of the device or the functional bandwidth of the ear , by lowering high frequency energy to a region that is more likely to provide and/or benefit from audible sound .
while there are no published studies suggesting one approach to be superior to another , frequency lowering technology has been found to improve audibility and speech understanding of high frequency sounds .
commercially available types of frequency lowering signal processing include frequency transposition ( widex ) , nonlinear frequency compression ( phonak ) , and frequency translation ( starkey ) .
these processors are commercially labelled as audibility extender , soundrecover , and spectral iq , respectively .
this review has highlighted that cisplatin ototoxicity is a frequent adverse event of cisplatin chemotherapy that may negatively affect the quality of life of patients with cancer .
the different molecular and cellular mechanisms involved in cisplatin - associated ototoxicity highlight the complexity of this condition and the consequent difficulty in identifying an effective otoprotective agent .
the varying incidence rates reported in both adults and paediatrics may be due to the different audiological tests employed in the monitoring of the cancer patient 's hearing status and therefore highlight the importance of the use of extended high frequency audiometry and dpoaes in ototoxicity monitoring .
an audiological monitoring programme comprising a team of health care professionals , knowledgeable about cisplatin ototoxicity , may therefore serve to improve evidence - based service delivery to these patients .
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cisplatin is an effective drug used in the treatment of many cancers , yet its ototoxic potential places cancer patients , exposed to this drug , at risk of hearing loss , thus negatively impacting further on a patient 's quality of life .
it is paramount for health care practitioners managing such patients to be aware of cisplatin 's ototoxic properties and the clinical signs to identify patients at risk of developing hearing loss .
english peer - reviewed articles from january 1975 to july 2015 were assessed from pubmed , science direct , and ebscohost .
seventy - nine articles and two books were identified for this review , using mesh terms and keywords such as ototoxicity , cisplatin , hearing loss , and ototoxicity monitoring .
this review provides an up - to - date overview of cisplatin - associated ototoxicity , namely , its clinical features , incidence rates , and molecular and cellular mechanisms and risk factors , to health care practitioners managing the patient with cancer , and highlights the need for a team - based approach to complement an audiological monitoring programme to mitigate any further loss in the quality of life of affected patients , as there is currently no otoprotective agent recommended routinely for the prevention of cisplatin - associated ototoxicity .
it also sets the platform for effective dialogue towards policy formulation and strengthening of health systems in developing countries .
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1. Introduction
2. Method
3. Conclusion
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the synthetic modification
of proteins enables the construction
of biomolecular hybrids that can be used to study protein function , deliver potent therapeutics to cellular targets , and build new materials .
the synthesis of these constructs requires a suite of chemoselective
bioconjugation reactions that proceed under mild , aqueous conditions
in the presence of the native functional groups that are present on
protein surfaces .
the most common methods for protein modification
target the nucleophilic side - chains of lysine and cysteine . however , these strategies can
result in complex product mixtures , as lysine is typically found in
high abundance on the protein surface and
uniquely reactive cysteine labeling sites can be difficult to install
in many instances ( such as thiol proteases and proteins produced via
the eukaryotic secretory pathway , for example ) . many newer approaches
for the site - selective modification of proteins
involve the introduction of artificial amino acids with reactivities
that are orthogonal to those of the native
amino acids . along these lines ,
a number of powerful methods have
been developed for the selective modification of azide , alkyne , alkene , carbonyl , and aniline moieties .
however , the difficulty
of introducing a non - canonical amino acid can limit the application
of these methods .
complementary approaches rely on the site - selective
modification of native amino acids by enzymes .
in addition , a reliable method for the modification of c - terminal
thioesters with n - terminal cysteines , termed native chemical
ligation , has been developed by kent and co - workers .
this method has been used for the semi- and total synthesis of complex
protein substrates , including the chemical synthesis of a single glycoform
of human erythropoietin . as an alternative
strategy ,
we and others have developed methods
for the selective modification of the n - terminal amino group .
methods that target the n - terminus can offer significant advantages
for bioconjugate preparation , as they can be used for a wide range
of protein targets produced by virtually any expression system . conceptually
powerful as they are , however , these methods can be hampered by long
reaction times , often require large excesses of reagent , and/or involve
at least two - steps for the attachment of synthetic molecules .
we have
therefore sought to develop new techniques that can achieve n - terminal
modification with similarly high positional selectivity , but with
significantly improved efficiency .
herein , we report an oxidative
coupling pathway that can preferentially
modify the n - terminus of proteins with fast kinetics .
peptide substrates
were first used to screen reaction conditions and identify the site
of modification . a peptide panel with varying n - terminal residues
was then evaluated to determine the sequence specificity of the reaction ,
leading to the identification of proline as the optimal n - terminal
amino acid .
the reaction was next applied to protein substrates , showing
similarly high levels of conversion when an n - terminal proline residue
was present .
this mild bioconjugation reaction enables the facile ,
rapid modification of proteins to create a well - defined and stable
linkage in a single position , and thus should be useful for many different
applications in chemical biology and the construction of biomolecular
materials .
we have previously reported
the chemoselective coupling of aniline
moieties on proteins to electron - rich aromatic rings , such as o - aminophenols , at slightly acidic ph ( 6.06.5 ) .
these reactions require the addition of naio4 or k3fe(cn)6 as a terminal oxidant , with the latter
reagent exhibiting improved compatibility with glycoproteins and substrates
with free sulfhydryl groups . the use of ferricyanide as the oxidant
also yields a single reaction product ( 1 ) , whereas periodate
leads to the formation of a ring contracted species as a competing
pathway .
the ferricyanide - based reactions
are presumed to involve an o - iminoquinone as the
reactive intermediate , as suggested in scheme 1 , or could involve the corresponding o - quinone after
imine hydrolysis . taken together , the oxidative coupling strategies
have demonstrated excellent functional group compatibility and the
ability to join large unprotected biomolecules at low concentrations ,
as demonstrated for the coupling of peptides , polymers , and nucleic
acids to specific locations on viral capsids and antibody fc domains . while these coupling reactions
were found to be highly aniline - selective
under the conditions used , several studies have reported the reaction
of o - aminophenols and o - catechols
with native amino acids , dating back to 1949 .
in addition , recent work by messersmith has shown the ability of
proteins to be coupled to o - quinone moieties present
on polydopamine - coated surfaces .
these reports
suggested that secondary coupling pathways could be developed to achieve
the modification of native amino acids with o - aminophenols
( scheme 1 ) , and thus initial experiments were
designed to identify the optimal reaction conditions for achieving
this with complex molecules . in our previous
work
, we noted that low amounts of background reactivity could be
observed in aniline - based oxidative coupling reactions when higher
ph conditions ( > ph 6.5 ) were used . in
an initial effort to characterize this alternative reaction pathway ,
conditions and reaction times
were first screened to increase the
reaction yields for peptides that did not contain aniline groups .
angiotensin i and melittin were used as substrates , as they contain
many reactive amino acids , including lys , arg , his , trp , and tyr .
the peptides were exposed to 2-amino - p - cresol using
k3fe(cn)6 as the oxidant .
the reaction ph was
varied from 5.5 to 8.5 , and the reaction mixtures were analyzed using
maldi - tof ms ( supporting information figure
s1 ) .
the level of modification increased with the basicity of the
reaction , with near quantitative modification of angiotensin i after
20 min at ph 7.5 and higher . throughout these initial investigations ,
it was noted that angiotensin and melittin showed significant differences
in reactivity , with angiotensin consistently demonstrating better
conversion .
ms / ms analysis of the angiotensin product was used to
identify the participating residue , and revealed that the n - terminal
amino group was responsible for the observed reactivity ( supporting information figure s2 ) . as further
confirmation of the site - selectivity ,
several peptide substrates were
screened for reactivity ( figure 1a and supporting information figure s3 ) .
consistent
with the n - terminal reaction selectivity , the only peptide that did
not react had a pyroglutamate in this position , and therefore no free
amino group .
( a )
modification of commercially available peptides was monitored by maldi - tof
ms .
( b ) a positional scan of the n - terminal amino acid was
evaluated .
peptides with the sequence xadswag were tested for reactivity with 2-amino - p - cresol .
the reactions were run with 100 m peptide , 200 m
aminophenol , and 5 mm ferricyanide at ph 7.5 and analyzed by lc ms .
shown is the average percent modification with error bars representing
the standard deviation of three reactions .
the same peptides were
used for a screen of coupling partner equivalents ( c ) and a time screen
( d ) .
most buffers did not alter the reactivity , but
imidazole and buffers containing a morpholine or piperazine ring ( pipes ,
hepes , hepps , and mops ) significantly impeded the reaction ( supporting information figure s4 ) .
this is possibly
due to small amounts of buffer impurities that react competitively
with the oxidized intermediate .
the time course of the reaction was
also investigated ( supporting information figure s5 ) .
in addition , it was found that the peptides could be modified
using naio4 as the oxidant , or 4-methylcatechol as the
coupling partner ( supporting information figures s67 ) . both of these reactions showed the same dependence
on ph ; however , moderate levels of modification were still observed
at acidic ph ( 5.56.5 ) when using these alternative coupling
conditions .
these observations suggested that the peptides were reacting
with the o - quinone intermediate formed in
situ from either the catechol or the iminoquinone precursor
( after imine hydrolysis ) .
given the differential
reactivity observed on peptide substrates , we synthesized peptides
with varied n - terminal residues ( xadswag ) to determine the specificity of the reaction .
the base sequence
was selected to increase the mass of the peptide , impart water solubility ,
and include a tryptophan residue for quantitation using uv monitoring .
the peptides were synthesized on the solid phase using standard fmoc
synthesis , cleaved from the resin , and purified by hplc .
after purification ,
the peptides were resuspended in phosphate buffer , ph 7.5 , adjusted
to a concentration of 1 mm , and stored at 20 c until
use .
to assess the effect of the n - terminal residue on reactivity ,
the peptides ( 100 m ) were reacted with 2 equiv of 2-amino - p - cresol ( 200 m ) in the presence of k3fe(cn)6 ( 5 mm ) in phosphate buffer , ph 7.5 ( figure 1b ) .
ms ( see supporting information figure s8 for representative ms data
for the modified peptides ) .
most n - terminal amino acids showed good - to - high
levels of conversion ( 6090% ) , but proline stood out as the
only residue that showed nearly complete modification ( 90100% ) .
a second observation of this screen was the fact that tryptophan ,
tyrosine , and methionine residues were not oxidized by the ferricyanide
reagent , consistent with our previous report of oxidative coupling
with this oxidant ( see supporting information figure s8 ) .
however , free cysteine residues can be oxidized to various
species , potentially including disulfides and sulfenic acids , and
thus it is recommended that they be protected as disulfides before
oxidative coupling is attempted ( vide infra ) .
to optimize the reagent ratios ( specifically the equivalents of o - aminophenol ) , the peptides ( 100 m ) were reacted
with 110 equiv of the o - aminophenol ( 1001000
m ) in the presence of ferricyanide ( 10 mm ) . after 30 min , the
reactions were quenched with excess tris(2-carboxyethyl)phosphine
( tcep ) .
it was demonstrated that conversion was highest using 25
equiv of the coupling partner ( figure 1c ) .
using more than 5 equiv of
this was most likely due to the ability of
the aminophenol to react with itself at higher concentrations ( 1
mm ) . consistent with this , when using 10 equiv of the o - aminophenol , a byproduct was observed with a mass that corresponded
to the condensation of 3 aminophenols ( 344 da ) .
we also investigated
the differences in coupling rates for representative
n - termini .
the reaction of 2-amino - p - cresol with
three different peptides was monitored over the course of 1 h ( figure 1d ) .
the peptides ( 100 m ) were reacted with
2 equiv of the o - aminophenol ( 200 m ) in the
presence of ferricyanide ( 5 mm ) , and aliquots were quenched with excess
tcep at the indicated time points .
the proline terminal peptide not
only reached the highest level of conversion , but also did so in a
significantly shorter time than the other termini . despite efforts
to optimize conditions for all n - termini
the reaction of n - terminal
amines with o - aminophenols
was characterized using small molecule mimics .
the methyl esters of
phenylalanine ( h - phe - ome ) and proline ( h - pro - ome ) were coupled to
2-amino - p - cresol using ferricyanide at ph 7.5 .
the
crude products were characterized using two - dimensional nmr and high - resolution
mass spectrometry .
the primary amine of h - phe - ome formed p - iminoquinone product 2 , which was analogous to the
one formed with aniline coupling partners ( scheme 1 , supporting information figure
s9 ) . however , the secondary amine of proline prevented the formation
of the p - iminoquinone tautomer , and thus favored o - quinone product 3 ( scheme 1 , supporting information figure
s10 ) .
given the different linkage obtained with proline , we verified
the stability of the product to a variety of conditions .
the proline
terminal peptide , padswag , was first modified with 2-amino - p - cresol .
after purification , the modified peptide ( 100
m ) was exposed to reductants , nucleophiles and acidic and basic
ph ( 10 mm additives or buffer ) .
no loss of product was
observed under any of the conditions tested , demonstrating the hydrolytic
stability of the product ( supporting information figure s11 ) .
the ability of the linkage to withstand these conditions
renders this method quite useful for the construction of biomolecular
materials for a variety of applications . with a view toward in vivo applications ,
current efforts are examining the
stability of the linkage in blood plasma , as well as evaluating the
intrinsic immunogenicity of the o - quinone group . in the process of characterizing the reaction products ,
it was observed
that the colored products absorbed light at wavelengths greater than
500 nm ( with max between 505 and 525 nm depending
on the amine coupling partner ) . as the starting coupling partners
and ferricyanide did not absorb at these wavelengths , this unique
absorbance provided a means to monitor the reaction progress . the
different amine coupling partners ( p - toluidine , h - pro - ome ,
and h - phe - ome )
were reacted with 4-methylcatechol in the presence
of 10 mm ferricyanide , and the absorbance of the resulting solution
was monitored at 520 nm to determine the relative rates of reactivity
( figure 2a ; for unnormalized data see supporting information figure s12 ) .
the catechol
substrate was used for these studies to simplify the reaction pathway
by eliminating the imine hydrolysis step .
the reactions were run under
pseudo - first order conditions with 0.1 mm catechol and 1 mm amine
coupling partner . when the reaction was carried out at ph 6.0 ,
the
reaction with the proline analogue reached completion nearly as rapidly
( 2 min ) , but the reaction with the primary aliphatic amine
of phenylalanine required longer reaction times ( 10 min ) .
( a ) amine coupling partners were reacted with 4-methylcatechol
as a model substrate .
reactions were run under pseudo - first
order conditions with 100 m catechol , 1 mm amine , 10 mm ferricyanide
in 50 mm phosphate buffer .
( b ) a peptide containing both an n - terminal
proline and a p - aminophenylalanine residue ( pad(paf)swag ) was tested for reactivity with 2 equiv of 2-amino - p - cresol at ph 6 .
the remainder of the reaction was purified
and then reacted with 2 equiv of the aminophenol at ph 7.5 and analyzed
by lc ms . by quantifying product
formation by absorbance
, we were also able
to measure the second - order rate constant for the proline - based coupling
( supporting information figure s13 ) .
the
reaction of 1 equiv of h - pro - ome ( 100 m ) with 1 equiv of 4-methylcatechol
( 100 m ) and 100 equiv of k3fe(cn)6 ( 10
mm ) was performed in triplicate at 25 c .
the second - order rate
constant for the coupling was determined to be 44 4 m s. while proline reacted rapidly with the electron - rich
coupling partner , the small molecule studies indicated that aniline
should react faster .
the rate for the aniline reaction was too fast
under these conditions to determine the second - order rate constant
accurately . given the differences in reactivity observed
at ph 6.0 and 7.5 ,
we hypothesized that it would be possible to modify the aniline side
chain of p - aminophenylalanine ( paf ) and the n - terminal proline amine sequentially . to test this hypothesis ,
we synthesized a peptide containing both reactive moieties ( pad(paf)swag ) .
only one modification was observed when the peptide
was reacted with 2-amino - p - cresol at ph 6.0 ( figure 2b ) .
ms / ms analysis of the modified peptide confirmed
that the single modification occurred on the aniline side chain ( supporting information figure s14 ) . after this
step , the peptide was purified and subsequently reacted with 2-amino - p - cresol at ph 7.5 .
reaction at the higher ph enabled a
second modification of the peptide substrate , at the n - terminal proline
residue .
the differential reactivity
was investigated by comparing the reactivity of a protein containing
a paf residue to proteins without the artificial
amino acid .
the paf residue was introduced into the
coat protein of bacteriophage ms2 , which self - assembles into a spherical ,
hollow protein shell .
myoglobin and a mutant of the tobacco mosaic
virus ( tmv ) coat protein were used as native protein substrates .
reactions
with 2-amino - p - cresol were either performed on the
isolated , individual proteins or with the aniline containing protein
mixed with the native protein substrate ( supporting
information figure s15 ) .
addition of the aniline containing
protein to the native protein decreased the n - terminal reactivity ,
indicating that the aniline residues react more rapidly than n - terminal
residues with the o - aminophenols .
in addition , ms2
showed significantly higher reactivity at all of the phs tested , confirming
preference for aniline residues .
the oxidative coupling reaction
with n - terminal amino groups was
first tested on proteins with native n - termini .
several proteins were
reacted with o - aminophenol - functionalized 5 kda peg
under the optimized reaction conditions ( figure 3 ) .
the native proteins showed moderate levels of reactivity , which
could be attributed to inaccessible n - termini or simply to the less
reactive n - terminal residues . to test
if proline terminal proteins
were more reactive , a proline residue was introduced to the n - terminus
of gfp and the tobacco mosaic virus ( tmv ) coat protein .
the n - terminus
of tmv was also slightly extended from the native sequence ( addition
of pag ) .
the proline - gfp was treated with a variety of conditions
to determine the specificity of the reaction ( figure 4a ) . only at basic ph in the presence of both the o - aminophenol substrate and
the oxidant was modification observed .
additionally , the proline - terminal variant showed significantly improved
reactivity compared to that of the wild - type n - terminus .
these high
levels of modification were maintained even when only 12 equiv
of the o - aminophenol peg was used .
the site of modification
was confirmed to be the n - terminal proline by lc
ms / ms analysis
of a tryptic digest of proline - gfp modified with 2-amino - p - cresol ( supporting information figure
s16 ) .
( a )
the n - terminus of several proteins was pegylated using o - aminophenol - functionalized 5 kda peg and ferricyanide .
( b ) modification
of wild type proteins with 5 kda o - aminophenol - peg
was monitored by sds - page .
( a ) a proline
was introduced to the n - terminus of gfp .
the proline terminal
variant showed much higher levels of modification than the wild - type
protein .
the band doubling is due to a gel artifact , and appears in all
lanes .
( b ) mutants of tmv were reacted with 5 equiv of 2-amino - p - cresol and 1 mm k3fe(cn)6 for 30 min and analyzed by lc ms .
reaction conditions for both native and proline terminal
proteins
were optimized by evaluating reactivity with myoglobin and proline - gfp .
the reaction time , buffer , and ph were screened ( supporting information figures s17 and s18 ) .
similar to the
results obtained with peptide substrates , the reaction reached its
highest level of conversion after about 1530 min .
in addition ,
most buffer salts tested were compatible with the reaction with the
exception of buffers containing morpholine ( mops ) or piperazine moieties
( hepes ) , as was observed with peptide substrates .
these buffers decreased
the level of modification slightly , but did not completely inhibit
reactivity .
the effect of reaction ph was tested using both k3fe(cn)6 and naio4 as the oxidants .
little
reactivity was observed at acidic ph , and the level of conversion
increased between ph 7.0 and 8.0 . at higher reaction ph ( 8.0 )
a second modification was observed , indicating that lysines may also
participate in the reaction .
however , it is also possible that under
more forcing conditions , such as higher reaction ph or increased concentration
of aminophenol substrate , the aminophenol reacts with both itself
and the n - terminal amino group resulting in double modification of
the n - terminus .
n - terminal mutants of tmv were also evaluated
for their reactivity
with o - aminophenols .
the tmv monomers assemble into
well - known double disk structures , displaying 34 copies of the n - terminal
groups on their peripheries .
two n - terminal
mutants ( pag and ag ) were reacted with 5 equiv of 2-amino - p - cresol ( 100 m ) and 1 mm k3fe(cn)6 for 30 min .
ms demonstrated that the
proline terminal mutant reached nearly complete conversion , while
the alanine terminal mutant showed low levels of modification under
these coupling conditions ( figure 4b , see supporting information figure s19 for wider mass
range and ion series ) .
the compatibility of the reaction with
cysteine residues was also
tested using tmv . a single cysteine residue ( s123c )
was introduced
into the tmv coat protein with a proline n - terminus ( pag s123c tmv ) .
this mutant was reacted with 2-amino - p - cresol and
analyzed by lc
the cysteine residue also reacted with the o - aminophenol , resulting in two modifications .
however , it was found that the n - terminal proline could be modified
selectively if the cysteine was first capped ( figure 5a , b , see supporting information figures s2122 for wider mass range and ion series ) . to do
this ,
the cysteine residue was protected as a disulfide bond by reaction
with 5,5-dithiobis(2-nitrobenzoic acid ) ( dtnb , ellman s
reagent ) .
after the oxidative coupling step the disulfide bond was
readily reduced by tcep , leaving the free cysteine and the modified
n - terminus .
alternatively , the cysteine residue was modified with
a maleimide , followed by modification at the n - terminus with an o - aminophenol reagent .
a ) pag s123c tmv was reacted with small molecule substrates and analyzed
by lc
cysteine residues were protected as a disulfide using
ellman s reagent ( dtnb ) before oxidative coupling .
subsequent
reduction of the disulfide resulted in selective modification of the
n - terminus .
the n - terminal
proline was then modified with a rhodamine - functionalized o - aminophenol .
this strategy allowed for the direct , dual modification of
the
protein at both the cysteine residue and the n - terminus .
two fluorophores
paired for frster resonance energy transfer ( fret , alexa fluor
488 c5-maleimide and o - aminophenol functionalized
rhodamine b ) were thus conjugated to tmv using this strategy to create
a templated array of chromophores for light harvesting applications .
the free cysteine was first quantitatively labeled with an alexa
fluor maleimide ( supporting information figure s22 ) .
the n - terminal proline was then coupled to a fluorescent o - aminophenol resulting in 50% modification of the
tmv monomers with both fluorphores ( figure 5b ) .
complete modification of the n - terminus was not observed as tmv
precipitated from solution with increasing levels of modification
with the rhodamine dye . in current experiments
, we are using this
dual - labeling strategy to introduce more soluble chromophores .
the oxidative coupling
reaction was also compared to the reaction
of protein amines with activated esters .
this acylation methodology
is commonly employed , and can be targeted to the n - terminus by controlling
the reaction ph in some cases .
the reactions
were compared on creatine kinase , a protein with a native proline
n - terminus .
reaction with 15 equiv of o - aminophenol
peg resulted in good levels of modification of creatine kinase ( 5060% ) ,
while reaction with 15 equiv of n - hydroxysuccinimide
( nhs ) peg resulted in low levels of modification ( 525% , figure 6 ) .
only when a vast excess of the nhs peg was used
were moderate levels of modification achieved . as was the case with
proline - gfp ,
some over modification was observed under the oxidative
coupling conditions when using five or more equivalents of aminophenol .
this could result from dimerization of the oxidized species before
protein coupling , but has yet to be characterized due to the low abundance
of this product . in any case , lowering the reaction ph slightly or
using fewer equivalents of the o - aminophenol substrate
prevented the over modification from occurring .
the modification of the
n - terminus of creatine kinase with aminophenol
peg was compared to the reaction of creatine kinase with nhs peg .
in this study , we have identified conditions
for the oxidative
coupling of o - aminophenols to n - terminal amino acids .
proline residues work particularly well with this strategy , and are
therefore strongly recommended when using it .
these groups can be
introduced readily in n - terminal positions using site - directed mutagenesis
and escherichia coli expression , especially
since the methionine residue resulting from the start codon is cleaved
when proline is in the second position .
the fast kinetics of the reaction allow it to be successful even
at low reagent and substrate concentrations , and suggest that it can
be used for sterically demanding bioconjugations .
the oxidative
coupling strategy reported here offers two distinct
advantages over other n - terminal labeling methods .
first , the modification
occurs in a single step and does not require initial oxidation of
the n - terminus .
second , the fast second - order kinetics allow for low
concentrations of the coupling partners to be used .
however , to achieve
high levels of modification on protein substrates , proline was required
as the n - terminal residue .
this new protein modification strategy is currently being explored
in our lab for the generation of protein - based materials . in the larger
context ,
new techniques for the introduction of a single functional
group in a specific position on a protein surface are always in demand .
the ability of the n - terminal oxidative coupling method to achieve
this in a single , brief reaction step is highly advantageous , and
the fact that it can be combined with cysteine modification chemistry
provides new opportunities for complex bioconjugate synthesis .
|
the
synthetic modification of proteins plays an important role
in chemical biology and biomaterials science .
these fields provide
a constant need for chemical tools that can introduce new functionality
in specific locations on protein surfaces . in this work
, an oxidative
strategy is demonstrated for the efficient modification of n - terminal
residues on peptides and n - terminal proline residues on proteins .
the strategy uses o - aminophenols or o - catechols that are oxidized to active coupling species in
situ using potassium ferricyanide .
peptide screening results
have
revealed that many n - terminal amino acids can participate in this
reaction , and that proline residues are particularly reactive .
when
applied to protein substrates , the reaction shows a stronger requirement
for the proline group .
key advantages of the reaction include its
fast second - order kinetics and ability to achieve site - selective modification
in a single step using low concentrations of reagent .
although free
cysteines are also modified by the coupling reaction , they can be
protected through disulfide formation and then liberated after n - terminal
coupling is complete .
this allows access to doubly functionalized
bioconjugates that can be difficult to access using other methods .
|
Introduction
Results and Discussion
Conclusion
|
many studies over the past 15 years have focused on discovering solvents that help promote efficient cc bond formation under benign reaction conditions .
new solvents in dielsalder(2 ) and barbier reactions have been sought to help streamline the one - pot transformations further , reducing synthetic steps and organic waste .
polar media often accelerate reactions of this type which has led to the use of water as an acceptable solvent for such organic conversions .
recent contributions from our laboratory to this field have centered on coupling reactions between propargyl aldehydes and allyl or propargyl halides under barbier conditions to form homoallylic and homopropargylic propargyl alcohols .
these compounds find wide use as synthetic templates and show a propensity for oxy - cope rearrangements ( scheme 1 ) .
the use of -chloropropargylphenyl sulfide ( 4 ) , coupled with a propargyl aldehyde should offer an easy route into formation of enediyne and epoxydiyne skeletal structures ( scheme 2 ) .
previous studies revealed that formation of 2 or 3 under indium - promoted barbier conditions is solvent dependent .
use of an organic solvent for the coupling reaction results in a mixture of 2 and 3 while the use of water as a solvent yields 2 as the sole product .
it is possible that water protonates the coupled product to form 2 quickly , quenching the alkoxide intermediate , thereby suppressing the possible oxy - cope pathway shown in scheme 1 .
it may also be possible that the increased polarity of the solvent itself stabilizes the anion formed upon coupling , thus raising the relative rate of cope rearrangement in this system . with a less polar , aprotic solvent ,
the anion will not be quenched , nor will it be stabilized to the same extent as witnessed in water .
a combination of these phenomena may be why an oxy - cope rearrangement occurs under less polar conditions , forming product 3 ( scheme 1 ) .
a coupling reaction between 1 and 4 ( scheme 2 ) was conducted under aqueous conditions to form 2 ( y = sph ) , with good regioselectivity ; however , the rate of this reaction is surprisingly slow .
although reactions of the type shown in scheme 1 are reported in the literature to be accelerated by polar solvents such as water , coupling reactions between 1 and 4 , as shown in scheme 2 , are quite slow . indeed ,
when water is used as the solvent , reactions often require 1036 h for completion .
this oil has a density greater than that of water so it is presumed to be an organometallic species formed by interaction of the indium metal with the halide species .
isolation of the oil followed by workup in an acidic thf / h2o mixture leads to isolation of propargylphenyl sulfide and , to a smaller extent , the aldehyde .
we surmise that low solubility of this organometallic complex leads to poor reagent mixing , slowing the barbier coupling under aqueous conditions .
second , indium - promoted coupling reactions conducted in water become more acidic as the reaction proceeds .
we believe it is possible that the aldehyde is in equilibrium with its corresponding hydrate species , reducing the electrophilicity of the carbon center .
a combination of these two reasons would lead to a decrease in the rate of reaction between 1 and 4 when conducted in an aqueous solvent .
the reaction proceeds more quickly ( 812 h ) in dmf , a less polar solvent , due to better solubility of all reagents , however , a mixture of 2 and 3 was isolated .
it was originally expected that a mixture of water and dmf would lead to faster reaction times while still maintaining regiocontrol of product formation . the use of a water / dmf solvent system , however , resulted in a mixture of products with water constituting up to 50% v / v of the solvent system .
when water exceeds 50% v / v of the solvent system , only product 2 is isolated , but the rate of reaction slows perceptively once again . to help expedite this reaction sequence ,
we began a search for solvent with polarity similar to , or greater than that of water .
it was expected that an extremely polar organic solvent would promote the same type of acceleration normally witnessed in barbier couplings carried out under aqueous conditions .
an organic solvent could also help solubilize the intermediate and reduce possible formation of the hydrate species , thus allowing for a faster coupling reaction to occur .
if increased polarity stabilizes the anion formed by coupling of 1 and 4 , oxy - cope rearrangement should also be suppressed . with a dielectric constant of 186.9 at 25 c , significantly greater than that of water ( 78.37(7 ) ) , and dmf ( 38.3(7b ) ) ,
n - methylformamide ( nmf ) has found occasional use in previous reactions requiring polar organic solvents ; however , it has historically found more widespread use as an organic reagent . with a pka of approximately 24,(7d )
nmf is reactive under a range of organometallic reagent conditions , limiting its use as a solvent .
indium - promoted coupling reactions , however , have been shown to be accelerated under aqueous conditions,(1 ) and the relative acidity of nmf , being higher than that of water , should not hinder the proposed barbier couplings proposed within this paper .
we were especially intrigued by the fact that the dielectric constant of nmf increases to 220 as the temperature is lowered from 25 to 0 c .
we were interested in examining what effect the increase in dielectric constant at lower temperature could have on a barbier - style reaction of the type shown in scheme 2 among others .
this paper reports on the use of nmf in barbier - style reactions with a focus on rates of reaction , stereochemistry , and its use to form enediyne and epoxydiyne skeletal structures.(9 )
although nmf has characteristics similar to those of dmf , it also has characteristics similar to water , such as a polar xh bond.(7 ) thus , having found a very polar solvent with properties similar to both water and organic systems , we hypothesized that the 1,2-coupling product could be formed in high yield under short reaction times .
a series of reactions was set up to test this hypothesis . as shown in table 1 , use of either water or
nmf resulted in formation of the 1,2-product ( 2 ) solely with no trace of 3 detected in the product mixture .
although the yield of product was moderate to high in either solvent , product formation in reactions conducted in nmf proceeded at a much greater rate .
we believe that there are two possible reasons behind the rate increase of coupling reactions carried out in nmf .
the ratio of reagent was as follows : 1.0:1.5:1.1 ( aldehyde / halide / indium ) .
the reaction was run at 0.1 m with respect to indium . in the lewis acid catalyzed reaction , 10 molar % of the lewis acid was used .
all species completely dissolved in nmf , unlike what was observed for reactions in water . as such , we believe mixing is much more efficient , allowing the reaction to proceed at a faster rate .
has also been noted in many literature examples to lead to rate enhancement in indium - promoted barbier reactions .
we believe a mixture of better solubility coupled with greater solvent polarity results in an additive effect toward increasing the rate of reactions conducted in nmf when compared to water .
h - bonding capabilities of nmf may explain the greater regioselectivity for reactions conducted in nmf when compared to those observed in dmf and thf ; however , the h - bonding capacity of nmf is much lower than that of water.(7d ) increased solvent polarity , thus increasing the stability of the intermediate anion and raising the relative energy of the oxy - cope rearrangement , may seem more likely at this time . in an effort to exclude the solvent as a direct participant in intermediate formation when nmf is used , nmr spectroscopy studies were performed to determine if a possible hemiaminal or imminium species was forming in situ ( figure 1 ) .
the imminium functionality carries a positive charge and would increase the electrophilicity of the reactive carbon .
mixing of 1 and nmf under sonication for periods of up to 24 h , however , revealed the presence of only 1 and nmf by nmr spectroscopy . possible formation of hemiaminal or imminium species . the presence or absence of a lewis acid has shown to help control stereoselectivity in indium - promoted cc bond - forming reactions .
previous studies in our laboratory have shown that the addition of indium(iii ) chloride as a lewis acid favors formation of the syn - product in indium - promoted cc bond - forming reactions between 4 and aldehyde functional groups . in the absence of the lewis acid ,
the anti - product is favored ( figure 2).(5b ) it was our hope that similar effects would be witnessed in the present system , especially while using nmf as the reaction solvent , and we were pleased to see this trend continue .
absence of a lewis acid in the reagent mixture favored a nonchelated pathway , leading to formation of anti-2 .
use of indium chloride to control stereochemistry.(5b ) the presence of a lewis acid in the reaction mixture favored a chelated pathway , leading to formation of syn-2 ( figure 3 ) .
diastereoselectivities were determined either by direct measurement of vicinal coupling constants in the hydroxyl sulfide or by corresponding conversion to the epoxide compounds as shown in figure 4.(11 ) general j values for hydroxyl sulfides and epoxide structures .
entries 1 , 5 , and 13 ( table 1 ) describe reactions run in nmf in the absence of a lewis acid .
good anti - selectivity is witnessed with ratios as high as 15:85 ( syn / anti ) . in all cases , with
the exception of r = phenyl , systems , with and without incl3 , run in nmf gave better selectivity than systems run in water .
both systems showed poor selectivity when r = tms for reasons of which we are not quite sure . when incl3 was added as a lewis acid , the chelated route was favored in ratios as high as 90:10 ( syn : anti , entry 15 ) .
n - methylformamide was shown to increase the rate of reaction compared with water and offered better stereocontrol in the systems studied .
enediyne and epoxydiyne functionalities contain either a ( z)-3-ene-1,5-diyne or ( z)-3-epoxy-1,5-diyne unit which leads to their noted activity.(9 ) these unique functional groups cyclize to form a p - benzyne- or p - benzyne - like intermediate that interacts with dna by abstracting hydrogen.(9 ) the dna diradical then either couples with itself , or cleaves , stopping replication .
natural product enediyne and epoxydiynes have found use as anticarcinogenic compounds.(9 ) this area of study is slowed , however , by complicated access into the skeletal systems required.(9 ) literature procedures that find use in epoxy alkyne formation such as halohydrin cylization,(12)m - cpba,(13 ) or oxone(14 ) have been shown to be successful , but limited examples are known with these systems at present . under the barbier coupling conditions described in this paper , formation of compound 2 offers an efficient and controlled method for the formation of either an enediyne or epoxydiyne skeletal structure under benign conditions by allowing easy conversion to either the cis - epoxydiyne ( from syn-2 , scheme 3 ) or the cis - enediyne ( from anti-2 , scheme 4 ) .
facile synthesis of epoxydiyne and enediyne functional groups under the reported conditions would allow for their use as template structures in organic synthesis , opening access to more sophisticated units for further exploration . in the following pages , we describe the transformation of 1 into either an epoxydiyne ( 5 ) or enediyne ( 6 ) skeletal structure in two steps with high regio- and stereocontrol using nmf as a new solvent to form 2 under barbier conditions , followed by a one - step transformation to yield either 5 or 6 . as shown in scheme 3 , syn-2 can adopt the correct conformation to form a cis - oxirane upon reaction with either me3obf4 or tloet .
conversion of the hydroxy sulfide starting material ( 2 ) to the epoxyalkyne product ( 5 ) was quite successful as shown in table 2 . in all cases ,
this fact is worth mentioning as barbier reactions with propargyl halides can lead to rearrangement of the organometallic intermediate into an allenyl product .
reagent 4 has been previously shown to offer one of the few examples of a barbier reaction with a propargyl halide where rearrangement does not occur.(5b ) use of tloet(17 ) gave yields similar to that of me3obf4(18 ) in all cases of oxirane formation , with stereoselectivity preserved under both sets of conditions .
use of the anti-2 allows formation of the ( e)-alkene ( the alkyne functional groups are cis to each other ) functional group by the pathway shown in scheme 4 .
formation of the enediyne product was possible under slightly acidic conditions . a catalytic amount of p - tsoh is added to anti-2 in a mixture of water and thf ( 10% water ) with stirring for 37 h. the reaction was also conducted under basic conditions after transformation of the oh group to a tosylate .
yields were quite low under basic conditions , presumably due to the competing acidity of the alkynyl proton , leading to decomposition and side product formation . due to low yields ,
results of the acid - catalyzed conversion of anti-2 to ( e)-6 are shown in table 3 .
a slight amount of isomerization was witnessed under acidic conditions ( about 3% of the product mixture ) , but overall , good stereocontrol was realized in these conversions , allowing for easy entry into the cis - enediyne ( ( e)-alkene ) skeletal structure in a two - step sequence .
the assignment of e- and z - isomers was made by comparison of the -vinyl proton shift .
alignment of the -vinyl proton and the heteroatom cis to each other gives rise to a lower field proton resonance than trans alignment of these two groups ( figure 5).(19 ) the assignments for the enediyne products are compared correctly with the stereochemistry of compounds 2 and 3 within this study .
coupled with the original indium - promoted barbier reaction , the transformation of starting materials 1 and 4 into enediyne or epoxydiyne compounds proceeded in two steps in an overall 6080% overall yield , offering an efficient , benign , and good - yielding formation of these important skeletal structures .
the use of n - methylformamide is shown to be a nice alternative to aqueous conditions in indium promoted coupling reactions between propargyl aldehydes ( 1 ) and -chloropropargylphenyl sulfide ( 4 ) .
this paper introduces the use of nmf as s viable solvent in a barbier coupling reaction , revealing increased rates and better selectivity when compared to reactions conducted in water and other organic solvents .
the increased efficiency of the barbier coupling reaction as described in this paper allows for facile formation of epoxy - and enediyne skeletal structures in an overall two - step procedure .
the introduction of nmf as an alternative solvent in barbier reactions will open new pathways to organic synthetic chemists . further studies to test other halides and aldehyde functional groups under barbier coupling reaction conditions in nmf are currently underway and will be reported in due time .
tetrahydrofuran was purified by distillation under an argon atmosphere over sodium and benzophenone prior to use .
radial chromatography was performed using a chromatatron . to a solution of 30 mmol of alkyne in 80 ml of thf that had been cooled to 60 c under nitrogen
was added 15 ml of n - buli ( 2.0 m in thf ) with stirring . to this solution
was added n , n - dimethylformamide ( 4.66 ml , 60 mmol ) dropwise over 10 min .
after 20 min of further stirring , the solution was added to a mixture of 75 ml of tert - butyl methyl ether and 160 ml of 10% kh2po4 solution which had been previously cooled to 0 c .
the entire solution was stirred at 0 c for 30 min and then allowed to warm to room temperature .
the organic layer was dried over mgso4 , and the solvent removed under vacuum to give a clear oil .
all propargyl aldehydes were used without further purification and could be stored at 0 c for 23 weeks .
yields for this reaction ranged from 85 to 95% . a magnetically stirred solution of aldehyde ( 1 ) ( 2.0 mmol ) in deionized water ( 22 ml ) was treated with -chloropropargylphenyl sulfide ( 702 mg , 3.0 mmol ) and indium powder ( 228 mg , 2.0 mmol ) .
the solution was allowed to proceed at room temperature until no 1 was seen ( tlc analysis ) .
the layers were separated , and the aqueous phase was extracted with dichloromethane ( 2 10 ml ) .
the combined organic layers were dried over mgso4 and evaporated to leave a dark yellow to brown oil .
purification was accomplished by flash chromatography or radial chromatography on silica gel ( hexanesethyl acetate ) to give a mixture of hydroxy sulfides ( 2)(11 ) as a light yellow oil .
purification was accomplished using radial chromatography on silica gel ( 40:1 hexanesethyl acetate ) to give a mixture of diastereomeric hydroxy sulfides .
stereoselectivity was determined upon transformation to the epoxide product : yield = 471 mg ( 1.76 mmol ) = 83% ; h nmr ( 300 mhz , cdcl3 ) 0.91.5 ( m , 7h ) , 1.82 ( d , j = 1.7 hz , 0.2h ) , 1.87 ( d , j = 1.6 hz , 0.8h ) , 2.13 ( t , j = 6.5 hz , 2h ) , 4.09 ( m , 1h ) , 4.86 ( m , 1h ) , 7.17.8 ( m , 5h ) , the oh proton was not detected ; c nmr ( 75 mhz , cdcl3 ) 15.5 , 18.2 , 21.3 , 34.2 , ( 41.5 , 42.9 ) , ( 65.0 , 66.1 ) , 71.3 , 78.5 , 80.2 , 84.7 , 125.4 , 126.1 , 126.3 , 127.6 , 128.0 , 136.4 ; ms m / z ( m ) calcd 258.1078 , obsd 258.0998 .
calcd for c16h18os : c , 74.38 ; h , 7.02 . found : c , 74.61 ; h , 6.99 .
separation was accomplished using radial chromatography on silica gel ( 35:1 hexanesethyl acetate ) allowing isolation of two hydroxyl sulfide diastereomers in a 20:80 syn / anti ratio : total yield = 489 mg ( 1.78 mmol ) = 80% .
anti isomer : yield = 391 mg ( 1.41 mmol ) ; h nmr ( 300 mhz , cdcl3 ) 1.94 ( d , j = 1.8 hz , 1h ) , 4.10 ( dd , j = 1.8 , 4.7 hz , 1h ) , 5.11 ( d , j = 4.7 hz , 1h ) , 7.27.7 ( m , 10h ) , the oh proton was not detected ; c nmr ( 75 mhz , cdcl3 ) 43.8 , 66.0 , 68.0 , 81.3 , 87.2 , 93.6 , 121.7 , 125.8 ( 2 ) , 127.3 ( 3 ) , 128.1 , 128.3(2 ) , 129.3 , 129.5 , 136.1 .
h nmr ( 300 mhz , cdcl3 ) 1.83 ( d , j = 1.8 hz , 1h ) , 3.95 ( dd , j = 1.8 , 8.1 hz , 1h ) , 4.95 ( d , j = 8.1 hz , 1h ) , 7.27.7 ( m , 10h ) , the oh proton was not detected ; c nmr ( 75 mhz , cdcl3 ) 44.1 , 65.8 , 68.0 , 81.3 , 87.2 , 93.6 , 121.7 , 125.8 ( 2 ) , 127.3 ( 3 ) , 128.1 , 128.3(2 ) , 129.3 , 129.5 , 136.1 .
separation was accomplished using radial chromatography on silica gel ( 35:1 hexanesethyl acetate ) allowing isolation of two hydroxyl sulfide diastereomers in a 50:50 syn / anti ratio : total yield = 418 mg ( 1.54 mmol ) = 70% ; anti isomer : yield = 209 mg ( 7.7 mmol ) ; h nmr ( 300 mhz , cdcl3 ) 0.24 ( s , 9h ) , 1.84 ( d , j = 1.6 hz , 1h ) , 4.23 ( dd , j = 1.6 , 5.3 hz , 1h ) , 4.80 ( d , j = 5.3 hz , 1h ) , 7.17.4 ( m , 5h ) , the oh proton was not detected ; c nmr ( 75 mhz , cdcl3 ) 0.28(3 ) , 44.4 , 66.5 , 67.5 , 74.1 , 84.3 , 86.4 , 125.0 , 125.3 , 126.5 ( 2 ) , 127.3 , 136.1 ; ms m / z ( m ) calcd 274.0848 , obsd 274.0819 .
calcd for c15h18ossi : c , 65.64 ; h , 6.61 . found : c , 65.17 ; h , 6.55 .
syn isomer : yield = 209 mg ( 7.7 mmol ) = 70% ; h nmr ( 300 mhz , cdcl3 ) 0.24 ( s , 9h ) , 1.79 ( d , j = 1.7 hz , 1h ) , 4.15 ( dd , j = 1.7 , 7.8 hz , 1h ) , 4.69 ( d , j = 7.8 hz , 1h ) , 7.17.4 ( m , 5h ) , the oh proton was not detected ; c nmr ( 75 mhz , cdcl3 ) 0.28(3 ) , 44.1 , 67.9 , 68.1 , 75.0 , 83.2 , 86.9 , 125.0 , 125.3 , 126.5 ( 2 ) , 127.3 , 136.1 .
separation was accomplished using radial chromatography on silica gel ( 35:1 hexanesethyl acetate ) to give a mixture of diastereomeric hydroxy sulfides : yield = 633 mg ( 1.69 mmol ) = 77% ; h nmr ( 300 mhz , cdcl3 ) 0.25 ( s , 6h ) , 0.97 ( s , 9h ) , 1.25 ( m , 2h ) ,
1.802.00 ( m , 3h ) , 3.83 ( t , j = 6.5 , 2h ) , 4.11 ( m , 1h ) , 4.99 ( m , 1h ) , 7.17.4 ( m , 5h ) , the oh proton was not detected ; c nmr ( 75 mhz , cdcl3 ) 0.28 ( 2 ) , 14.5 , 15.1 , 21.8 ( 2 ) , 21.9 33.4 , 47.0 , 64.6 , 65.1 , 67.9 , 75.2 , 81.9 , 87.3 , 125.4 , 126.6 ( 2 ) , 127.1 , 128.4 , 135.9 ; ms m / z ( m+ ) calcd 374.1736 , obsd 365.1633 ( loss of h2o ) .
the general procedure was followed as described in section i.a with the exception that 1 mmol of incl3 was added to the reaction mixture for every 1 mmol of aldehyde used .
separation was accomplished using radial chromatography on silica gel ( 40:1 hexanesethyl acetate ) to give a mixture of diastereomeric hydroxy sulfides .
stereoselectivity was determined upon transformation to the epoxide product : yield = 437 mg ( 1.63 mmol ) = 77% .
separation was accomplished using radial chromatography on silica gel ( 35:1 hexanesethyl acetate ) allowing isolation of two hydroxyl sulfide diastereomers in a 75:25 syn / anti ratio : yield = total = 458 mg ( 1.65 mmol ) = 75% .
anti isomer : yield = 115 mg ( 0.41 mmol ) ; relevant h nmr shifts ( 300 mhz , cdcl3 ) 1.94 ( d , j = 1.8 hz , 1h ) , 4.12 ( dd , j = 1,8 , 4.5 hz ,
syn isomer : yield = 343 mg ( 1.24 mmol ) ; relevant h nmr shifts ( 300 mhz , cdcl3 ) 1.83 ( d , j = 1.8 hz , 1h ) , 3.92 ( dd , j = 1.8 , 8.0 hz , 1h ) , 4.94 ( d , j = 8.1 hz , 1h ) processing and analysis of this reaction proceeded as described in section i.b . separation was accomplished using radial chromatography on silica gel ( 35:1 hexanesethyl acetate ) allowing isolation of two hydroxyl sulfide diastereomers in a 60:40 syn / anti ratio : yield = total = 434 mg ( 1.58 mmol ) = 72% .
anti isomer : yield = 174 mg ( 0.63 mmol ) ; relevant h nmr shifts ( 300 mhz , cdcl3 ) 4.25 ( dd , j = 1.7 , 5.5 hz , 1h ) , 4.77 ( d , j = 5.5 hz , 1h ) .
syn isomer : yield = 260 mg ( 0.95 mmol ) ; relevant h nmr shifts ( 300 mhz , cdcl3 ) 4.15 ( dd , j = 1.5 , 7.8 hz , 1h ) , 4.71 ( d , j = 7.8 hz , 1h ) .
separation was accomplished using radial chromatography on silica gel ( 35:1 hexanesethyl acetate ) to give a mixture of diastereomeric hydroxy sulfides .
stereoselectivity was determined upon transformation to the epoxide product : yield 583 mg ( 1.55 mmol ) = 71% .
analysis as shown in section i.a.4 . a magnetically stirred solution of aldehyde 1 ( 2.0 mmol ) in nmf ( 22 ml ) was treated with -chloropropargylphenyl sulfide ( 702 mg , 3.0 mmol ) and indium powder ( 228 mg , 2.0 mmol ) .
the solution was allowed to proceed at room temperature until no 1 was seen ( tlc analysis ) .
the layers were separated , and the aqueous phase was extracted with dichloromethane ( 2 10 ml ) .
the combined organic layers were washed with 1 n hcl ( 3 20 ml ) , dried over mgso4 , and evaporated to leave a dark yellow to brown oil .
purification was accomplished by flash chromatography or radial chromatography on silica gel ( hexanesethyl acetate ) to give a mixture of hydroxy sulfides ( 2)(11 ) as a light yellow oil .
separation was accomplished using radial chromatography on silica gel ( 40:1 hexanesethyl acetate ) to give a mixture of diastereomeric hydroxy sulfides .
stereoselectivity was determined upon transformation to the epoxide product : yield = 522 mg ( 2.02 mmol ) = 92% .
separation was accomplished using radial chromatography on silica gel ( 35:1 hexanesethyl acetate ) allowing isolation of two hydroxyl sulfide diastereomers in a 20:80 syn / anti ratio : yield = total = 556 mg ( 2.00 mmol ) = 91% .
anti isomer : yield = 445 mg ( 1.60 mmol ) ; relevant h nmr shifts ( 300 mhz , cdcl3 ) 4.10 ( dd , j = 1.8 , 4.7 hz , 1h ) , 5.11 ( d , j = 4.7 hz , 1h ) .
syn isomer : yield = 111 mg ( 0.40 mmol ) ; relevant h nmr shifts ( 300 mhz , cdcl3 ) 3.95 ( dd , j = 1.8 , 8.1 hz , 1h ) , 4.95 ( d , j = 8.1 hz , 1h ) .
separation was accomplished using radial chromatography on silica gel ( 35:1 hexanesethyl acetate ) allowing isolation of two hydroxyl sulfide diastereomers in a 40:60 syn / anti ratio : yield = total = 566 mg ( 2.06 mmol ) = 94% .
anti isomer : yield = 340 mg ( 1.24 mmol ) ; relevant h nmr shifts ( 300 mhz , cdcl3 ) 4.25 ( dd , j = 1.7 , 5.5 hz , 1h ) , 4.77 ( d , j = 5.5 hz , 1h ) .
syn isomer : yield = 226 mg ( 0.82 mmol ) ; relevant h nmr shifts ( 300 mhz , cdcl3 ) 4.15 ( dd , j = 1.5 , 7.8 hz , 1h ) , 4.71 ( d , j = 7.8 hz , 1h ) .
separation was accomplished using radial chromatography on silica gel ( 35:1 hexanesethyl acetate ) to give a mixture of diastereomeric hydroxy sulfides .
stereoselectivity was determined upon transformation to the epoxide product : yield = 717 mg ( 1.91 mmol ) = 87% .
the general procedure was followed as described in section i.c with the exception that 1 mmol of incl3 was added to the reaction mixture for every 1 mmol of aldehyde used .
separation was accomplished using radial chromatography on silica gel ( 40:1 hexanesethyl acetate ) to give a mixture of diastereomeric hydroxy sulfides .
stereoselectivity was determined upon transformation to the epoxide product : yield = 527 mg ( 2.05 mmol ) = 93% .
separation was accomplished using radial chromatography on silica gel ( 35:1 hexanesethyl acetate ) allowing isolation of two hydroxyl sulfide diastereomers in a 85:15 syn / anti ratio : yield = total = 568 mg ( 2.05 mmol ) = 93% .
anti isomer : yield = 85 mg ( 0.31 mmol ) ; relevant h nmr shifts ( 300 mhz , cdcl3 ) 4.13 ( dd , j = 1.8 , 4.7 hz , 1h ) , 5.08 ( d , j = 4.7 hz , 1h ) .
syn isomer : yield = 483 mg ( 1.74 mmol ) ; relevant h nmr shifts ( 300 mhz , cdcl3 ) 3.92 ( dd , j = 1.8 , 8.1 hz , 1h ) , 4.97 ( d , j = 8.1 hz , 1h ) .
separation was accomplished using radial chromatography on silica gel ( 35:1 hexanesethyl acetate ) allowing isolation of two hydroxyl sulfide diastereomers in a 70:30 syn / anti ratio : yield = total = 548 mg ( 2.00 mmol ) = 91% .
anti isomer : : yield = 164 mg ( 0.60 mmol ) ; relevant h nmr shifts ( 300 mhz , cdcl3 ) 4.23 ( dd , j = 1.7 , 5.5 hz , 1h ) , 4.80 ( d , j = 5.5 hz , 1h ) .
syn isomer : yield = 384 mg ( 1.40 mmol ) ; relevant h nmr shifts ( 300 mhz , cdcl3 ) 4.15 ( dd , j = 1.5 , 7.8 hz , 1h ) , 4.68 ( d , j = 7.8 hz , 1h ) .
separation was accomplished using radial chromatography on silica gel ( 35:1 hexanesethyl acetate ) to give a mixture of diastereomeric hydroxy sulfides .
stereoselectivity was determined upon transformation to the epoxide product : yield = 732 mg ( 1.96 mmol ) = 89% .
a solution of the hydroxy sulfide mixture ( 85:15 syn / anti ) ( 100 mg , 0.39 mmol ) in dichloromethane ( 10 ml ) was treated with trimethyloxonium tetrafluoroborate ( 90 mg ( 0.60 mmol ) .
the solution was stirred at room temperature for 8 h and diluted with 7% sodium hydroxide solution ( aqueous , 8 ml ) .
purification was accomplished by radial chromatography on silica gel ( elution with 80:1 hexanesethyl acetate ) to give a mixture of diastereomers as a colorless oil in an 85:15 syn / anti ratio : yield = 52 mg ( 0.35 mmol ) = 90% ; h nmr ( 300 mhz , cdcl3 ) 0.9 1.5 ( m , 7h ) , 1.82 ( d , j = 1.8 hz , 0.15h ) , 1.86 ( d , j = 1.9 hz , 0.85h ) , 2.17 ( m , 2h ) , 3.25 ( d , j = 4.6 hz , 0.85h ) , 3.40 ( d , j = 2.3 hz , 0.15h ) , 3.50 ( dd , j = 1.8 , 2.3 hz , 0.15h ) , 3.61 ( dd , j = 1.9 , 4.6 hz , 0.85h ) ; c nmr ( 75 mhz , cdcl3 ) ( 14.0 , 14.1 ) , ( 17.2 , 17.4 ) , 21.6 , ( 31.7 , 31.8 ) , 44.5 , ( 48.2 , 48.3 ) , 66.7 , ( 77.5 , 77.9 ) , ( 79.5 , 79.7 ) , ( 85.2 , 85.5 )
calcd for c10h12o : c , 81.04 ; h , 8.16 . found : c , 80.99 ; h , 8.19 .
a solution of the hydroxy sulfide mixture ( 85:15 syn / anti ) ( 200 mg 0.72 mmol ) in dichloromethane ( 20 ml ) was treated with trimethyloxonium tetrafluoroborate ( 180 mg ( 1.20 mmol ) .
the solution was stirred at room temperature for 12 h and diluted with 7% sodium hydroxide solution ( aqueous , 15 ml ) .
purification was accomplished by radial chromatography on silica gel ( elution with 100:1 hexanesethyl acetate ) allowing isolation of two diastereomers as colorless oils in an 85:15 syn / anti ratio : yield = total = 110 mg ( 0.64 mmol ) = 89% .
anti isomer : yield = 12 mg ( 0.07 mmol ) ; h nmr ( 300 mhz , cdcl3 ) 2.44 ( d , j = 1.8 hz , 1h ) , 3.51 ( dd , j = 1.8 , 2.4 hz , 1h ) , 3.97 ( d , j = 2.4 hz , 1h ) , 7.157.25 ( m , 5h ) ; c nmr ( 75 mhz , cdcl3 ) 48.2 , 53.1 , 66.2 , 80.3 , 87.3 , 91.7 , 123.5 , 128.4 ( 2 ) , 129.0 , 133.1 ( 2 ) .
calcd for c12h8o = c , 85.69 h , 4.79 . found : c , 86.00 h , 4.71 .
syn isomer : yield = 98 mg ( 0.57 mmol ) ; h nmr ( 300 mhz , cdcl3 ) 2.43 ( d , j = 1.8 hz , 1h ) , 3.46 ( dd , j = 1.8 , 4.7 hz , 1h ) , 4.15 ( d , j = 4.7 hz , 1h ) , 7.157.23 ( m , 5h ) ; c nmr ( 75 mhz , cdcl3 ) 48.7 , 53.1 , 66.5 , 80.1 , 87.3 , 92.0 , 123.0 , 128.3 ( 2 ) , 128.7 , 132.3(2 ) . a solution of the hydroxy sulfide mixture ( 70:30 syn / anti ) ( 200 mg 0.73 mmol ) in dichloromethane ( 20 ml ) was treated with trimethyloxonium tetrafluoroborate ( 180 mg ( 1.20 mmol ) .
the solution was stirred at room temperature for 12 h and diluted with 7% sodium hydroxide solution ( aqueous , 15 ml ) .
purification was accomplished by radial chromatography on silica gel ( elution with 100:1 hexanesethyl acetate ) allowing isolation of two diastereomers as colorless oils in an 70:30 syn / anti ratio : yield = total = 108 mg ( 0.66 mmol ) = 90% ; anti isomer : yield = 32 mg ( 0.30 mmol ) ; h nmr ( 300 mhz , cdcl3 ) 0.13 ( s , 9h ) ; 2.21 ( d , j = 2.0 hz , 1h ) , 3.25 ( dd , j = 2.0 , 2.3 hz , 1h ) , 3.55 ( d , j = 2.3 hz , 1h ) ; c nmr ( 75 mhz , cdcl3 ) 0.10 ( 3 ) , 44.9 , 49.9 , 69.9 , 81.7 , 86.0 , 102.0 ; ms m / z ( m ) calcd = 164.0657 , obsd = 164.0656 . syn isomer : yield = 76 mg ( 0.46 mmol ) ; h nmr ( 300 mhz , cdcl3 ) 0.13 ( s , 9h ) , 2.19 ( d , j = 2.0 hz , 1h ) , 3.32 ( dd , j = 2.0 , 4.9 hz , 1h ) , 3.63 ( d , j = 4.9 hz , 1h ) ; c nmr ( 75 mhz , cdcl3 ) 0.10 ( 3 ) , 45.1 , 49.3 , 70.1 , 81.5 , 85.6 , 101.2 . a solution of the hydroxy sulfide mixture ( 90:10 syn / anti ) ( 250 mg 0.67 mmol ) in dichloromethane ( 20 ml ) was treated with trimethyloxonium tetrafluoroborate ( 150 mg ( 1.00 mmol ) .
the solution was stirred at room temperature for 12 h and diluted with 7% sodium hydroxide solution ( aqueous , 12 ml ) .
purification was accomplished by radial chromatography on silica gel ( elution with 100:1 hexanesethyl acetate ) to give a mixture of two diastereomers as a colorless oil in a 90:10 syn / anti ratio : yield = 148 mg ( 0.56 mmol ) = 83% ; h nmr ( 300 mhz , cdcl3 ) 0.22 ( s , 6h ) ; 0.97 ( s , 9h ) , 1.27 ( m , 2h ) , 1.82.0 ( m , 3h ) , 3.23 ( d , j = 4.4 hz , 0.9h ) , 3.31 ( d , j = 2.5 hz , 0.1h ) , 3.53 ( dd , j = 1.9 , 2.5 hz , 0.1 hz ) , 3.69 ( dd , j = 1.9 , 4.4 hz , 0.9h ) , 3.83 ( m , 2h ) ; c nmr ( 75 mhz , cdcl3 ) 0.28 ( 2 ) , 14.5 , 14.7 , 21.8 ( 2 ) , ( 22.0 , 22.3 ) , ( 33.9 , 34.2 ) , ( 43.8 , 44.0 ) , ( 47.1 , 47.9 ) , ( 64.7 , 65.0 ) , 67.3 , ( 78.7 , 79.1 ) , 81.8 , ( 85.4 , 85.8 ) ; ms m / z ( m ) calcd = 264.1546 , obsd = 264.1545
calcd for c15h24osi : c , 68.13 ; h , 9.15 . found : c , 67.79 ; h , 9.4 .
a solution of the hydroxy sulfide mixture ( 85:15 syn / anti ) ( 200 mg 0.72 mmol ) in chloroform ( 20 ml ) was treated with tloet ( 234 mg , 0.93 mmol ) .
after 20 min of stirring , the insolubles were removed by filtration through a short celite pad . the resulting organic solution was washed with a saturated nahco3 solution ( aq ) and a saturated nacl solution ( aq ) . the organic layer was dried and concentrated .
purification and analysis of products proceeded in the manner previously described ( section ii.a ) .
subsequent hydroxy sulfides were treated in a fashion similar to that in ii.b.1 and analyzed in the manner previously described in section ii.a .
a solution of the hydroxy sulfide mixture ( 25:75 syn / anti ) ( 100 mg , 0.39 mmol ) in a 9:1 thf / h2o mixture ( 10 ml ) was treated with a catalytic amount of p - toluenesulfonic acid .
the solution was stirred at room temperature for 812 h and diluted with 10% sodium bicarbonate solution ( aqueous , 10 ml ) .
after 5 min of stirring , 20 ml of dichloromethane was added to the solution with stirring for 10 min .
the organic layers were combined , washed with 3 10 ml of water , dried over mgso4 , and concentrated .
purification was accomplished by radial chromatography on silica gel ( elution with 25:1 hexanesethyl acetate ) to give a mixture of diastereomers as a colorless oil in a 73:27 cis / trans ratio : yield = 82 mg ( 0.34 mmol ) = 87% ; h nmr ( 300 mhz , cdcl3 ) 0.901.51 ( m , 7h ) , 2.07 ( m , 2h ) , 2.63 ( s , 0.27h ) , 2.71 ( s , 0.73h ) , 5.90 ( s , 0.73h ) , 6.21 ( s , 0.27h ) 7.107.25 ( m , 5h ) ; c nmr ( 75 mhz , cdcl3 ) ( 13.9 , 14.1 ) , 17.1 , 22.9 , 31.6 , 77.2 , ( 79.5 , 80.3 ) , ( 83.1 , 83.7 ) , ( 90.5 , 91.7 ) , ( 113.3 , 113.9 ) , 125.1 , 128.4(2 ) , 129.3(2 ) , 134.0 , ( 145.9 , 146.2 ) ; ms m / z ( m ) calcd 240.0973 , obsd 240.0973 .
calcd for c16h16s : c , 79.95 ; h , 6.71 . found : c , 80.09 ; h , 6.60 .
a solution of the hydroxy sulfide ( 200 mg 0.72 mmol ) in a 9:1 thf / h2o mixture ( 20 ml ) was treated with a catalytic amount of p - toluenesulfonic acid .
the solution was stirred at room temperature for 1015 h and diluted with 10% sodium bicarbonate solution ( aqueous , 20 ml ) .
after 5 min of stirring , 30 ml of dichloromethane was added to the solution with stirring for 10 min .
the organic layers were combined , washed with 3 10 ml of water , dried over mgso4 , and concentrated .
purification was accomplished by radial chromatography on silica gel ( elution with 25:1 hexanesethyl acetate ) to give the product as a colorless oil in a 97:3 e / z ratio ( the minor isomer was detected by gcms ; the minor isomer was not seen by nmr ) : ield = 156 mg ( 0.60 mmol ) = 83% ; h nmr ( 300 mhz , cdcl3 ) 0.2.95 ( s , 1h ) , 6.01 ( s , 1h ) , 7.077.30 ( m , 10h ) : c nmr ( 75 mhz , cdcl3 ) 81.7 , 82.9 , 89.3 , 92.6 , 114.0 , 122.0 , 125.7 , 128.1 ( 2 ) , 128.3 , 128.8 ( 2 ) , 129.5 ( 2 ) , 133.6 ( 2 ) , 134.0 , 144.6 .
a solution of the hydroxy sulfide mixture ( 20:80 syn / anti ) ( 200 mg , 0.72 mmol ) in a 9:1 thf / h2o mixture ( 20 ml ) was treated with a catalytic amount of p - toluenesulfonic acid .
the solution was stirred at room temperature for 1015 h and diluted with 10% sodium bicarbonate solution ( aqueous , 20 ml ) .
after 5 min of stirring , 30 ml of dichloromethane was added to the solution with stirring for 10 min .
the organic layers were combined , washed with 3 10 ml of water , dried over mgso4 , and concentrated .
purification was accomplished by radial chromatography on silica gel ( elution with 25:1 hexanesethyl acetate ) to give a mixture of diastereomers as a colorless oil in a 77:23 e / z ratio : yield = 160 mg ( 0.61 mmol ) = 85% ; h nmr ( 300 mhz , cdcl3 ) 0.2.95 ( s , 0.77h ) , 3.01 ( s , 0.23h ) , 6.04 ( s , 0.77h ) , 6.35 ( s , 0.27h ) , 7.077.30 ( m , 10h ) : c nmr ( 75 mhz , cdcl3 ) ( 81.5 , 81.7 ) , 83.0 , 89.3 , ( 92.6 , 93.0 ) , ( 114.0 , 119.2 ) , 122.0 , 125.7 , 128.1 ( 2 ) , ( 128.3 , 128.5 ) , 128.8 ( 2 ) , 129.5 ( 2 ) , 133.6 ( 2 ) , ( 134.0 , 134.5 ) , ( 144.6 , 145.1 ) . a solution of the hydroxy sulfide ( 200 mg 0.73 mmol ) in a 9:1 thf / h2o mixture ( 20 ml ) was treated with a catalytic amount of p - toluenesulfonic acid .
the solution was stirred at room temperature for 1015 h and diluted with 10% sodium bicarbonate solution ( aqueous , 20 ml ) .
after 5 min of stirring , 30 ml of dichloromethane was added to the solution with stirring for 10 min .
the organic layers were combined , washed with 3 10 ml of water , dried over mgso4 , and concentrated .
purification was accomplished by radial chromatography on silica gel ( elution with 15:1 hexanesethyl acetate ) to give the product as a colorless oil in a 97:3 e / z ratio ( the minor isomer was detected by gcms ; the minor isomer was not seen by nmr ) : yield = 123 mg ( 0.0.48 mmol ) = 65% ; h nmr ( 300 mhz , cdcl3 ) 0.0.11 ( s , 9h ) , 2.87 ( s , 1h ) , 6.20 ( s , 1h ) , 7.107.29 ( m , 5h ) ; c nmr ( 75 mhz , cdcl3 ) 0.09 ( 3 ) , 78.7 , 81.2 , 95.3 , 100.9 , 112.5 , 124.8 , 127.2(2 ) , 128.7(2 ) , 131.8 , 142.1 .
calcd for c15h16ssi : c , 70.25 ; h , 6.29 . found : c , 69.99 ; h , 6.43 .
a solution of the hydroxy sulfide mixture ( 40:60 syn / anti ) ( 200 mg 0.73 mmol ) in a 9:1 thf / h2o mixture ( 20 ml ) was treated with a catalytic amount of p - toluenesulfonic acid .
the solution was stirred at room temperature for 1015 h and diluted with 10% sodium bicarbonate solution ( aqueous , 20 ml ) .
after 5 min of stirring , 30 ml of dichloromethane was added to the solution with stirring for 10 min .
the organic layers were combined , washed with 3 10 ml of water , dried over mgso4 , and concentrated .
purification was accomplished by radial chromatography on silica gel ( elution with 15:1 hexanesethyl acetate ) to give a mixture of diastereomers as a colorless oil in a 50:50 e / z ratio : yield = 120 mg ( 0.48 mmol ) = 65% ; h nmr ( 300 mhz , cdcl3 ) 0.09 ( s , 4.5h ) 0.10 ( s , 4.5h ) 2.81 ( s , 0.5h ) , 2.87 ( s , 0.5h ) , 5.77 ( s , 0.5h ) , 6.20 ( s , 0.5h ) , 7.107.29 ( m , 5h ) ; c nmr ( 75 mhz , cdcl3 ) 0.09 ( 3 ) , ( 78.7 , 79.1 ) , ( 81.2 , 81.8 ) , ( 95.3 , 95.9 ) , 100.9 , ( 112.5 , 116.1 ) , 124.8 , 127.2(2 ) , 128.7(2 ) , ( 131.8 , 132.9 ) , ( 142.1 , 143.5 ) .
a solution of the hydroxy sulfide mixture ( 15:85 syn / anti ) ( 250 mg 0.67 mmol ) in a 9:1 thf / h2o mixture ( 10 ml ) was treated with a catalytic amount of p - toluenesulfonic acid .
the solution was stirred at room temperature for 812 h and diluted with 10% sodium bicarbonate solution ( aqueous , 10 ml ) .
after 5 min of stirring , 20 ml of dichloromethane was added to the solution with stirring for 10 min .
the organic layers were combined , washed with 3 10 ml of water , dried over mgso4 , and concentrated .
purification was accomplished by radial chromatography on silica gel ( elution with 10:1 hexanesethyl acetate ) to give a mixture of diastereomers as a colorless oil in an 82:18 cis / trans ratio : yield = 192 mg ( 0.54 mmol ) = 80% ; h nmr ( 300 mhz , cdcl3 ) 0.15 ( s , 6h ) , 0.93 ( s , 9h ) , 1.27 ( m , 2h ) , 2.11(m , 2h ) , 2.71 ( s , 0.82h ) , 2.80 ( s , 0.18h ) , 3.75 ( m , 2h ) , 5.93 ( s , 0.82h ) , 6.16 ( s , 0.18h ) , 7.077.26 ( m , 5h ) ; c nmr ( 75 mhz , cdcl3 ) 0.13(2 ) , ( 13.9 , 14.1 ) , ( 15.0 , 15.1 ) , 21.3(3 ) , 33.9 , ( 64.9 , 65.3 ) , ( 77.3 , 78.1 ) , ( 79.7 , 80.1 ) , ( 83.2 , 84.1 ) , ( 93.1 , 93.7 ) , ( 114.1 , 115.0 ) , 125.4 , 127.7(2 ) , 129.1 , 129.3 , ( 133.7 , 134.0 ) , ( 144.9 , 145.4 ) ; ms m / z ( m ) calcd 356.1630 , obsd 355.9891
calcd for c21h28ossi : c , 70.73 ; h , 7.91 . found : c , 70.81 ; h , 8.01 .
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indium - promoted coupling reactions between propargyl aldehydes ( 1 ) and -chloropropargylphenyl sulfide are reported .
although water has been shown to accelerate indium metal promoted reactions , the reverse pattern was observed in this series .
use of n - methylformamide ( nmf ) , which has not previously been a solvent known for use in indium - promoted reactions , afforded an acceleration of these barbier - style reactions compared to water .
indium - promoted reactions in this study also showed excellent regiocontrol and good stereocontrol , allowing for easy entry into the formation of epoxydiyne and enediyne skeletal structures .
this paper also describes use of the barbier coupled product ( 2 ) as a new , and easy , entry into the formation of enediyne and epoxydiyne skeletal structures .
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Indroduction
Results and Discussion
Conclusion
Experimental Section
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type 1 diabetes ( t1d ) is characterized by autoimmune destruction of pancreatic -cells , primarily mediated by t lymphocytes , resulting in insulin deficiency and lifelong exogenous insulin dependence ( 1 ) .
latent autoimmune diabetes in adults ( lada ) is a subtype of autoimmune diabetes , but the progression of autoimmune -cell destruction is slow ; therefore , patients with lada are not insulin dependent at the initial phase ( at least 6 months ) of disease onset
. clinical manifestation of lada shares the features of classical t1d and type 2 diabetes ( t2d ) ( 2 ) . t1d , lada , and t2d present a spectrum of diabetes .
they range from patients with classic childhood t1d characterized by autoimmune - mediated destruction of -cells with the lowest c - peptide levels to age - related exacerbation of glucose tolerance with the highest bmi seen in t2d ( 3 ) .
furthermore , patients with lada have susceptibility genes for both t1d and t2d ( 4 ) , suggesting that lada lies between t1d and t2d .
b cells infiltrate the islets of young nod mice , the commonly used mouse model for human t1d , and play a role in the initiation of -cell destruction by the autoimmune response ( 5 ) .
studies have shown that marginal zone b ( mzb ) cells and follicular b ( fob ) cells can effectively activate cd4 t cells and facilitate their proliferation by serving as important antigen - presenting cells , especially as antigen - specific antigen - presenting cells ( 6,7 ) .
b - cell targeted therapy has been shown to delay the fall of c - peptide levels in patients with recent - onset t1d ( 8) .
clinical trials have identified b cells as a possible therapeutic target for the prevention and reversal of t1d .
b - cell targeted therapy also has been successfully used in treating other autoimmune diseases , including rheumatoid arthritis ( 9 ) , systemic lupus erythematosus ( 10 ) , and multiple sclerosis ( 11 ) .
moreover , studies have shown that b cells are important in regulating both innate and adaptive immunity ( 12 ) . a subset of b cells expressing cd19cd5cd1d , which have a regulatory function in infection , inflammation , and autoimmune disease , recently has been identified .
these regulatory b cells can regulate t - cell responses and inhibit inflammation in an interleukin-10 ( il-10)dependent manner ( 1316 ) .
the maturation and expansion of b10 cells require il-21 and cd40 signals both in vivo and ex vivo ( 17,18 ) .
considerable evidence shows that patients with autoimmune diabetes have altered frequencies of circulating immune cells , including t cells ( 19 ) , dendritic cells ( 20 ) , natural killer cells ( 21,22 ) , and neutrophils ( 23 ) .
although studies have reported no significant change in total b cells in peripheral blood of patients with t1d ( 21,24 ) , little is known about b - cell subpopulations with distinct immune functions that may play a role in the diabetes spectrum of t1d , lada , and t2d .
we hypothesized that an altered phenotype of b - cell subsets is associated with autoimmune diabetes .
this study investigated a change of peripheral b - cell subsets in patients with t1d , lada , and t2d compared with healthy control subjects .
the results show altered frequencies of various b - cell subsets associated with autoimmune diabetes .
two hundred fifty - eight patients with t1d ( n = 81 , mean diabetes duration 3.1 3.5 years ) , lada ( n = 82 , 6.1 5.9 years ) , or t2d ( n = 95 , 3.0 3.7 years ) and 218 control subjects with normal glucose tolerance ( ngt ) ( table 1 ) were enrolled in the current study from the second xiangya hospital of central south university ( changsha , hunan , china ) .
diabetes was diagnosed according to world health organization ( who ) criteria ( 25 ) .
diagnosis of t1d was based on acute - onset ketosis or ketoacidosis with immediate insulin replacement therapy ; at least one positive islet autoantibody ( gad antibody [ gada ] , insulinoma - associated protein-2 antibody [ ia-2a ] , or zinc transporter 8 antibody [ znt8a ] ) ; or impaired c - peptide secretion .
lada inclusion criteria were 1 ) any positive islet autoantibody ( gada , ia-2a , znt8a ) , 2 ) age 30 years at onset of diabetes , 3 ) insulin independence for at least 6 months after diagnosis , and 4 ) no ketosis or ketoacidosis ( 2,26 ) .
diagnostic criteria of t2d were 1 ) typical history of hyperglycemia according to who criteria , 2 ) negative for islet autoantibodies , and 3 ) not requiring immediate insulin treatment .
healthy control subjects underwent a standardized 75-g oral glucose tolerance test for normoglycemia ( fasting plasma glucose [ fpg ] < 6.1 mmol / l , 2-h plasma glucose < 7.8
exclusion criteria for control subjects were secondary diabetes ; inflammation , infectious disease , or other autoimmune disease ; history of immunosuppressive medication or steroids for > 7 days ; pregnancy ; and malignant disease .
this study was approved by the ethical committee of the second xiangya hospital of central south university , and all subjects provided written informed consent for the study protocol .
anthropometric and metabolic data of participants data are % ( n ) , mean sd , and median ( 25th75th percentile ) .
compared after log transformation . * p < 0.05 compared with ngt . * * p < 0.01 compared with ngt . * * * p < 0.001 compared with ngt . p < 0.05 compared with t2d . p < 0.01 compared with t2d . p < 0.001 compared with t2d . p < 0.001 compared with t1d .
study physicians recorded body height and weight , waist circumference , hip circumference , and blood pressure .
fasting venous blood samples were tested for triglycerides ( tgs ) , total cholesterol ( tc ) , hdl cholesterol ( hdl - c ) , ldl cholesterol ( ldl - c ) , fpg , hemoglobin a1c ( hba1c ) , and fasting c - peptide ( fcp ) .
hba1c was measured by automated liquid chromatography ( variant ii hemoglobin testing system ; bio - rad laboratories , hercules , ca ) .
fcp was measured by a chemiluminescence method ( advia centaur ; siemens , munich , germany ) .
gada , ia-2a , and znt8a were measured by radioligand assays as previously described ( 27,28 ) .
the cutoff indices of positivity for gada , ia-2a , and znt8a were 18 units / ml ( who units ) , 3.3 units / ml , and 0.011 ( znt8a index ) , respectively , which were determined as the 99th percentile of 405 healthy control subjects .
the sensitivity of the current gada , ia-2a , and znt8a assays was 78% , 74% , and 70% , respectively , and the specificity was 96.7% , 96.7% , and 98.9% , respectively , in the islet autoantibody standardization program ( iasp2012 ) .
fresh venous blood samples were drawn into sodium heparin tubes from fasting subjects and processed within 2 h. the peripheral blood mononuclear cells were isolated by standard ficoll - paque plus density - gradient centrifugation and stained with various monoclonal antibodies ( allophycocyanin - cy7-cd19 [ hib19 ] , fluorescein isothiocyanate - cd5 [ ucht2 ] , allophycocyanin - cd1d [ 51.1 ] , peridinin chlorophyll protein - cy5.5-cd23 [ ebvcs-5 ] , and phycoerythrin - cy7-cd21 [ lt21 ] ; biolegend , san diego , ca ) to determine the percentage of b - cell subsets according to the manufacturer s protocol .
the stained cells were analyzed with a bd facscanto ii system , which was calibrated daily with appropriate single fluorochrome - stained samples .
fifty thousand lymphocytes were collected in a forward scatter / side scatter ( fsc - a / ssc - a ) lymphocyte gate and analyzed with flowjo version 7.6 software ( tree star , inc . ,
dead cells were excluded from the analysis on the basis of their forward- and side - light scatter properties and propidium iodide staining .
1 . the following antibodies were used to identify b - cell subsets : cd19 ( pan b marker ) , cd19cd23cd21 ( mzb cells ) , cd19cd23cd21 ( fob cells ) , cd19cd23cd21 ( transitional 2-marginal zone precursor [ t2-mzp ] b cells ) ( 29 ) , and cd19cd5cd1d ( b10 cells ) .
the intra - assay and interassay coefficients of variation for the percentages of cd19 were 3.71% and 12.15% , respectively .
the initial cd19 gate was derived from a lymphocyte gate ( defined on fsc and ssc ) followed by single - cell discrimination .
b : representative dot plot showing the gating strategy for mzb , fob , and t2-mzp b cells gated on cd19 b cells .
c : representative dot plot showing the gating strategy for b10 cells gated on cd19 b cells .
anova was performed to compare groups after adjustment for potentially confounding variables , including age , sex , and bmi .
all the significant differences between groups are the results of adjusted analyses unless stated otherwise .
associations of the frequencies of b - cell subsets with other parameters were estimated by pearson correlations or spearman nonparametric correlations .
for the statistical analysis , we used spss version 19.0 ( ibm corporation , chicago , il ) and graphpad prism 5 ( graphpad software , san diego , ca ) software .
two hundred fifty - eight patients with t1d ( n = 81 , mean diabetes duration 3.1 3.5 years ) , lada ( n = 82 , 6.1 5.9 years ) , or t2d ( n = 95 , 3.0 3.7 years ) and 218 control subjects with normal glucose tolerance ( ngt ) ( table 1 ) were enrolled in the current study from the second xiangya hospital of central south university ( changsha , hunan , china ) .
diabetes was diagnosed according to world health organization ( who ) criteria ( 25 ) .
diagnosis of t1d was based on acute - onset ketosis or ketoacidosis with immediate insulin replacement therapy ; at least one positive islet autoantibody ( gad antibody [ gada ] , insulinoma - associated protein-2 antibody [ ia-2a ] , or zinc transporter 8 antibody [ znt8a ] ) ; or impaired c - peptide secretion .
lada inclusion criteria were 1 ) any positive islet autoantibody ( gada , ia-2a , znt8a ) , 2 ) age 30 years at onset of diabetes , 3 ) insulin independence for at least 6 months after diagnosis , and 4 ) no ketosis or ketoacidosis ( 2,26 ) .
diagnostic criteria of t2d were 1 ) typical history of hyperglycemia according to who criteria , 2 ) negative for islet autoantibodies , and 3 ) not requiring immediate insulin treatment .
healthy control subjects underwent a standardized 75-g oral glucose tolerance test for normoglycemia ( fasting plasma glucose [ fpg ] < 6.1 mmol / l , 2-h plasma glucose < 7.8
exclusion criteria for control subjects were secondary diabetes ; inflammation , infectious disease , or other autoimmune disease ; history of immunosuppressive medication or steroids for > 7 days ; pregnancy ; and malignant disease .
this study was approved by the ethical committee of the second xiangya hospital of central south university , and all subjects provided written informed consent for the study protocol .
anthropometric and metabolic data of participants data are % ( n ) , mean sd , and median ( 25th75th percentile ) .
compared after log transformation . * p < 0.05 compared with ngt . * * p < 0.01 compared with ngt . * * * p < 0.001 compared with ngt . p < 0.05 compared with t2d . p < 0.01 compared with t2d . p < 0.001 compared with t2d . p < 0.001 compared with t1d .
study physicians recorded body height and weight , waist circumference , hip circumference , and blood pressure .
fasting venous blood samples were tested for triglycerides ( tgs ) , total cholesterol ( tc ) , hdl cholesterol ( hdl - c ) , ldl cholesterol ( ldl - c ) , fpg , hemoglobin a1c ( hba1c ) , and fasting c - peptide ( fcp ) .
hba1c was measured by automated liquid chromatography ( variant ii hemoglobin testing system ; bio - rad laboratories , hercules , ca ) .
fcp was measured by a chemiluminescence method ( advia centaur ; siemens , munich , germany ) .
gada , ia-2a , and znt8a were measured by radioligand assays as previously described ( 27,28 ) .
the cutoff indices of positivity for gada , ia-2a , and znt8a were 18 units / ml ( who units ) , 3.3 units / ml , and 0.011 ( znt8a index ) , respectively , which were determined as the 99th percentile of 405 healthy control subjects . the positive samples were tested twice for confirmation .
the sensitivity of the current gada , ia-2a , and znt8a assays was 78% , 74% , and 70% , respectively , and the specificity was 96.7% , 96.7% , and 98.9% , respectively , in the islet autoantibody standardization program ( iasp2012 ) .
fresh venous blood samples were drawn into sodium heparin tubes from fasting subjects and processed within 2 h. the peripheral blood mononuclear cells were isolated by standard ficoll - paque plus density - gradient centrifugation and stained with various monoclonal antibodies ( allophycocyanin - cy7-cd19 [ hib19 ] , fluorescein isothiocyanate - cd5 [ ucht2 ] , allophycocyanin - cd1d [ 51.1 ] , peridinin chlorophyll protein - cy5.5-cd23 [ ebvcs-5 ] , and phycoerythrin - cy7-cd21 [ lt21 ] ; biolegend , san diego , ca ) to determine the percentage of b - cell subsets according to the manufacturer s protocol .
the stained cells were analyzed with a bd facscanto ii system , which was calibrated daily with appropriate single fluorochrome - stained samples .
fifty thousand lymphocytes were collected in a forward scatter / side scatter ( fsc - a / ssc - a ) lymphocyte gate and analyzed with flowjo version 7.6 software ( tree star , inc . ,
dead cells were excluded from the analysis on the basis of their forward- and side - light scatter properties and propidium iodide staining .
1 . the following antibodies were used to identify b - cell subsets : cd19 ( pan b marker ) , cd19cd23cd21 ( mzb cells ) , cd19cd23cd21 ( fob cells ) , cd19cd23cd21 ( transitional 2-marginal zone precursor [ t2-mzp ] b cells ) ( 29 ) , and cd19cd5cd1d ( b10 cells ) .
the intra - assay and interassay coefficients of variation for the percentages of cd19 were 3.71% and 12.15% , respectively .
the initial cd19 gate was derived from a lymphocyte gate ( defined on fsc and ssc ) followed by single - cell discrimination .
b : representative dot plot showing the gating strategy for mzb , fob , and t2-mzp b cells gated on cd19 b cells .
c : representative dot plot showing the gating strategy for b10 cells gated on cd19 b cells .
anova was performed to compare groups after adjustment for potentially confounding variables , including age , sex , and bmi .
all the significant differences between groups are the results of adjusted analyses unless stated otherwise .
associations of the frequencies of b - cell subsets with other parameters were estimated by pearson correlations or spearman nonparametric correlations .
for the statistical analysis , we used spss version 19.0 ( ibm corporation , chicago , il ) and graphpad prism 5 ( graphpad software , san diego , ca ) software .
the control subjects with ngt were younger than patients with lada and t2d but significantly older than patients with t1d .
the percentage of males in the lada group was higher than in the ngt group .
the bmi of the ngt and lada groups was lower than the t2d group but higher than the t1d group .
patients with t2d or lada had higher systolic and diastolic blood pressures than patients with t1d and subjects with ngt . patients with t2d had distinctive tg , tc , ldl - c , and hdl - c levels compared with the other groups .
the fpg levels were similar in all the patients with diabetes but higher than in the subjects with ngt .
there was a significant decrease of fcp in the lada and t1d ( lowest ) groups compared with the t2d and ngt groups .
we did not find any significant changes in the frequency of cd19 b cells among the patient groups , regardless of age , sex , and bmi , compared with the ngt group ( p > 0.05 ) ( supplementary fig .
the frequencies of mzb cells in the t1d and lada groups were significantly higher than in the ngt and t2d groups ( t1d vs. ngt , t1d vs. t2d , lada vs. t2d p < 0.001 ; lada vs. ngt p < 0.01 ) ( fig .
we found that mzb cells were more frequent in the t1d group than in the lada group , but the differences were diminished after adjustment for age , sex , and bmi .
fob cells were less frequent in the t1d group compared with the other three groups ; however , the percentages of fob cells in the t1d and lada groups were decreased after adjustment for age , sex , and bmi compared with the ngt and t2d groups ( t1d vs. ngt , p < 0.001 ; t1d vs. t2d , lada vs. ngt , p < 0.01 ; lada vs. t2d , p < 0.05 ) ( fig . 2b ) .
we also assessed the percentages of t2-mzp b cells , which were reported to have a regulatory function ( 29 ) , and found these to be increased in the lada group compared with the ngt and t1d groups ( lada vs. ngt p < 0.001 ; t1d vs. lada p < 0.01 ) ( fig .
2c ) , whereas they were more frequent in the t2d group than in the ngt group ( t2d vs. ngt p < 0.001 ) ( fig .
the frequency of mzb ( a ) , fob ( b ) , t2-mzp b ( c ) , and b10 ( d ) cells gated on cd19 b cells .
p values refer to comparison of data after adjustment for age , sex , and bmi .
* p < 0.05 , * * p < 0.01 , * * * p < 0.001 . of note , regardless of the adjustment for age , sex , and bmi , the proportions of b10 cells were significantly decreased in all the patients with diabetes compared with the control subjects with ngt , but these were lowest in the t1d group ( t1d vs. ngt p < 0.001 ; t2d vs. ngt , lada vs. ngt p < 0.05 ) ( fig .
b10 cells were higher in the t2d and lada groups than in the t1d group ( p < 0.001 for both ) ( fig .
after matching for age and sex , these findings remained the same for the percentages of cd19 , mzb , b10 , and t2-mzp b cells after adjustment for bmi among groups ( supplementary table 1 and supplementary fig .
however , patients with lada but not those with t1d had decreased percentages of fob cells compared with the control subjects with ngt and the patients with t2d .
we analyzed whether the change of b - cell subsets had any association with the clinical manifestations and found that 1 ) mzb and fob cells were correlated with age , 2 ) t2-mzp b cells were associated with sex , and 3 ) b10 and mzb cells were correlated with bmi ( table 2 ) , regardless of the type of diabetes . of note , fpg was positively correlated with mzb and t2-mzp b cells but negatively correlated with b10 cells .
however , fcp showed positive correlations with fob and b10 cells but negative correlations with cd19 and mzb cells . in terms of lipid profile , we found that t2-mzp b cells were positively associated with tg and ldl - c levels but negatively associated with hdl - c level .
correlation between circulating b - cell subset frequencies and anthropometric and metabolic variables and islet autoantibodies correlation analyses between circulating b - cell subset frequencies ( after log transformation ) and anthropometric and metabolic variables were performed using pearson test .
in addition to the associations in all participants , we examined the correlations between the various b - cell subsets and clinical manifestations in the different types of diabetes . in patients with t1d , the proportion of cd19 b cells associated negatively with fcp ( r = 0.248 , p < 0.05 ) , and the mzb cells exhibited a negative correlation with age ( r = 0.498 , p < 0.001 ) , bmi ( r = 0.364 , p < 0.01 ) , and hba1c ( r = 0.324 , p < 0.01 ) . in patients with lada , cd19 b cells negatively correlated with bmi ( r = 0.498 , p < 0.001 ) and fcp ( r = 0.248 , p < 0.05 ) , whereas fob cells showed a positive correlation with age ( r = 0.236 , p < 0.05 ) .
finally , in patients with t2d , the percentage of t2-mzp b cells was positively associated with fcp ( r = 0.364 , p < 0.05 ) .
the control subjects with ngt were younger than patients with lada and t2d but significantly older than patients with t1d .
the percentage of males in the lada group was higher than in the ngt group .
the bmi of the ngt and lada groups was lower than the t2d group but higher than the t1d group .
patients with t2d or lada had higher systolic and diastolic blood pressures than patients with t1d and subjects with ngt . patients with t2d had distinctive tg , tc , ldl - c , and hdl - c levels compared with the other groups .
the fpg levels were similar in all the patients with diabetes but higher than in the subjects with ngt .
there was a significant decrease of fcp in the lada and t1d ( lowest ) groups compared with the t2d and ngt groups .
we did not find any significant changes in the frequency of cd19 b cells among the patient groups , regardless of age , sex , and bmi , compared with the ngt group ( p > 0.05 ) ( supplementary fig .
the frequencies of mzb cells in the t1d and lada groups were significantly higher than in the ngt and t2d groups ( t1d vs. ngt , t1d vs. t2d , lada vs. t2d p < 0.001 ; lada vs. ngt p < 0.01 ) ( fig .
we found that mzb cells were more frequent in the t1d group than in the lada group , but the differences were diminished after adjustment for age , sex , and bmi .
fob cells were less frequent in the t1d group compared with the other three groups ; however , the percentages of fob cells in the t1d and lada groups were decreased after adjustment for age , sex , and bmi compared with the ngt and t2d groups ( t1d vs. ngt , p < 0.001 ; t1d vs. t2d , lada vs. ngt , p < 0.01 ; lada vs. t2d , p < 0.05 ) ( fig . 2b ) .
we also assessed the percentages of t2-mzp b cells , which were reported to have a regulatory function ( 29 ) , and found these to be increased in the lada group compared with the ngt and t1d groups ( lada vs. ngt p < 0.001 ; t1d vs. lada p < 0.01 ) ( fig .
2c ) , whereas they were more frequent in the t2d group than in the ngt group ( t2d vs. ngt p < 0.001 ) ( fig .
the frequency of mzb ( a ) , fob ( b ) , t2-mzp b ( c ) , and b10 ( d ) cells gated on cd19 b cells .
p values refer to comparison of data after adjustment for age , sex , and bmi .
* p < 0.05 , * * p < 0.01 , * * * p < 0.001 . of note , regardless of the adjustment for age , sex , and bmi , the proportions of b10 cells were significantly decreased in all the patients with diabetes compared with the control subjects with ngt , but these were lowest in the t1d group ( t1d vs. ngt p < 0.001 ; t2d vs. ngt , lada vs. ngt p < 0.05 ) ( fig .
b10 cells were higher in the t2d and lada groups than in the t1d group ( p < 0.001 for both ) ( fig .
after matching for age and sex , these findings remained the same for the percentages of cd19 , mzb , b10 , and t2-mzp b cells after adjustment for bmi among groups ( supplementary table 1 and supplementary fig .
however , patients with lada but not those with t1d had decreased percentages of fob cells compared with the control subjects with ngt and the patients with t2d .
we analyzed whether the change of b - cell subsets had any association with the clinical manifestations and found that 1 ) mzb and fob cells were correlated with age , 2 ) t2-mzp b cells were associated with sex , and 3 ) b10 and mzb cells were correlated with bmi ( table 2 ) , regardless of the type of diabetes . of note , fpg was positively correlated with mzb and t2-mzp b cells but negatively correlated with b10 cells .
however , fcp showed positive correlations with fob and b10 cells but negative correlations with cd19 and mzb cells . in terms of lipid profile , we found that t2-mzp b cells were positively associated with tg and ldl - c levels but negatively associated with hdl - c level .
correlation between circulating b - cell subset frequencies and anthropometric and metabolic variables and islet autoantibodies correlation analyses between circulating b - cell subset frequencies ( after log transformation ) and anthropometric and metabolic variables were performed using pearson test .
in addition to the associations in all participants , we examined the correlations between the various b - cell subsets and clinical manifestations in the different types of diabetes . in patients with t1d ,
the proportion of cd19 b cells associated negatively with fcp ( r = 0.248 , p < 0.05 ) , and the mzb cells exhibited a negative correlation with age ( r = 0.498 , p < 0.001 ) , bmi ( r = 0.364 , p < 0.01 ) , and hba1c ( r = 0.324 , p < 0.01 ) . in patients with lada , cd19 b cells negatively correlated with bmi ( r = 0.498 , p < 0.001 ) and fcp ( r = 0.248 , p < 0.05 ) , whereas fob cells showed a positive correlation with age ( r = 0.236 , p < 0.05 ) .
finally , in patients with t2d , the percentage of t2-mzp b cells was positively associated with fcp ( r = 0.364 , p < 0.05 ) .
increasing evidence suggests that b cells are as important as t cells in the immunopathogenesis of autoimmune diabetes .
most , if not all , diabetogenic t cells are known to be t - helper 1 subsets , but little is known about b - cell subsets in autoimmune diabetes .
we investigated the phenotype and frequency of various b - cell subsets in the peripheral blood across the clinical spectrum of diabetes .
the major finding of this study was a distinct alteration of b - cell subsets across the diabetes spectrum and their association with clinical features .
we found no significant difference in the frequency of cd19 b cells , which is in accordance with the published studies ( 21,30 ) ; however , we did not find a negative correlation between total b cells and age , as previously reported ( 24,31 ) .
the current study went further than the examination of the frequency of total b cells , focusing on b - cell subsets with distinct immunological functions .
of note , we found that patients with t1d or lada had an increased frequency of mzb cells but decreased frequency of fob cells compared with control subjects with ngt and patients with t2d . in all groups , we found that fcp was negatively associated with mzb cells but positively associated with fob cells .
attanavanich and kearney ( 6 ) reported that mzb cells were more effective at activating naive cd4 t cells than fob cells .
mzb cells in nod mice were also shown to be important antigen - presenting cells for the activation of t cells in pancreatic - draining lymph nodes ( 32 ) . given the negative correlation between mzb cells and fcp , it is possible that mzb cells may promote -cell destruction in patients with t1d or lada .
decreased frequency of fob cells in the circulation of patients with t1d and lada suggests that some fob cells migrated from the blood to the pancreas or the draining lymph nodes as a result of the local inflammatory response .
along with the disease progression , more immune cells are known to infiltrate to the islets of both nod mice and patients with t1d ( 5,33 ) , but the patients with t1d in the current study have a relatively long history of diabetes ( 3.1 years ) .
thus , fob cells appear to be more important in initial rather than established pancreatic -cell autoimmunity ( 34 ) .
we showed that patients with t2d and lada more frequently had t2-mzp b cells than control subjects with ngt , which associates with parameters of blood pressure , lipidemia , and glycemia but not with hba1c or fcp .
these observations are consistent with a previous study showing that glycemia and lipidemia are associated with immune cells ( 35 ) . another interesting finding was the decrease of b10 cell frequency in all types of diabetes , being the lowest in t1d .
whether this phenotype is mediated through a common mechanism or different ones is not clear .
what is clear is that immune mechanisms are involved and that more studies , especially functional studies , are required .
although b cells are generally acknowledged for their function in humoral immune response by producing antibodies , they also have been demonstrated to play an immunoregulatory role in the prevention of autoimmune disease by producing il-10 and downregulating t - cell responses ( 13,14,36 ) .
the observed b10 cell reduction could contribute to the breakdown of self - tolerance that leads to -cell destruction in patients with t1d ( 24 ) or lada .
antigen - activated il-10producing b cells may selectively inhibit autoreactive t - cell responses to islet - specific antigen to maintain self - tolerance , and hyperglycemic nod mice and patients with t1d lack this population of regulatory b cells ( 37 ) .
in the clinical trial where b cells in patients with new - onset t1d were depleted , the patients showed a transient remission ( 8) .
however , blanket targeting of all b cells , as used in the clinical trial and in treating other autoimmune disorders , needs to be improved by selectively deleting pathogenic b cells while preserving regulatory b cells to promote tolerance .
our preclinical study showed that more regulatory b cells were regenerated after b - cell deletion in an animal model ( 38 ) .
furthermore , studies have shown that patients with long - standing t2d could have a secondary autoimmune response toward islet -cells ( 39 ) .
the reduction of b10 regulatory cells might contribute to the dysregulation of immune response in t2d .
a functional study on il-10producing b cells and further phenotyping will be the goal of future work . in the current study ,
although we currently can not identify a specific subset that could be used for diagnostic purposes , the results strongly imply that the change of b - cell subtypes is related to the pathogenesis of autoimmune diabetes .
therefore , it is important to reconsider the approach of b - cell targeted therapy in patients with t1d and aim to selectively remove pathogenic b cells but promote regulatory b cells . in summary ,
the current data demonstrate distinct differences in the frequencies of peripheral b - cell subsets in t1d and lada , which are associated with islet function and glycemia .
the findings support the notion that these immunological alterations are involved in loss of self - tolerance and -cell destruction .
the findings also suggest that , similar to regulatory t - cell therapy , transfusion of ex vivo expanded autologous b10 cells might open a new and effective therapeutic strategy in treating autoimmune diabetes .
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objectiveb lymphocytes play an important role in the immunopathogenesis of autoimmune diabetes .
we hypothesized that the altered b - cell subset phenotype is associated with autoimmune diabetes.research design and methodspatients with type 1 diabetes ( t1d ) ( n = 81 ) , latent autoimmune diabetes in adults ( lada ) ( n = 82 ) , or type 2 diabetes ( t2d ) ( n = 95 ) and healthy control subjects ( n = 218 ) with normal glucose tolerance ( ngt ) were recruited .
we determined the percentage of circulating b - lymphocyte subsets , including cd19+cd23cd21 + ( marginal zone b [ mzb ] ) , cd19+cd23+cd21 ( follicular b [ fob ] ) , and cd19+cd5+cd1dhi ( interleukin-10producing regulatory b [ b10 ] ) cells by flow cytometry.resultspatients with t1d or lada had increased percentages of mzb cells and decreased percentages of fob cells compared with healthy control subjects with ngt and patients with t2d .
moreover , patients with t1d showed the lowest frequency of b10 cells compared with patients with lada or t2d , whereas healthy control subjects expressed the highest frequency of b10 cells .
of note , the frequency of mzb cells was negatively associated and the frequency of fob cells was positively associated with fasting c - peptide ( fcp ) .
the frequency of b10 cells was positively correlated with fcp and negatively correlated with hemoglobin a1c.conclusionsthe data show that patients with t1d or lada express an altered frequency of b - cell subsets , which is associated with islet function and glycemia .
these findings suggest that b lymphocytes may be involved in loss of self - tolerance and -cell destruction and could be used as a biomarker and potential target for immunological intervention .
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Introduction
Research Design and Methods
Participants
C-Peptide and HbA
Islet Autoantibody Assays
Immunostaining and Flow Cytometry
Statistical Analysis
Results
Anthropometric and Metabolic Data
Change of B-Cell Subsets in Patients With Diabetes
B-Cell Subsets Are Associated With Clinical Diabetes
Conclusions
Supplementary Material
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adolescents in sub - saharan africa face high exposure to human immunodeficiency virus ( hiv ) infection . despite all efforts to prevent the disease , prevalence estimates in sub - saharan unaids ( i.e. , the joint united nations programme on hiv and aids ) priority countries range from 1.3 to 15.6 % among young women and 0.56.5 % among young men aged 1524 years ( unaids , 2011 ) .
higher prevalence estimates are associated with certain regions ( e.g. , eastern and southern africa ) and subpopulations ( e.g. , adolescent orphaned and/or married girls ; birdthistle et al . , 2008 ; operario , underhill , chuong , & cluver , 2011 ; pascoe et al . , 2010 ) .
in addition , united nations children s fund ( unicef ) estimated there were 2 million adolescents aged 1019 years living with hiv globally in 2009 ( unicef , 2011 ) .
kenya and zimbabwe were among countries with the highest numbers of infected adolescents with an estimated 136,000 ( prevalence estimate 1.5 % ) 1 and 104,000 ( prevalence estimate 3.1 % ) adolescents living with hiv , respectively ( unicef , 2011 ) .
the true measure of effectiveness for such interventions is reduction in hiv and other sexually transmitted infections ( stis ) .
however , very few adolescent prevention studies have used biomarker data as outcomes ( michielsen et al . , 2010 ) because of concerns about the difficulties in collecting these data in resource poor settings ( jukes , simmons , & bundy , 2008 ) , even when power analyses suggest that moderate intervention effects could be detected .
behavioral prevention scientists have instead relied on self - reported sexual behavior , which is subject to social desirability bias and likely to differ between study arms ( cowan & pettifor , 2009 ; kirby , obasi , & laris , 2006 ) .
widespread access to testing and treatment facilitated by the president s emergency plan for aids relief ( pepfar ; http://www.usaid.gov/what-we-do/global-health/hiv-and-aids ) have greatly enhanced opportunities to successfully incorporate biomarker procedures into adolescent hiv prevention research designs , since infected individuals identified through testing offered by studies may access free or subsidized hiv treatment and care ( stringer et al . , 2006 ; wools - kaloustian et al . , 2006 , 2009 )
moreover , including biomarker outcomes can greatly improve the credibility and legitimacy of hiv prevention research . to address the issue of feasibility , we present two case studies of prevention trials with biomarker data collection among adolescents in kenya and zimbabwe .
together these studies provide a valuable opportunity to examine the ethical and practical challenges of biomarker procedures within hiv prevention intervention clinical trials in developing countries .
specifically , we describe the challenges faced by investigators in the two cases in developing and implementing ethical procedures for informed consent , biomarker testing , and disclosure of test results .
we also present the practical challenges of collecting biological samples in resource - limited countries and compare sample collection by blood spots relative to venipuncture .
in addition , we examine difficulties with analyzing biological samples when the performance of assays in certain populations is unclear .
we also provide specific suggestions for further research and development in collecting and analyzing biomarker data for adolescent prevention research .
the primary goal of hiv prevention among adolescents ( ages 1019 years ) is to reduce new infections and related sexual risk factors [ world health organization ( who ) , 2006 ) .
although the hiv prevalence of adolescents ages 15 and older is monitored through demographic and health surveys ( dhs)2 in most sub - saharan africa countries , little is known about the prevalence of hiv in adolescents under 15 years .
moreover , these adolescents are rarely tested for hiv in clinical settings unless they present in poor health or with recurrent infections ( ferrand et al . , 2009 ) .
although a small number of recent randomized controlled trials have used biomarkers ( baird , mcintosh , & ozler , 2010 ; cowan et al .
; pronyk et al . , 2006 ; ross et al . , 2007 ) ,
most adolescent hiv prevention studies have relied on changes in self - reported sexual behavior to assess intervention effectiveness ( see reviews : cowan & pettifor , 2009 ; michielsen et al . ,
2010 ) , with audio computer - assisted self - interviews intended to improve veracity ( mensch , erulkar , & hewett , 2003 ) .
however , biomarker comparisons have suggested that self - reported measures have low validity ( cowan et al . , 2002 ; gavin et al . , 2006 ; mensch , hewett , gregory , & helleringer , 2008 ; palen et al . , 2008 ; plummer et al . , 2004 ) .
for example , in one study , only one of 16 youth with hiv admitted to ever having sex , and four of the 16 also had either chlamydia or gonorrhea ( cowan et al . , 2002 ) .
inclusion of a highly prevalent and reliable biomarker of sexual activity in conjunction with self - reported sexual behavior can help to improve reliability of this outcome measure .
hiv infection is not the most useful biomarker of adolescent sexual activity by itself because of the relatively low prevalence among younger adolescents and the potential for transmission through perinatal and non - sexual blood - borne routes ( amornkul et al .
, 2009 ; ferrand et al . , 2009 ; stover , walker , grassly , & marston , 2006 ) .
herpes simplex virus type 2 ( hsv-2 ) , the primary cause of genital herpes , is a commonly used biomarker of sexual activity because the presence of hsv-2 antibodies is highly associated with past sexual behavior ( tobian et al . , 2009 ; van de laar et al . , 1998 ) .
in contrast to other stis such as chlamydia , gonorrhea , syphilis and trichomoniasis , infection with hsv-2 is life - long , and , once established , there is currently no treatment to eliminate it ( geretti , 2006 ; gupta , warren , & wald , 2008 ) .
hsv-2 prevalence in the adult general population in sub - saharan africa is high , ranging from 30 to 80 % in women and from 10 to 50 % in men , as assessed by detection of hsv-2 antibodies [ amornkul et al . , 2009 ;
, 2005 ; munjoma et al . , 2010 ; national aids & sti control programme ( nascop ) , republic of kenya , 2009 ; tobian et al . , 2009 ; weiss , 2004 ] .
prevalence estimates for adolescents are only available through community - based studies and are lower .
one study in zimbabwe among adolescent females aged 1519 years reported that 12 % of participants tested positive for hsv-2 ( birdthistle et al .
a study in western kenya found hsv-2 prevalence to be 9 % for females and 4 % for males aged 1314 , and 28 % for females and 17 % for males aged 1519 years ( amornkul et al . , 2009 ) .
another study conducted in zimbabwe among adolescents whose mean age was 15 years reported a prevalence estimate of 0.2 % , suggesting a later stage of sexual debut ( cowan et al .
2012 ) evaluated the use of hsv-2 as a biomarker of sexual debut among young people aged 1024 years . following a comprehensive review of the literature
, the authors concluded that the use of hsv-2 as a biomarker for sexual debut is limited because of its low transmissibility and the fact that not all potential sexual partners are infected with the virus .
building on these findings , our study describes the challenges of collecting hiv and hsv-2 biological data as biomarkers for sexual activity among sub - saharan african youth in prevention trials .
we present two clinical trial case studies to examine important issues related to adolescent biomarker data collection in two clinical trials in kenya and zimbabwe ( hallfors et al . ,
both studies were school - based , cluster randomized trials testing school support ( including payment of school tuition and school uniforms ) as a structural hiv prevention intervention for adolescent orphans . in both studies , we collected blood samples from participants for hiv and hsv-2 testing , but they differed as follows : we collected samples by venipuncture with results disclosed to participants and their guardians in kenya , whereas in zimbabwe , we collected samples by finger pricks for dried blood spots ( dbs ) and results were not disclosed . the kenya sample ( n = 837 ) comprised young adolescent male ( 52 % ) and female ( 48 % ) orphans in grades 7 ( 61 % ) and 8 ( 39 % ) enrolled in school in siaya district , nyanza province .
their mean age was 14.9 ( sd 1.5 ; range 1121 years ) ; 22 % reported ever having had sex . among girls ,
hiv and hsv-2 prevalence was 1 % ( n = 10 ) and 3 % ( n = 28 ) , respectively . among those with positive hiv or hsv-2 test results , 70 % ( n = 7 ) and 64 % ( n = 18 ) , respectively , reported never having had sex .
the original sample for the zimbabwe study was orphan 6th grade girls ( n = 328 ) in manicaland province primary schools ; surveys were administered annually during 20072010 ( hallfors et al . , 2011 ) .
after securing a project renewal grant , we collected biomarker specimens and a final survey in march through june 2012 , when most participants were 1617 years old . from the original sample , we located 287 ( 88 % ) for the 2012 survey .
twenty - three percent reported ever having sex ; 18 % had ever been married and/or pregnant .
hiv and hsv-2 prevalence was 4 % ( n = 12 ) and 6 % ( n = 16 ) , respectively . among those with positive hiv or hsv-2 test results , 50 % ( n = 6 ) and 44 % ( n = 7 ) , respectively , reported never having had sex .
although the protection of adult participants in hiv prevention research is well developed ( national institute of mental health collaborative hiv / std prevention trial group , 2007 ) , we found that clear guidance regarding hiv and other sti testing and disclosure is lacking for children and adolescents .
as an example , our two clinical trials were peer - reviewed by two different nih scientific review panels ; one set of reviewers thought that it was an ethical imperative to disclose hsv-2 as well as hiv results to adolescents , whereas the other was concerned that disclosure of biomarker results might lead to stigma and abuse .
these divergent opinions led to discrepant protocols for disclosure , with one trial disclosing and the other not disclosing test results to participants .
we used the national hiv testing and counseling ( htc ) guidelines [ central statistical office ( cso ) & macro international inc .
, 2007 ; nascop , republic of kenya , 2008 ] for hiv in both countries .
given the lack of national guidelines for adolescent sti testing in kenya [ ministry of health ( moh ) , republic of kenya , 2006 ) , we developed a protocol in conjunction with the siaya district aids and std control office that closely followed hiv guidelines .
study protocols were reviewed and approved by irbs in the us , kenya and zimbabwe .
the kenya study was approved by the ministry of education and siaya district education office ; school head teachers identified orphans and organized meetings in which moi university research collaborators and a district education officer explained the study to students and their caregivers . caregivers and students
we invited a total of 923 orphans in grades 7 and 8 in 2011 to participate ; 849 ( 92 % ) consented and 837 ( 91 % ) completed both the student survey and biomarker testing .
following national guidelines , we required kenya study participants under age 18 to have an adult guardian accompany them for hiv rapid testing ( nascop , republic of kenya , 2008 ) . in some instances , however , the guardian was not available because of work , other commitments , or illness .
in such cases we permitted a relative , teacher or neighbor to stand - in for the guardian if they had a note from the guardian or if research staff were able to speak with the guardian by phone .
given the sensitive nature of hiv testing , the senior counselor interviewed all unclear or undocumented cases to determine whether or not to proceed with specimen collection and htc .
we visited clinics several times to give participants the opportunity to return for testing with their guardian or authorized proxy .
although not required , all participants ages 18 and older were accompanied by an adult for support in case of a positive hiv test result . in accordance with national guidelines
, we gave referrals to publicly funded hiv care and treatment centers to all adolescents with positive hiv test results , while hiv negative participants were counseled on risk reduction ( nascop , republic of kenya , 2008 ) .
we disclosed results to both guardians and participants if the latter were under age 18 and to the participants alone if they were 18 years old according to the study protocol . in zimbabwe , with permission from the provincial education officer , the consortium principal investigator and a research associate held meetings at study schools to explain the continuing study to parents / guardians , participants , and key school staff .
we obtained written parent / guardian consent and participant assent for continued voluntary participation in the study , including separate checkbox consent for survey and blood sample collection .
guardians raised few concerns , although they did ask why we were not disclosing test results .
participants who were married or 18 years or older provided their own consent . in deference to local cultural norms and to prevent potential spousal abuse , we prepared a second consent form for husbands to provide consent for their wives participation .
most of the wives gave their own consent for the survey and did not ask their husbands for consent . in four cases ,
the husband signed a consent form and in five additional cases , the husband refused and the wife did not take the survey . although asking husbands for permission for their wives study participation is not consistent with us culture , it appears to be a useful protocol accommodation for some young wives in rural zimbabwe .
we consistently relied on local researchers and key informants to make such culturally sensitive decisions .
after 4 months , we found 88 % of the original sample who consented to the survey ; 85 % consented to blood specimen collection .
of those not surveyed , two participants had died in childbirth , 2 % refused , and approximately 10 % of the sample could not be found . of those not consenting to blood specimen collection ( n = 8) , six were married and refused for religious reasons or husband s objection .
two were consented for the survey by school officials who refused to provide additional consent for the blood collection , and no guardian was available to give permission .
hiv testing is technically far more advanced for widespread use in sub - saharan africa than hsv-2 testing .
rapid hiv antibody tests are now widely used and accepted ( moh , republic of kenya , 2006 ) , and sequential algorithms for retesting have been adopted in kenya and zimbabwe for rapid testing ( cso & macro international inc . , 2007 ; nascop , republic of kenya , 2008 ; who , 2004 ) .
rapid hiv tests meet high performance standards , sample collection procedures are simple , tests are easy to perform , and interpretation of test results is reasonably clear . in contrast , hsv-2 serologic testing presents some important challenges .
although a number of hsv-2 type - specific serologic tests are available , only a limited number have been tested among sub - saharan african populations .
these include the herpeselect hsv-2 enzyme - linked immunosorbent assay ( elisa ; focus diagnostics , cypress , ca ) and kalon hsv-2 elisa ( kalon biological ltd . , guildford , uk ; biraro , mayaud , morrow , grosskurth , & weiss , 2011 ) .
these tests require skilled laboratory staff , specialized equipment , and a constant source of electricity .
they are appropriate for use with large batches of samples as in sentinel surveys and are inexpensive , costing about $ 3 per test .
although the literature reports variable performance of these tests with respect to sensitivity and specificity in sub - saharan populations , kalon is often found to be superior to focus herpeselect ( see biraro et al . , 2011 for a review of the performance of hsv-2 tests in sub - saharan africa ;
2008 ; ngayo , friedrich , holmes , bukusi , & morrow , 2010 ; smith et al . , 2009 ; van dyck et al . , 2004 ) .
when using the manufacturer s instructions ( index cutoff value = 1.1 for both tests ) , kalon tends to have lower sensitivity but higher specificity than focus herpeselect ( delany - moretlwe et al .
, 2010 ; gamiel et al . , 2008 ; ngayo et al . , 2010 ; smith et al . , 2009 ; van dyck et al . ,
the low specificity of focus herpeselect is particularly problematic when disclosing results to research participants because of an unacceptably high rate of false positives when prevalence is low ( gamiel et al . , 2008 ; mark et al . , 2008 ; ngayo et al
, 2010 ; van dyck et al . , 2004 ) . increasing the assay cut - off values is one strategy for improving the performance of both the kalon and focus herpeselect tests .
a number of studies have shown that increasing the cut - off value for focus herpeselect to 3.5 increases specificity , although sensitivity is necessarily reduced ( delany - moretlwe et al . , 2010 ; smith et al . ,
other studies similarly improved performance at cut - off values of 3.2 and 3.3 ( delany - moretlwe et al . , 2010 ; gamiel et al . , 2008 ; ngayo et al . ,
although findings are mixed , increasing the cut - off value for the kalon test to 1.5 has also been shown to increase specificity , with an attendant reduction in sensitivity ( gamiel et al . , 2008 ; ngayo et al .
in our studies , the kenya laboratory used the kalon assay , and the zimbabwe laboratory used the focus test . at the time of our studies , neither assay had been validated for dbs by the manufacturer in african populations .
the efficiency of dbs for hsv-2 testing with each assay must be evaluated in specific populations prior to use ( hogrefe , ernst , & su , 2002 ) . because the kalon test had not been validated for hsv-2 testing with dbs samples , specimens in the kenya study were obtained by venipuncture .
the zimbabwe laboratory had validated the focus test with dbs ( mudzori & mutsogoro , 2006 ) . in both studies
, we used a modified algorithm to interpret results : samples with initial index values < 0.9 were reported as negative and no further testing was conducted .
samples with initial index values > 2.5 were defined as high positive and were reported as positive without additional testing .
samples with initial index values from 1.5 to 2.5 were considered low positive , and those with initial index values between 0.9 and 1.5 were considered equivocal ; all low positives and equivocals were retested in duplicate . if both retest results had index values > 1.5 , the final result was reported as positive . if both retest results were < 0.9 , the final result was negative .
if retest results were equivocal ( 0.91.5 ) or discordant , the final result was indeterminate .
although test performance was improved by increasing the cut - off and expanding the range of results for repeat testing , the performance characteristics of tests to detect antibodies to hsv-2 are imperfect . based on a recent systematic review of hsv-2 antibody test performance in sub - saharan africa ( biraro et al . , 2011 ) , sensitivity for the kalon test used in kenya was estimated at 94 % and specificity at 92 % with the higher cut - off value of 1.5 .
the predictive values of all diagnostic tests are strongly influenced by the prevalence of infection in the population , and in low prevalence situations , even a very good test will have a poor positive predictive value ( ppv ) . using the sensitivity and specificity values for the kalon test under the testing conditions employed in our kenya study ,
the ppv was estimated at 26 % with a 3 % hsv-2 prevalence .
that is , we can be confident that about one in four participants with positive test results truly had hsv-2 antibodies present in their blood sample ; however , as many as three in four could have had a false positive test result .
in kenya , we conducted sample collection and htc primarily in local public health facilities near the schools . eighteen facilities participated .
in two sites , with permission from the school headmasters and district education officer , we collected blood samples and conducted htc at the school .
venipuncture was conducted by six skilled phlebotomists trained in htc . to avoid obtaining two separate blood specimens ( finger prick for rapid hiv testing and venipuncture for hsv-2 testing )
the process , including counseling , blood draw , hiv testing , disclosure of results took on average 20 min .
the process was longer for participants with discordant results who needed a tie - breaker test and for participants with hiv positive results .
we offered all participants and their guardians a snack either before or after specimen collection .
we stored blood samples for hsv-2 testing in labeled vacutainer tubes in a cooler box without ice and transported them to a local laboratory within 24 h for serum processing .
sera were stored at the local laboratory in a freezer at 20 c until transport on ice weekly to the hsv-2 testing laboratory .
the logistics of specimen collection , processing and transport for this study were challenging but feasible . in comparison ,
we did not require guardians to be present because hiv results were not provided to participants .
sampling and dbs preparation were much less obvious to others nearby , helping to safeguard participant privacy and reduce concerns about witchcraft or other conspiracy theories regarding hiv / aids and blood , which circulate in sub - saharan african communities ( tenkorang , gyimah , maticka - tyndale , & adjei , 2011 ) .
blood from the finger prick was dropped on five circles on a dbs filter card labeled with the participant s study i d .
some participants may have been dehydrated or not very well nourished , making it difficult to fill all circles , but adequate samples were collected from all participants .
specimens were dried , stored with a desiccant , and transported to the testing lab at ambient temperatures .
dbs samples are easy to store and transport and require no processing or refrigeration ( see table 1 for a summary of the study procedures).table 1comparison of biomarker testing procedures in two research studieskenyazimbabweethical considerationswritten guardian consent if < 18/adolescent assentwritten guardian consent / adolescent assentresults disclosed to guardian and adolescentno results disclosedreferrals for carenot applicablecollecting and analyzing biological samples methodvenipuncturedried blood spot hiv testrapid testrapid test hsv testkalon elisafocus elisafeasibility of collection methods time20 min3 min temperature for specimen transportcooler with no iceambienttesting costsreference labreference lab bench fee , supplies , storage , labor us$ 5,784bench fee , supplies , labor us$ 8,000field site lab sample processing , labor , supplies , storage us$ 3,611additional fee us$ 480 other supplies us$ 100 transportation us$ 375 total us$ 9,919total us$ 8,480
elisa enzyme - linked immunosorbent assay comparison of biomarker testing procedures in two research studies
elisa enzyme - linked immunosorbent assay
given the experience we have described , we conclude that it is feasible to obtain hiv and hsv-2 biomarker data for adolescent hiv prevention intervention studies . in particular
the us pepfar program has accomplished considerable staff training in sub - saharan africa ; it has also stimulated the development of well - equipped laboratories , as well as useful tools for testing and protocols for improving test sensitivity , specificity , and cost ( stringer et al . , 2006 ) .
in addition , it has stimulated the development of national guidelines ( particularly for adults ) for the disclosure of results . for hiv
incorporation of hsv-2 serology results poses considerable challenges , particularly if results are disclosed to participants .
based on our experience , there are two reasons why we do not recommend disclosing hsv-2 serology results to adolescent human subjects in sub - saharan africa .
first , in populations with low or moderate prevalence of infection , as might be expected for adolescents , the potential for false positive test results is substantial in combination with relatively minor imperfections in test specificity .
hsv-2 infection is often a silent disease , insofar as only 1025 % of people with antibodies for the condition are aware that they have genital herpes ( fleming et al . , 1997
; leone , fleming , gilsenan , li , & justus , 2004 ; sizemore , lakeman , whitley , hughes , & hook , 2006 ) .
adolescents without symptoms may deny ever having had sexual intercourse3 , further complicating the clinical picture and raising concerns about false positives with the potential for psychological and social harm related to participation in the study .
second , local departments of health and sti authorities in sub - saharan african countries were much less familiar with hsv-2 pathology and treatment than with hiv and some other stis . even in cases where subjects acknowledge sexual exposure or genital herpes symptoms , local health clinics may not be prepared to provide appropriate treatment or care . given these vexing problems , the wisdom of disclosing hsv-2 results to adolescents after testing within a clinical prevention trial is questionable . in particular , decisions about whether to inform adolescent participants in prevention trials about hsv-2 test results should be made only after prevalence data are available and the predictive values of the test can be accurately assessed . in light of the feedback from the nih study section that raised concerns about possible stigma and abuse of hiv - positive participants in zimbabwe , as well as our own concerns ,
we recommend that more research be conducted to study the consequences of disclosure to adolescents in sub - saharan africa .
a review of the literature indicates that sub - saharan adolescent prevention studies with biomarker outcomes have taken a variety of approaches to disclosure . in some studies , counselors disclosed participants hiv results immediately after rapid testing ( baird , garfein , mcintosh , & ozler , 2012 ; dalal et al . , 2012 ; medley et al . , 2012 ) .
in other studies , participants could choose to receive their results after testing , either immediately or a few weeks later ( amornkul et al .
, 2009 ; jewkes et al . , 2006 ; ross et al . , 2007 ) .
in still other studies , the research did not provide test results but voluntary counseling and testing ( vct ) was made freely available for study participants ( birdthistle et al .
none of these studies , however , examined whether adolescents experienced any untoward outcomes with testing and disclosure , whether hiv positive youth enrolled in care or took steps to prevent transmission , whether hiv negative youth benefited from prevention counseling and engaged in subsequent testing , or if adolescents and their guardians felt it was appropriate to learn their status in the context of a research study . from our kenya study , the problem of stigma did not appear to be a major obstacle to adolescent testing and disclosure in our study area .
perhaps this is because hiv prevalence is among the highest in the country , and few families have been unaffected by hiv / aids .
moreover , high hiv prevalence has drawn research , education , and hiv counseling and testing campaigns to the area .
for example , in our kenya clinical trial , more than 50 % of participants ( all in grade 7 and 8) reported that they had previously been tested for hiv , mostly through a home - based testing initiative of the kenya medical research institute ( kemri ) and us centers for disease control and prevention ( cdc ) .
although our study provided hiv test results to youth , we relied on phlebotomist / nurse counselors to refer youth and their guardians for care , following national guidelines .
the onus of making contact with the clinic was left up to the individual and was not tracked by the system of care ; and follow up to make sure that hiv positive youth accessed care was well beyond the scope of our study . since it is unknown whether youth experience significant mental trauma and social prejudice , and whether they actually engage in the system of care , we recommend that further research be conducted to examine the perceptions and behaviors of youth and particularly newly diagnosed youth after disclosure of their test results to ascertain the consequences of this aspect of research participation and whether it can be improved .
this is particularly important for adolescents , since results are disclosed in the presence of their guardian .
regarding procedures for collecting biomarkers , we conclude that finger - sticks and dbs are overwhelmingly superior to venipuncture in sample collection efficiency and reduced burden on participants and community institutions .
manufacturer validations of dbs for hsv-2 are urgently needed , however , for this procedure to be more widely accessible to researchers . because blood collection for dbs is minimally invasive , this procedure has excellent potential for widespread acceptability and consequent high participation in school and community research sites .
in addition , rapid hiv testing in sub - saharan africa is typically conducted by finger prick , making this the preferred way for trained african health workers and counselors to participate in research data collection ( who , 2004 ) .
given the complex decisions required for biomarkers , we recommend that behavioral prevention scientists interested in using biomarker data collaborate from the early planning stages with biomedical scientists who have expert knowledge in hsv-2 , as well as hiv , laboratory tests and testing procedures .
we found that team members with such expertise provide valuable assistance in selecting qualified in - country laboratories , developing appropriate budgets , protocols , quality assurance and algorithms for interpreting results , and helping research staff to bridge communication with in - country laboratory staff .
further , we recommend that hsv-2 test kit manufacturers determine optimal cut - off standards for sub - saharan populations , and that researchers who conduct hsv-2 testing explicitly define the testing cutoffs they use in publications .
there is now a substantial literature documenting validity problems when using manufacturers cutoffs with african populations potentially inflating prevalence findings ( gamiel et al . , 2008 ; ngayo et al . , 2010 ; van dyck et al . , 2004
this suggests the need for action to improve assay validation practices on the part of manufacturers as well as methodological details from researchers documenting their findings . when conducting collaborative international research , it is important to recognize that alternative ethical systems exist .
thus , for international research we recommend inclusion of team members who are knowledgeable about local contexts . indeed
, successful collaborative research partnerships respect local cultures , values and practices ; negotiate effectively within these systems ; and incorporate into study designs ethical practices that are appropriate and sensitive to local cultural contexts ( e.g. , in our case , providing for husband consent for married participants ; christakis , 1992 ; emanuel , wendler , killen , & grady , 2004 ) .
behavioral prevention scientists traditionally have relied on self - reported sexual behavior survey item measures to evaluate adolescent hiv prevention interventions .
our experiences conducting research with orphan adolescents in kenya and zimbabwe suggest that collection of hiv and sti test results even in rural , resource - poor settings in sub - saharan africa is a feasible addition to the behavioral research toolkit .
the use of sti biomarkers can greatly improve the validity of findings from adolescent behavioral intervention trials .
research is urgently needed to examine the risks and benefits of hiv testing and disclosure of test results in the context of a research study for adolescents . further development and implementation of sti biomarker assessment techniques particularly pertaining to using dbs is needed to advance hiv prevention science .
|
self - report of sexual behavior among adolescents is notoriously inconsistent , yet such measures are commonly used as outcomes for human immunodeficiency virus ( hiv ) prevention intervention trials .
there has been a growing interest in the use of hiv and other sexually transmitted disease biomarkers as more valid measures of intervention impact in high hiv prevalence areas , particularly in sub - saharan africa .
we examine the challenges , benefits , and feasibility of including hiv and herpes simplex virus type 2 ( hsv-2 ) biomarker data , with details about different data collection and disclosure methods from two adolescent prevention trials in kenya and zimbabwe . in kenya ,
whole blood samples were collected using venipuncture ; adult guardians were present during biomarker procedures and test results were disclosed to participants and their guardians . in contrast , in zimbabwe , samples were collected using finger pricks for dried blood spots ( dbs ) ; guardians were not present during biomarker procedures , and results were not disclosed to participants and/or their guardians . in both countries ,
prevalence in the study samples was low .
although the standard of care for testing for hiv and other sexually transmitted infections includes disclosure in the presence of a guardian for adolescents under age 18 , we conclude that more research about the risks and benefits of disclosure to adolescents in the context of a clinical trial is needed .
notably , current serological diagnosis for hsv-2 has a low positive predictive value when prevalence is low , resulting in an unacceptable proportion of false positives and serious concerns about disclosing test results to adolescents within a trial .
we also conclude that the dbs approach is more convenient and efficient than venipuncture for field research , although both approaches are feasible .
manufacturer validation studies using dbs for hsv-2 , however , are needed for widespread use .
|
Introduction
Brief Description of Case Studies
Ethical Considerations Including Consenting Procedures
Collecting and Analyzing Biological Samples
Feasibility Comparison of Sample Collection Methods
Discussion
|
nanobiomaterials are characterized by constituent
particles and/or surface features less than 100 nm
in at least one dimension
.
starting with photolithography and dry etching in the
1980 's to high - resolution electron beam lithography and other technologies
in the 1990 's
, nanotechnology allows for making surface structures for
cell engineering and has led to an increasing application in healthcare over the last decades .
nanolayers are used to enhance the
surface biocompatibility of polymeric drug delivery systems ,
control the release of substances such as antibiotics or growth factors
, act as gene - delivery
vehicles , or serve as robust light emitters for
cellular labeling and tracking
[ semiconductor nanocrystals , quantum dots ( qds ) ]
.
nanotechnology is also applied to modify and improve the surface
structure in orthopaedic implants to promote their
osseous integration . however , there are also side effects of nano- and
microparticles in vivo .
micro- and nanoparticles released by friction of
articulating partners from artificial joints are a major
reason for aseptic
implant loosening in orthopaedic surgery and may lead to severe
peri - implant
osteolysis ( particle disease )
.
in addition ,
nanoparticles can induce or promote allergic or inflammatory
reactions or
influence hemolysis and blood coagulation
[ 57 ] .
although the cytocompatibility of a biomaterial
is strongly influenced
by its chemical composition , surface topography plays a
crucial role for cell - surface
interactions .
material surface
properties have been studied intensively , but
still lack from reliable data about cytocompatibility .
especially , the
superordinate principles of cellular responses to
surfaces with a defined
topography are not well known and poorly understood .
because many variables
influence cellular interactions to surface structures ,
it is difficult to draw
conclusions and formulate general principles for
nano- and microstructured
surfaces .
this review summarizes recent data of effects by nano- and
microstructured biomaterials and particles in vitro designed for
orthopaedic application to get a solid
framework outlining the critical interactions that govern the
cytocompatibility .
because biomaterials in orthopaedics are predominantly
applied on bone , this review is focussed on
the interactions of osteoblasts
and bone - marrow - derived
cells with structured biomaterials .
osteoblasts and osteoclasts are mainly
responsible for the osteointegration of
nanostructured biomaterials in orthopaedics .
osteoblasts derive from
mesenchymal progenitor cells which are localized mainly
in the bone marrow and
periosteum .
they are characterized by cuboidal
and flat morphology ( diameter about
20 m ) , present a large
amount of rough endoplasmatic reticlum and a large golgi apparatus ,
and are potent to produce osteoid , a collagen i rich matrix
.
in addition , these mononuclear cells are also responsible
for osteoid calcification ( hydroxyapatite ) .
typical marker proteins for
osteoblasts are cbfa1/runx2 , osteocalcin , osteopontin , osteonectin , bone
sialoprotein ( bsp ) , osteoprotegerin
( opg ) , collagen i , and alkaline phosphates
( alp ) .
figure 1
gives a brief summary of the expression
of several markers during osteoblast differentiation .
osteocytes act in a paracrine and mechanosensory manner , and
can activate osetoblasts and osteoclasts .
the latter cell type derived from the
hematopoietic line , has multiple nuclei and is
responsible for bone resorption .
its ruffled border is flanked
by a sealing zone which facilitates local acidification
and removal of bony
matrix such as ca , h3po4 , and
h2co3
by endocytosis .
osteoclasts express
high levels of tartrate - resistant acid phosphatase
( trap ) and cathepsin k. the
interaction between osteoblasts and osteoclasts is complex . during differentiation ,
the ostoblast progenitors express receptor
activator of nuclear
factor ligand ( rankl ) and macrophage colony - stimulating factor
( m - csf ) which are strong stimuli for osteoclastogenesis .
in contrast ,
osteoprotegerin ( opg ) is a potent inhibitor
of osteoclasts .
moreover , the
interactions between osteoblasts and osteoclasts in vivo are regulated
by several hormones and cytokines , including parathyroid hormone ( pth ) ,
calcitonin , and il-6 .
it is generally accepted that the
three - dimensional surface topography
( size , shape , surface texture ) is one of
the most important parameters that influence cellular reactions
[ 2 , 1119 ] .
although many studies have investigated
cellular reaction to different surface pattern ,
the significance of macro
structure studies on bone cell behavior is
questionable since in vivo adhesion
structures ( e.g. , cell membranes ,
basement membranes ) are comprised of much
smaller nanometer scale features [ 20 , 21 ] .
the immature bone is characterized by an average
inorganic grain size of
1050 nm whereas mature bone has an average
inorganic grain size of 2050 nm
( 25 nm in diameter )
.
considering these parameters , modern
implants for bone application have been
designed with a smooth surface at the
nanometer level .
it was surprising that
some of these have induced the formation of peri - implant
fibrous tissue and
implant loosening in vivo , while other implants with a higher
degree of roughness showed significant better osteoconductive properties
[ 2325 ] .
there are various methods to modify the degree of
roughness as well as surface energy
and topography in orthopaedic implants
.
typically applied techniques to enhance
the degree of roughness and promote
the osteointegrative properties of
biometals ( e.g. , ti , cocrmo , ss ) are chemical
etching or anodization and
also sand - blasting , sputter - coating , and machine - tooling .
the lack of knowledge in cellular reaction to
nanostructered biomaterials is based
to a great extent on
the difficulty in varying surface chemistry and
topography independently .
moreover ,
the use of different cell lineages and culture
conditions makes it difficult to
compare results from different investigators
[ 2631 ]
( table 1 ) .
there is also a lack of consensus concerning the
proper representation of implant surface topography
.
one major misunderstanding is the practice of defining a
surface by its manufacturing process instead of concisely defining the
topographic measurements [ 17 , 33 ] .
considering these limitations for interpretation ,
the
following review gives an overview of cellular
reactions to surface structures
of different orthopaedic biomaterials .
the first step after exposure of any biomaterial
to a biological environment results in the rapid
adsorption of proteins to its
surface .
the composition , type , amount , and
conformation of adsorbed proteins regulate the
secondary phenomena such as
cellular adherence and protein exchange
[ 3537 ]
and also following cellular reactions such as migration ,
proliferation , and differentiation .
the potency for biomaterials to adsorb
proteins is influenced by its physiochemical
characteristics such as surface energy or hydrophobicity ,
and is also dependent on
the local environment ( ph ,
concentration of ions , composition and
functional groups of proteins , strength
of solution , temperature )
( vroman effect )
( figures 2 , and 3 ) . for inorganic nanocrystals and microstructured
surfaces
there are at least two approaches to change
their hydrophobic surfaces :
a ligand exchange reaction can replace the original
hydrophobic surface with
bifunctional coupling molecules or an inorganic
coating such as silica ( 1 ) or
an encapsulation of nanocrystals in an
amphiphile organic coating ( 2 ) .
the first phase of protein adsorption onto a
biomaterial 's surface is characterized
by the attachment of small rapidly diffusing
proteins , followed by a progressive replacement by larger
proteins with a high
affinity to the substrate . here
, especially proteins
with arg - gly - asp ( rgd )
containing sequences such as fibronectin or
vitronectin act as cell receptors
and have chemotactic or adhesive properties to bone cells .
in addition , these rgd - peptides
also have a strong effect on matrix maturation
and biomineralization [ 4648 ] .
after conditioning of a naked biomaterial by protein adsorption , cells attach rapidly
on the protein - coated surface .
besides the influence of proteins , the
cellular attachement to a nanostructed surface
is also influenced by its physiochemical
properties , especially by the outer functional groups
[ 30 , 50 , 51 ] .
schweikl et al .
showed on self - assembly monolayers that the
osteoblast proliferation on hydrocarbon
chains , terminated by ch3 ,
was as high as on amino groups
( nh2 )
and hydrophilic oxidized surfaces , but significantly lower
on fluorocarbon
( cf3 )
groups .
mller et al
.
showed that
3-aminopropyl triethoxysilane ( apts )
presents amine functional groups which allow for
grafting rgd tripeptides and
that the rgd - apts hybrid promotes cell adhesion ,
spreading , and cytoskeletal
organization . here , the zetal potential ( differences in potentials
between the surface of a tightly
bounded layer and a diffuse layer )
and the interfacial tension ( wettability ) of
a surface is crucial [ 54 , 55 ] .
it was demonstrated for cpti surfaces that the contact angle ( ca ) ,
parameter for
wettability , increases linearly with the average roughness when
the angles were
higher than 45 , but decreases linearly with roughness
when the angle was less
than 45 .
recent data examining osteoblast
response to controlled surface chemistries indicate that
hydrophilic surfaces
( high number of polar components ) improve cell attachment
and matrix synthesis and also the osteogenic
potency compared to hydrophobic
surfaces [ 5759 ] .
compared ti alloys and cocr alloys towards protein
absorptive properties and cell attachment with an
osteoblast precursor cell
line .
they found no significant
differences between ti alloys and cocr ,
but significantly greater cell adhesion
rates for the ti implants and concluded that cell
adhesion is a result of
higher hydrophilicity of ti alloys . in
contrast
, other data showed that a low degree
of wettability promotes protein
adhesion and also cellular attachment to a biomaterial
,
and mller et al .
found no direct correlation between the
wettability of the material surface and the osteoblast attachment and
proliferation rate .
also qu et al .
found no significant differences of cell attachement
on various titanium surfaces with different degrees of wettabilities
( hydrophobic acid - etched , coarse - blasted large
grit acid - etched , hydrophilic modified
acid - etched , and modified coarse - blasted large grit ,
acid - etched
) on mg68 cells .
heating ( oxygen / atm ) or peroxide treatment of biometals
result in a thicker oxide layer
and a more hydrophilic surface .
.
showed that
heat - treated titanium
surfaces changed the wettability ( more hydrophilic )
but does not significantly
affect the fibronectin and albumin adsorption as well as
the initial osteoblast
precursor cell attachment in vitro .
emphasized that the rate
of protein correlates more with changes in chemical
composition than
with changes in wettability in metal surfaces .
they showed that
a preheating of ti6al4v specimen does not only lead to a
thicker oxide layer but also results in
an enrichment of v and al within
the surface oxide .
in contrast ,
post - treatment with butanol after preheating
reduces the content of v , but not in al ,
and significantly increases the rate
of fibronectin adsorption up to 2040%
.
compared to the cellular attachment phase , the following adhesion
phase lasts longer and
involves various proteins and molecules
( figure 2 ) .
as a link between cell and
biomaterial , the interactions of a surface topography
and serum proteins are
crucial for the cytocompatibility of a biomaterial .
especially , the adsorption
of adhesion proteins , such as fibronectin and vitronectin , from serum
containing solutions and integrin - mediated signaling has been
demonstrated to
mediate cell adhesion and spreading
. it has been shown that nanotube or nanoparticle surfaces
created by anodization have
promoted osteoblast adhesion up to three times
compared to unanodized ti .
these results were confirmed by the group of webster
and other investigators [ 6871 ]
who demonstrated that the initial
attachment of osteoblasts onto the surface of biometals such as cpti ,
ti6al4v , and cocrmo is enhanced by submicron to nanometer
consistent particles compared
to metals composed of respective micron particles .
one possible explanation of
this phenomenon is the higher amount of particle binding sites for
osteoblast adhesion at the surfaces of nanophase metals
compared to micron particle size
metals .
the theory of enhanced protein and cell binding capacities
by larger
surface areas / roughness degrees was also confirmed for porous
ha materials .
another example of the significance of surface structures
for protein binding and
osteoblast attachment is the helical rosette nanotubes
( hrn ) which can build
self - assembly surface structures .
it was demonstrated that
a significant change
of hrn coverage by heating correlated with the protein - binding
and osteoblast
adhesion potency in titanium surfaces [ 73 ,
74 ] .
it is evident that not only the surface topography
influences protein deposition and
cell adherence but also proteins and cells modify the surface
properties of a
defined surface .
based on a surface analysis of the different
biometal specimen
before and after cell cultivation , we showed previously
that a cell attachment and/or protein
precipitation increase the roughness in polished biomaterials
( steel , ti6al4v ,
and cocr ) .
for porous coated cocr surfaces , we found only slight and no
relevant changes in roughness whereas cell cultivation onto
sandblasted ti6al4v
lead to a strong decrease in specimen roughness .
both , the increase in roughness after
cell culturing in the different biometals
and the decrease in roughness of sandblasted ti6al4v could be
explained by the
dense cellular growth and accumulation of debris in depth of
the structured surfaces
and/or protein deposition as shown by other investigators
[ 75 , 76 ] .
in addition , not only the amount but also the type of
protein adsorption by a
surface is crucial for cellular adherence and following reactions such as
migration and differentiation . as an example , ti surfaces ( ra :
0.370.01 m ) adsorp
fibronectin in higher concentrations
compared to albumin , and fibronectin - coated ti surface promoted
more osteoblast attachments
in comparison to albumin - coated ti surfaces
.
these results correspond to the data of other authors
who showed excellent osteoconductive properties after
fibronectin adsorption
onto a biomaterials ' surface [ 7880 ] . based on irm and tem analysis ,
the closest distance
of cells to a surface ( glass ) was
found to be approximately 10 nm [ 81 ,
82 ] .
historically , results from chicken
fibroblasts have lead to a classification of three different types of
separation .
( 1 ) focal contacts ( fc ) : approximately
1015 nm separation from the substrate under the
peripheral regions of the leading lamellae
( appearing black in tem ) .
fc act as
an interface between intra and extracellular components and occur linearly
beneath the associated cytoplasmic stress fibres
[ 83 ,
84 ] .
they are tenacious adhesion sites that
remain attached to the substratum even when cells are forcibly detached ,
indicating their function as anchorage
structures .
( 2 ) close contacts : corresponding to approximately
30 nm separation ( broader grey areas
in tem ) .
( 3 ) greater separation : corresponding to approximately
100140 nm ( white
regions in tem ) .
it is evident that not only fc appear soon after cellular attachment but also that ( -catenin - positive ) adherence
junctions occur within 14 hours
for grooved ti - based substrates .
these observations underline the high
significance of an early intercellular communication soon
after adherence to a
surface .
the mechanisms of initial cellular adherence to a surface are
different from long - term adherence as shown by a lack of statistical
correlation between short - term adhesion ( strength of cell attachment
and early
adhesion ) and long - term adhesion ( strength of cell - matrix interface )
forces [ 14 , 15 , 86 ] .
.
showed that the cultivation time has an
influence on the long - term adhesion in biometal surfaces according to
td
( t ) = atb ,
a being independent of b ( td : time - dependend
adhesion index , a : surface - dependent parameter , b : substrat - independent
exponent , 0.5+/-0.03 ) . for polylactides ( plla ) , it was shown on oct-1 osteoblast - like
cells that cell
adhesion but not the proliferation could be enhanced by nanoscale
and microscale
roughness compared to smooth surfaces .
in addition ,
there is evidence that fc
show a dynamic behavior which allows for cellular migration and motility .
linear plla fibres with length scales of 0.52 m ,
constructed by electrospinning , have shown cellular contact
guidance and enhanced
osteoblastic differentiation . here , cell morphology revealed that cells
grown on fibres had smaller projected areas than those on planar
surfaces .
also other polymers such as plga have been shown to
be effective in enhancing osteoblast differentiation in vitro
.
diener et al . demonstrated
on mg-63 osteoblastic cells
that fc adhesion was smaller on ti and ss than on collagen - coated glass
coverslips and that all fc showed a mobility of focal adhesions .
however ,
anselme et al . found higher
adhesions on ti6al4v
substrates than on noncollagen - covered glass samples ,
and emphasized that substrates
with various surface compositions
but with the same surface topography did not
induce significant differences of adhesion .
based on the knowledge of protein adsorption and its effects
on cellular attachment and adherence , a selective surface coating
of nanostructured
surfaces with rgd or collagen proteins offer a promising solution
to improve
the number of osteoblasts adhered on artificial surfaces
[ 53 , 95102 ] .
imprinting
surfaces technology with deposition of specific
protein - recognition sites can
help to promote osteoblastic growth
and differentiation [ 103106 ] .
protein - recognition
can be based on a protein - ligand binding and/or electron donor - acceptor
interactions or other types of binding forces .
integrin
51 and 5
v3 subunits competitively
bind to rgd - sites of fibronectin [ 107 ,
108 ] .
dependent on the
surface topography and
chemistry of the
biomaterial , fibronectin
undergoes changes in structure including
modulation in functional activity and
shift in integrin binding capacity .
based on the data of self - assembled monolayers ,
it was shown that integrin subunits
show selective binding capacities to different
terminal groups .
integrin 51 shows a
strong affinity to oh and
nh2 surfaces , whereas
51 and
5v3 bind also to cooh but show poor binding capacities
on ch3
surfaces [ 109113 ] .
furthermore , some data show that oh
and nh2 surfaces can up - regulate
osteoblast - specific gene expression but also
matrix mineralization compared
with cooh and
ch3
functional groups [ 47 , 112 ] .
cell migration and proliferation is the attachment
following phase between the cell
and the material surface .
it is evident for designing
nanostructured implants
that cells use the nanotopography of a substrate for
orientation and migration [ 117119 ] .
although
it is known that bone cells
align along defined substrate morphologies ( contact
guidance ) , the detailed relation between ordered
nanotopography and cell
behavior remains unknown in detail .
for the first time , in 1964 it was shown
that convex surfaces enhance cellular overlap , while
grooves minimize cellular
overlap .
as pre - requisite to reach a defined cell colonization during
directed tissue formation , structured nanophase surfaces
lead to a predictable
osteoblast orientation and migration on these surfaces
[ 17 , 121 , 122 ] .
interaction between the ecm and associated
changes in the
orientation of the cytoskeleton are crucial for cell
metabolism of cells and
morphology due to actin - myosin tension structures
.
anisotropic topographies ( e.g. ,
topographical grooves , chemically patterned stripes ,
or curved surfaces of a
fibre ) are potent to exert morphological
as well as physiochemical
features on cells at the same time , indicative for the
complex environmental
influence on cells .
focal contacts are important structures for cellular
adherence onto a surface but may
also delay migration and mobility of the cells .
it was shown that bone - derived cells ( mg63 cells ) respond to
a nanoscale roughness by a higher cell thickness and a
delayed appearance of
focal contacts .
especially , nanoporous
ti - oxide surfaces promote cellular
spreading and induce numerous filopods and osteoblastic differentiation
[ 124 ,
125 ] . on
electrochemically
microstructured hexagonal
pattern , mg63-cells go inside
30100 m but not in 10 m
cavities .
most authors report a
parallel orientation of cells cultured on
polished ( smooth ) surfaces [ 57 , 114 , 126 ]
( figure 4 ) .
another method to not only enhance cellular adherence but also to
promote osteoblastic differentiation and
biomineralization of biometals is a surface anodization ,
for example , by -glycerophosphate sodium and calcium
acetate [ 6671 ] .
cellular adhesion via fc may strengthen the linkage
between cell and ecm and
also impair the ability to dynamically remodelling
the ecm and influence the
migration rate . for
collagen - coated coverslips , focal adhesion of mg-63
osteoblastic cells moved
with a speed of 60 nm / min , whereas the speed was
reduced in ti and more in ss
surfaces .
another study
on nb2o5-coated
polished cpti samples showed that mc3t3-e1-osteoblast
migration was fastest on
smooth surfaces ( ra = 7 nm ) , whereas adhesion strength ,
spreading area , and
collagen - i synthesis were promoted by intermediate
roughness ( ra = 15 nm ) .
however ,
it was surprising that higher degrees of roughness
( ra = 40 nm ) were rather
peaked and reduced the speed of adhesion process in the
same study . besides the surface properties of a biomaterial ,
the cellular migration rate is
dependent on the cell type and its differentiation stage .
a higher migration
rate is associated with a lower level of osteoblast
differentiation .
cells with
a low motility are characterized by a strong formation of fc
while motile cells
form less adhesive structures .
it was found that mature
osteoblasts spread out
and form a greater number of fc when settled on smoother surfaces
.
although
cellular spreading is higher on
smoother surfaces , some data indicate that the
alp - expression is higher for
rough isotropic surfaces ( electro - erosion , acid - etching ,
sandblasted ) compared
to smoother substrates ( machine tooling , polishing )
. considering recent publications , there
is no or only week statistical significance that there
is a difference between
the initial number of adherent cells and following proliferation of cells
cultured onto a biometal or ceramic nano-/microscale surface
in vitro .
however ,
some authors emphasize that
the influence of functional chemical groups for cellular migration and
proliferation are stronger than general surface
properties such as wettability .
especially a tio2-layer
seems to promote
cellular growth and proliferation on nanostructured
biometals [ 128 , 129 ] .
other examples for a promotion of cell - to - bone contact in vitro
and also in vivo are
machine - etched ti - surfaces ( e.g. , osteotite )
, defined sand - blasted
implants [ 124 , 125 , 131 ] , and hydroxyapatite ( ha ) coatings ,
for example , by plama - spray techniques [ 132134 ] .
recent studies investigating the response of adherent
cells to nanography surfaces
indicate that different cell phenotypes have different
levels of sensitivities [ 117 , 135137 ] . here
,
osteoblasts react to features as
low as to the 10 nm dimensions , which is comparable in size to a single
collagen fibre .
moreover , the qualitative and quantitative kinetics
in gene and protein expression is
strongly influenced by topography and physiochemistry
of a defined surface .
microporous
ha surfaces seem to promote a high number of fc and
increased levels of alp but
short actin stress fibres compared to nonmicroporous ha surfaces
[ 72 , 139 ] .
there is also evidence that ti and ha
surfaces can activate early intracellular signalling
pathways as shown by
expression of relevant molecules such as
- and 1-integrin , fak , erk followed
by c - jun and c - fos genes for proliferation and alp for differentiation
[ 139 , 140 ] .
however , hallgren et al .
found no significant histomorphometric
and biomechanical differences between nanopatterned and control implants .
.
showed that microfabricated
discontinuous - edge surfaces ( des ) ,
repeated open square boxes with a depth of
10 m , alter osteoblast adherence and migration
but enhance cell multilayering ,
matrix deposition and mineralization when compared to smooth controls .
in contrast to our data ,
anselme et al . found
higher proliferation
rates on ss compared to ti6al4v .
however , bigerelle et al .
demonstrated that neither
material composition nor surface roughness amplitude influence cell
proliferation , whereas they found a very significant influence
on manufacturing
process and surface topography for long - term adherence
and proliferation in vitro .
our in vitro results confirm the
well known osteogenic in
vivo properties of ti implants , which may be based on
surface factors observed
on its outer tio2-layer
[ 143146 ] .
mller et al .
demonstrated the ability of osteoblasts
to grow into an open - porous ti implant ( metal foam ) and li et al
.
also demonstrated that mc3t3-e1 cells
attach to and are able to divide well in the inner surface of
a highly porous
trabecular ti6al4v implant .
some in vitro studies demonstrated an
enhanced total protein and collagen production ,
as well as increased alp
activity of osteoblasts cultured on nanoparticulate
metals ( cpti , ti6al4v , and
cocrmo ) indicating advantages for nanostructured surfaces
for osteointegration [ 1 , 149 , 150 ] . based on the
data of redey et al . ,
it can be concluded that the low
attachment and collagen production rates are related to a low
wettability of a
nanosurface .
nanotextured surfaces of ti surfaces
prepared by chemical etching
have upregulated the expression of bsp and
op .
as demonstrated by qu et al . ,
the expression of the bone - associated
genes such as alp , oc , type - i - collagen , osteoprotegerin , and
glyceraldehyde-3-phosphate - dehydrogenase is promoted
by modsla ti surfaces .
some data also suggest that fluoride - modified
ti surfaces can stimulate
osteoblastic differentiation compared to
unmodified titanium surfaces [ 151 ,
152 ] .
.
showed in their in vitro experiments that nanophase biometals induce
significantly greater calcium and phosphorus
deposition by osteoblasts and also allow for
calcium and phosphorous precipitation from
culture media without osteoblasts in
contrast to microphase ti6al4v and cocrmo .
furthermore , the authors found
advantages in mineral precipitation without osteoblast for
tial4v but no differences
in dependency to the type of ti ( wrought , microphase ,
or nanophase ) .
it was
evident that the increased calcium and phosphorus
mineral content correlated to
greater amounts of underlying aluminium content on
ti6al4v surfaces . although some data indicate
that nanostructured ti alloys promote non - cell - mediated ca / po4-mineral
deposition from culture media compared to cocrmo substrates ,
the greatest cell - dependend calcium and
phosphorus mineral deposition occurred
on nanophase cocrmo .
it is evident that micropattern collagen films or
scaffolds promote not
only cellular adhesion but also allow for
an osteoblastic differentiation and
biocalcification in vitro [ 153155 ] .
for ha- and dcpp - coated , ti
surfaces the ca / p ratio influence the biomineralization
rate in vitro .
besides the osteoblast - promoting effects
of defined substrates and
surface topographies , some data also allocate
an inflammatory response induced
by nano- or microstructured biomaterials .
it was shown in many studies that
cell - biomaterial interactions can activate macrophages
which results in the synthesis of proinflammatory
agents such as tnf , ifn , il-1 and -6 ,
rankl and no [ 157159 ] .
some data have
shown proinflammatory effects of different
biomaterials which increase with the
degree of surface roughness .
here ,
macrophage inflammatory protein-1 , tnf ,
monocyte chemoattractant protein-1 , and
members of the interleukine and leukotriene family
play a crucial role in
biometal - induced inflammations
[ 160164 ] .
most studies report about an
enhanced expression of pro - inflammatory
cytokines and chemokines by cells
attached to rougher surfaces .
some data also
indicate that anionic and neutral hydrophilic surfaces
increase macrophage - monocyte apoptosis and reduce macrophage
fusion to modulate
inflammatory responses to implanted materials
.
however , adverse cellular effects seen with
metallic implants may
also be attributed to corrosion products or to
the separation of metal ions
( fe , cr , ni ) which may have a major impact on cellular survival and
differentiation [ 166168 ] .
those studies which suggest that a cell - mediated metal ion
release by biometals that
did not affect
the cell viability or proliferation are
characterized by short cultivation periods or
other conditions which limit the
reliability of data [ 169171 ] . up to date , only few authors report about no significant influence
of the cellular adherence and expression of osteoblast
proteins by different
biometals and surfaces such as alp expression
[ 172 ,
173 ] .
in contrast to the great opportunity enhancing
biocompatibility and osteogenic potency of surfaces applied on bone by
nanotechnology , micro- and nanoscaled particles released by friction of
artificial joints can induce severe
inflammation and may lead to osteolysis and
implant failure [ 174 ,
175 ]
( figure 5 ,
table 2 ) . there is a wide
range in particles size and morphology produced by
simulators for artificial
joints .
particles released from metal - metal ( crcomo alloys ) are
predominantly chromium oxide particles or cocrmo with
varying ratios of co and cr .
they show a round to oval morphology
and also a substantial number of needle - shaped
particles were found during the first circles .
emphasize the
importance of particle size
as a critical factor in osteoblasts proliferation
and viability in vitro .
some data indicate
that in contrast to ti - surfaces
nano- and mircoparticles induce an
inflammatory response although titanium is
one of the biometals with the highest degree of
cytocompatibility . as shown by miyanishi et al .
,
the release of vegf may play a crucial role in the
pathogenesis of ti - induced osteolysis .
some data indicate that
phagocytosis of ti particles is not a precondition for
an inflammatory response
such as a release of tnf or il-6 in
cultured macrophages .
it is evident that a binding of the macrophage cd11b / cd18 ( macrophage
mac-1 receptors / receptor of complement cr3bi ,
can also bind to icam-1 and
icam-2 ) by integrin - specific antibodies
also increased the release of tnf and il-6 in macrophages .
this finding also suggests
that the complement system plays a role in the pathogenesis
of particle - induced
inflammation , too .
especially , uhmwpe particles with
a size range of
0.11.0 m have been shown to be most reactive for
macrophage activation and cytokine
secretion in bone marrow cells [ 179 ,
180 ]
. however , not only the particle size but also the particle volume
( number ) is a critical factor for particle - mediated
release of cytokines by
macrophages .
green et al .
demonstrated for pe
that the cell - particle ratios of 1 : 100
( size 0.497.2 m ) and 1 : 10
( size : 0.494.2 m ) induced
significant stronger release
of tnf and il-1 in macrophages .
the authors conclude that especially
particles in the phagocytosable size range of
0.310 m appear to be the most
biologically active ones .
the latter statement was also confirmed for silicon carbide ( sic )
particles and biometals such as cpti ,
ti6al4v and uhmwpe [ 184 , 185 ] .
granchi et al .
investigated the in vitro
effects of al2o3
and uhmwpe particles in an
osteoblast - osteoclast co - culture system .
both particles did not affect either
cell viability or tnf and gm - csf release ,
whereas il6 release was dependent on
the particle concentration .
uhmwpe particles
increased the release of rankl
from osteoblasts and induced large amounts
of multinucleated trap - positive
giant cells in an osteoblast - osteoclast
co - culture system .
also ,
carbon - based particles with low wear
factors such as p25-cvd showed a high degree
of cytocompatibility in vitro . howling et al .
demonstrated
on fibroblasts and monocytes that p25-cvd
particles < 100 nm were significantly less cytotoxic
to both cell types than
cocr metal wear particles .
while the classical water - suspendable
nanoc60 nanocrystal is apparently
cytotoxic to various cell lines , the closely related
fully hydroxylated ,
c60(oh)24
, is nontoxic ,
thus producing no cellular
response .
also ,
functionalized single - walled carbon nanotubes are nontoxic
to cells in culture [ 198200 ] .
there is evidence that not only particle
size and chemical content but also the concentration strongly influence
cellular reactions in vitro .
wilke
et al .
showed a positive correlation between the
release of proinflammatory cytokines
( il-6 , -1 , and tnf ) and amounts of
ti6al4v - particles
( 10 ,
10 , 10 ,
and 10 particles / ml )
by human bone marrow cells over 2 weeks .
some in vitro data also indicate that ti particles induce a stronger
fibroblastic differentiation signal than uhmwpe
in monocytes and other cells [ 182184 ] .
.
showed that particles
of high - density polyethylene
( hdp ) and ti6al4v induced significantly more
proinflammatory mediators ( il-1 ,
il-6 , tnf ) and bone resorption compared to
al2o3
and zro2 in vivo .
based on these data ,
it can be assumed that ceramics show a high
degree of cytocompatibiltiy . for ha especially , particles with a size
< 53 m inhibit cellular proliferation ,
especially in osteoblasts and lead
to a decrease in tgf1 and a significant increase in pge2 and ldh
concentration , but did not influence the tnf
or alp titer in vitro .
it could be concluded that larger ha particles may
be compatible with bone cells while smaller - sized
ha particles can both activate
the osteoclasts and decrease the cell
population of the osteoblasts in vitro .
numerous variables influence the biocompatibility and osteogenic
potency of nanostructured biomaterials in
vitro and in vivo .
besides the locotypical
environment in vivo or in vitro , the surface
structure and the composition of a
biomaterial affects cellular attachment ,
adherence , proliferation and migration ,
and also
differentiation and survival of defined cell types .
here , information about typical
parameters such as chemical composition ,
surface structure ( topography , geometry ,
roughness , particle size ) , surface energy ,
hydrophobicity , and the degree of
solubility in aqueous solutions of a biomaterial
will help to value and grade a
defined implant concerning its osteblast promoting potency . considering recent publications , we could assume some general
principles of cytocompatiblity and cell - surface
interactions in nano- and
microstructured surfaces .
( 1 ) wettability of a nanosurface influences significantly
protein adsorption , which is a prerequisite
of cellular adherence in serum containing
solutions .
( 2 ) nanostructured surfaces enhance the surface area of
biomaterials and promote cellular adherence . ( 3 ) the chemical outer functional
groups of a nanosurface significantly influence cellular
migration , proliferation , and differentiation but direct
correlations between
distinct parameters and cell functions are not entirely cleared .
( 4 ) the formation of fc underly a
dynamic process and influence the motility and migration of cells . ( 5 ) a higher degree of differentiation
is corresponding to a decreased cellular motility .
( 6 ) phagocytable particles with a size
< 10 m induce the strongest
cellular response with regard to releasing
inflammatory cytokines .
( 7 ) although ti has a high degree of
cytocompatibility in vitro , phagocytable
ti particles can induce a fibroblastic
differentiation .
|
cell - surface interactions play a crucial role for biomaterial application in orthopaedics .
it is evident that not only the chemical composition of solid substances influence cellular adherence ,
migration , proliferation and differentiation but also the surface topography of a biomaterial .
the progressive application of nanostructured surfaces in medicine has gained increasing interest
to improve the cytocompatibility and osteointegration of orthopaedic implants .
therefore , the
understanding of cell - surface interactions is of major interest for these substances . in this review
,
we elucidate the principle mechanisms of nano- and microscale cell - surface interactions in vitro for
different cell types onto typical orthopaedic biomaterials such as titanium ( ti ) ,
cobalt - chrome - molybdenum ( cocrmo ) alloys , stainless steel ( ss ) , as well as synthetic polymers
( uhmwpe , xlpe , peek , plla ) .
in addition , effects of nano- and microscaled particles and their
significance in orthopaedics were reviewed .
the significance for the cytocompatibility
of nanobiomaterials is discussed critically .
|
1. INTRODUCTION
2. BONE CELLS
3. CYTOCOMPATIBILITY OF MICRO- AND
NANOSTRUCTURED SURFACES
|
approximately 40 million people are infected with the human immunodeficiency virus ( hiv ) worldwide ( unaids 2006 ) .
infections by this lentivirus from the family retroviridae are characterized by a long asymptomatic period after host immune defenses control the initial infection . during this subsequent chronic phase of infection , hiv slowly diminishes the host
the virus replicates by hijacking the intracellular molecular machinery to transcribe viral rna , and eventually the productively infected cells die ( fauci 1988 ) . over time , the density of virions in the blood stream increases and the immune system functioning becomes progressively compromised , leaving hiv - infected individuals increasingly susceptible to opportunistic infections .
being blood - borne , hiv is transmitted via contact with the blood of an infected individual : through transfusions , needle - sharing , sexual contact or from mother to child during childbirth or breast - feeding .
initially there were concerns that hiv might be vector - borne ( e.g. , transmitted via mosquitoes ) but it has since become widely accepted that such transmission does not occur at any significant level ( bockarie and paru 1996 ) .
this current belief stems both from epidemiological data and experimental studies that directly examine the potential for hiv transmission via arthropods ( lawrence 1987 ; lifson 1988 ; bockarie and paru 1996 )
. why is hiv not vector - borne ( throughout this article we use the term vector synonymously with
arthropod ) ? the majority of medical scientists , when asked this question , will offer the following explanations ( bockarie and paru 1996 ) : ( i ) hiv concentrations in the blood are too low during human infection to permit vector transmission ; ( ii ) hiv is unable to survive long enough outside of humans ( or primates ) for vector transmission ; ( iii ) hiv is not able to replicate within arthropod vectors .
each of these explanations has empirical support ( lifson 1988 ; bockarie and paru 1996 ) and thus all three are good explanations for the lack of vector transmission in hiv .
if most arthropods pick up very little hiv when feeding on humans , and if the level of hiv in ( or on ) these vectors quickly decays , then no significant vector transmission is expected to occur .
the above findings provide a satisfying proximate explanation for the lack of vector transmission in hiv .
given the current characteristics of hiv , these three features of the virus make it unlikely that vector transmission will occur . in this article , we approach this question from an evolutionary perspective . therefore , rather than taking the characteristics of hiv as given , we are interested in understanding why hiv has evolved these particular features and not others instead . for example :
( i ) why has hiv not evolved a replication strategy that results in viral concentrations high enough to allow vector transmission ? ( ii ) why has hiv not evolved to be more durable and thus to survive longer outside of humans ?
( iii ) why has hiv not evolved the ability to replicate in arthropod vectors ?
asking such questions might strike many readers as strange since much of evolutionary biology proceeds by asking why certain features of organisms are as we observe them rather than by asking why some features are absent .
this is only natural , since it is difficult to decide which , among the infinite number of missing features , should be the focus of study .
it is useful to view such questions as falling on a continuum , from questions about the lack of traits that are virtually non - existent in all taxa ( e.g. , why do dogs not have wheeled appendages ) to questions about the lack of traits that are common in some populations or species but not others ( e.g. , why do german shepherds not have curly hair ? ) .
our contention is that , by asking such questions about the absence of some traits , we can gain deeper insight into biology . to quote philosopher arthur eddington ( 1927 ) , the contemplation in natural science of a wider domain than the actual leads to a far better understanding of the actual . . as in all science , however , not all questions are interesting , and this serves as our primary guide for focusing on some missing features and not others . in particular , we focus on the lack of vector transmission in hiv , because of the profound epidemiological significance of its absence .
an arthropod - transmissible form of hiv would clearly exacerbate the already devastating impact of the disease , opening up routes of transmission to groups previously at low - risk ( e.g. , children ) .
therefore , it is worth asking why , from an evolutionary standpoint , this has not occurred ( weiss 2001 ) .
more to the point , if there are conditions under which hiv could have evolved vector transmission we would do well to understand these , not only from the standpoint of scientific curiosity , but also to prevent such an outcome in the future . in this article , we address the question of why hiv lacks vector transmission , both through a consideration of available empirical data and through the construction of mathematical models . although our results are necessarily speculative , we believe that they shed some light on the evolutionary biology of hiv , and on the evolutionary biology of blood - borne pathogens more generally .
there are two broad reasons why a trait of interest ( in this case vector - transmission ) might not evolve .
first , the necessary genetic variation for the trait might arise only very rarely ( if at all ) .
for instance , the evolution of rna viruses , such as hiv , could be strongly constrained by the size of their genome ( holmes 2003 ) .
second , the necessary form and strength of natural selection might not be present for the trait to evolve , at least over the timescale under consideration .
thus , from an evolutionary standpoint , hiv is not vector - borne because either the necessary genetic variation for such transmission has never arisen , or the necessary selective factors that would make such variants increase in frequency over the relevant timescale have not occurred .
our evolutionary explanation for why hiv is not vector - borne will be sought within these two possibilities . before beginning to consider these explanations
there are two different processes that might result in vector - transmission , and that are often lumped under this single heading .
this occurs when the arthropod acts solely as a means of physical transport of viral particles between hosts ( e.g. , having viral particles in and around mouthparts ) .
this occurs when the virus replicates within the arthropod vector during the time period between feeding events .
how likely is it that hiv has not evolved vector transmission because of a lack of appropriate genetic variation ? the most direct way to assess this possibility is to determine if genetic variants capable of vector transmission are currently present in the hiv population .
unfortunately , performing such an assay on all genotypes within the population would be next to impossible .
furthermore , if variants capable of vector transmission are selectively disadvantageous , then their frequency in the population might be extremely low . nevertheless , there are some studies available that take this approach as far as is possible . a second approach to addressing this issue
if some of hivs close relatives have evolved vector - transmission , then the premise that genetic variation for this route of transmission in hiv does not exist would be significantly weakened .
below we review both types of evidence , for both mechanical and biological transmission . mechanical transmission :
a number of studies working directly with hiv have assayed its ability to be transmitted mechanically by arthropods .
such transmission is often difficult to assess under natural conditions , and therefore most studies have employed artificial experimental setups , using a variety of arthropods including mosquitoes , stable flies , and tabanids ( e.g. , horse flies and deer flies ) .
for example , hiv was found to be viable for up to 10 days in african soft ticks , and perhaps even up to 14 days ( humphrey - smith et al .
1989 found that hiv can remain infectious for up to 8 days in the gut of cimex hemipterus .
most research on this topic has stressed the need for large bloodmeal size , however , because hiv tends not to have a very high viral titre during infection in humans ( lifson 1988 ; webb et al .
this has led to a focus on arthropods , like ticks , whose bloodmeal sizes are often 70 times larger than that of mosquitoes ( humphrey - smith et al . 1993 ) .
it has also been suggested that squashing mosquitoes while they are feeding , and subsequently scratching the area ( which might result in lacerations ) could increase the likelihood of transmission as well ( siemens 1987 ) .
other studies of closely related viruses suggest that , in principle , there is no obvious barrier to mechanical transmission of hiv by arthropods .
in fact , it has also been suggested that mechanical vector transmission might be the route through which hiv was initially transmitted to humans ( eigen et al .
at least three other retroviruses can be transmitted mechanically , including bovine leukemia virus , friend murine leukemia virus , and equine infectious anemia virus ( foil and issel 1991 ; humphrey - smith et al .
the latter ( equine infectious anemia virus ) is believed to be a close relative of hiv ( mcclure et al .
1988 ) , and epidemiological evidence suggests that vector transmission might play a significant role in its transmission ( foil and issel 1991 ) .
interestingly , however , all three of these retroviruses tend to reach viral titres in their hosts that are much higher than those typical of hiv ( foil and issel 1991 ) .
it has also been suggested that some hepatitis viruses can be transmitted mechanically by arthropods ( jupp et al .
even if this does occur , however , the viral levels of hiv in humans are thought to be about 10 to 100 times lower than that of some hepatitis viruses ( foil and issel 1991 ) , again implicating hivs low titre during infection of humans as the primary reason that mechanical transmission does not occur in this virus . biological transmission : despite evidence that mechanical transmission of hiv by arthropods can occur , there is no evidence that biological transmission is possible .
( 1993 ) found that hiv can remain viable in ticks for up to 2 weeks , they failed to find any evidence that hiv can replicate in these vectors .
evidence has been reported of hiv - related nucleic acids being found in tsetse flies from central africa ( becker et al .
1986 ) , but this has been controversial , and epidemiological evidence is not indicative of vector transmission ( noireau et al .
furthermore , experiments using cell cultures from arthropods have demonstrated that hiv is not capable of replicating in these cells ( srinivasan et al .
in fact , no evidence exists to date that any retrovirus is capable of biological transmission by arthropods ( webb et al .
1989 ; foil and issel 1991 ; kuno 2004 ; kuno and chang 2005 ) .
it remains unclear exactly why such replication is not possible , but functional constraints on receptor use in arthropods versus mammals provides one proximate explanation ( van den heuvel et al .
alternatively , it remains possible that appropriate genetic variation can arise , but that selection simply does not favor the spread of biological transmission in hiv or other retroviruses .
the above empirical findings suggest that , in principle , hiv is capable of being transmitted mechanically . in practice , however , the typical hiv viral titre in the bloodstream of humans is too low for significant vector - borne transmission to occur ( lifson 1988 ; webb et al .
there is evidence of genetic variation for differences in viremia in hiv - infected patients ( kanki et al .
1999 ) and this therefore suggests that mechanical transmission should be evolutionarily feasible if it were selectively advantageous .
thus , we are forced to ask : why have genetic variants of hiv that induce higher viremia , and thus that transmit mechanically via arthropods , not increased in frequency ?
on the other hand , there is no evidence to suggest that biological transmission can occur , even in principle .
this might be because the relevant genetic variation for such transmission has not appeared ( or perhaps is not possible ) .
alternatively , perhaps such variation does occasionally arise , but that such strains are acted against by natural selection . in this section
we use some mathematical calculations to elucidate potential reasons why selection might not favor increased viremia and thus mechanical vector transmission in hiv .
mechanical transmission : to understand the form and strength of selection shaping the evolution of hiv viremia it is helpful to quantify the important properties of hiv epidemiology in terms of a mathematical model . at present hiv infection is increasing in prevalence in the human population ( unaids 2006 ) .
the simplest description of this is exponential growth in the number of infected individuals ; where y(t ) is the number of hiv - positive individuals at time t , and r is the per capita growth rate .
although the rate of increase of hiv appears to be slowing in recent years , exponential growth nevertheless provides a useful benchmark for its initial spread in humans .
the per capita growth rate will depend on various properties of hiv , including its mode of transmission . to derive an expression for the per capita growth rate of hiv - positive individuals in terms of underlying epidemiological parameters , we first need to specify a model for the epidemiological dynamics .
this model will allow for both sexual transmission of hiv as well as insect transmission , and therefore it will also track the number of insects carrying hiv . using w(t ) for the number of insects carrying hiv at time t , and y(a , t ) as the number of hiv - positive people who were infected a years ago , we specify the dynamics as with boundary condition . in equations ( 2 ) , v is the population size of insects free of hiv , a is the insect - biting rate , b1 is the probability of an insect picking up hiv , given it feeds on an infected human , and is the per capita loss rate of infected insects ( which subsumes both insect mortality and the decay of hiv stores in or on the insect ) .
note that , although many insects display a characteristic time lag between feeding events , for simplicity we have ignored this . also note that , for simplicity , equations ( 2 ) implicitly assume that the likelihood of an insect picking up hiv from an infected human is constant across all infection ages ( i.e. , it does not depend on a ) .
the parameter b2 is the probability that an insect - carrying hiv infects a human when feeding , and x is the number of hiv - negative people .
we assume that the number of susceptible insects and people are both constant over the timescale of interest because hiv infection is still increasing in prevalence in the human population ( unaids 2006 ) .
the parameter is the transmission rate through sexual contact of hiv , and is assumed to be independent of infection age .
our assumption is justified on the basis that we are concerned with average viremia throughout the entire infection , and viral loads during the acute infection phase are strongly correlated with viral loads during the subsequent chronic phase ( kelley et al . 2007 ) .
results in appendix 1 also show that the main conclusions are not altered by relaxing this assumption .
lastly , (a ) is a function describing the mortality rate of hiv - positive people as a function of infection age .
in particular , we will suppose that (a ) has the form where is the time during the infection at which aids develops .
this model is analyzed in appendix 1 to show that the asymptotic per capita rate of increase , r , is defined implicitly , as a function of various epidemiological parameters , by the equation : some of the parameters in equation ( 4 ) will depend on the level of viremia in humans , and therefore viremia will affect the per capita rate of spread of hiv . in particular , the amount of hiv picked up by an arthropod during a feeding , b1 , is expected to increase with viremia . similarly , evidence suggests that sexual transmission rate , , also increases with viremia ( operskalski et al . 1997
lastly , evidence also shows that high levels of viremia lead to a more rapid development of aids , and thus a lower value of in untreated patients ( mellors et al .
with these specifications , our question about the evolution of mechanical transmission of hiv can now be cast in population - genetic terms .
suppose the predominant strain of hiv is one that gives rise to a viremia too low for vector transmission ( i.e. , b1 0 ) .
also suppose that a mutant strain arises that produces a viremia high enough for mechanical vector transmission .
assuming that multiple infections do not occur ( an assumption that we relax below ) , the rate of change in frequency , p , of this mutant strain is ( day and gandon 2007 ) where ra is the per capita growth rate of the original strain and rb is the per capita growth rate of the mutant strain . specifically , ra and rb are implicitly defined , from equation ( 4 ) , as respectively .
equations ( 6 ) make the implicit assumption that the increased viremia of the mutant strain does not significantly increase its sexual transmission rate .
more precisely , sexual transmission must be a concave function of viremia with an optimal viral load that is lower than for vector transmission for the following argument to hold .
empirical evidence in support of this functional form for hiv exists ( quinn et al .
, a comparison of equations ( 6a ) and ( 6b ) reveals that rb will be smaller than ra ( i.e. , the mutant strain will decrease in frequency ) and hiv will be predominately sexually transmitted , when the following conditions hold : vector mortality , or the hiv decay rate , is high ( i.e. , large ) , vector population size is small ( i.e. , small v ) , vector biting rate is small ( i.e. , small a ) , or the hiv transfer rates to and from vectors , b1 and b2 , are small . indeed , rb is not only less than ra in such situations ( as in fig .
1 ) , but often negative as well , meaning that vector transmissible strains will not only decrease in frequency but in absolute numbers . therefore ,
some or all of the above conditions must hold if this analysis is to explain why hiv has not evolved mechanical vector transmission .
conversely , if the opposite conditions hold , then the mutant will increase in frequency , and both sexual and mechanical vector transmission will play a significant role in the disease s epidemiology ( fig .
1 ) . sexual transmission rate ( solid line ) and vector transmission rate ( dashed line ) as a function of viremia , . the resulting per capita growth rate based on equation ( 4 ) is also plotted ( growth rate is negative where is falls below the horizontal axis , meaning that such strains can never increase in number ) .
b denote the sexually transmitted and vector transmitted genotypes considered in model ( 5 ) of the text .
parameter values : 0 = 1/65 , a = 1 , v = 100 , x = 5 , = 15 .
these considerations provide the conditions required for the spread of a mutant with mechanical vector transmission , but they do not tell us the time frame over which such spread occurs .
for example , it would be useful to know by how much arthropods must increase the overall transmission rate of hiv if such strains are to have increased significantly in frequency during the period of time in which hiv has been evolving in humans .
the calculations in appendix 2 demonstrate that the factor by which vectors must increase the overall transmission rate of hiv , in order for the relative frequency of the mutant , p/(1 p ) , to increase by a factor of k over a period of t years , is given by all parameters in equation ( 7 ) can be estimated except k , t , and b ( appendix 2 ) , yielding fig .
the factor by which vectors must increase the overall transmission rate of hiv in order for a vector transmissible virus to increase in relative frequency by a factor of k , as a function of the amount of time over which evolution occurs ( between 30 and 50 years for hiv in humans ) .
solid lines assume that the increased viremia caused by the vector - transmissible virus decreases the time until the development of aids from a = 8 to b = 5 years .
the parameter k has very little effect over several orders of magnitude , meaning that the benefit of vector transmission required for it to evolve over 3050 years is determined largely by the value of b .
first , note that changes in k ( the amount of increase that must occur for evolutionary change to be deemed
, we can view any of these curves as general requirements for significant evolution of vector transmission to have occurred during the past 30 to 50 years .
also , as expected , the curves are decreasing , reflecting the fact that a smaller benefit of vector transmission is required to generate significant evolution if evolution has longer to act .
more interestingly we can see that , if the increased viremia that allows for vector transmission also results in the development of aids after only 5 years as opposed to 8 years , then arthropods would need to cause a doubling of hiv transmission rate for appreciable evolution to have occurred . on the other hand , if increased viremia results in the development of aids after only 1 year , then arthropods would need to increase hiv transmission rate by 5- or 6-fold for appreciable evolution to have occurred .
unfortunately , there are currently no estimates available of these parameters , but these calculations nevertheless suggest that significant mechanical transmission could have evolved within the last 3050 years under biologically plausible conditions . therefore , the lack of vector transmission in hiv can not immediately be attributed to an insufficient evolutionary history of hiv in humans . biological transmission : although there is no evidence suggesting that the required genetic variation for biological transmission is possible in hiv ( or any other retrovirus ; foil and issel 1991 ; kuno 2004 ; kuno and chang 2005 ; webb et al .
1989 ) , it is nevertheless instructive to consider whether there might also be reasons associated with the nature and strength of selection for why such transmission has not evolved . equation ( 5 ) can again be used in this context , with equation ( 4 ) again defining the per capita growth rate for different strains of hiv .
biological transmission need not require an increased viremia in humans , however , because the pathogen would replicate to transmissible levels once in the arthropod vector . as a result , strains that are capable of biological vector transmission need not result in the more rapid development of aids . without some associated cost , however
, biological vector transmission would clearly enhance the growth rate of hiv and thus would readily evolve .
thus , if an absence of such transmission is to be explained in terms of selection ( as opposed to an explanation based on a lack of genetic variation ) then there must be some associated cost .
there are at least two biologically plausible mechanisms through which such a cost might arise .
first , effective biological vector transmission might require evolutionary changes that reduce hivs capacity for sexual transmission . in this case
, the cost stems from an evolutionary trade - off between these two transmission routes .
the sexually transmitted form would have a growth rate defined by whereas the vector - transmitted form would have a growth rate defined by the parameter values in equations ( 8)
could readily be such that the growth rate of the vector - transmitted strain was less than that of the sexually transmitted strain .
there is , however , no a priori reason why this would be expected rather than the reverse .
therefore , it does not provide a very compelling answer as to why biological transmission has not evolved in hiv , particularly given that it is absent in all other retroviruses as well .
the second way in which a cost of biological transmission might arise is through a conflict between natural selection acting on transmission of hiv between hosts , and natural selection acting on the virus replicative capacity within a host .
it is well documented in hiv ( and other retroviruses ) that extensive genetic variation arises within an infected host via mutation .
if vector - transmissible strains suffer a cost in terms of their replicative ability within humans , then this within - host natural selection acting against vector transmission might be enough to prevent its spread .
modeling the evolutionary consequences of this in hiv is difficult because each infected human will harbor a suite of genetic variants , some of which will be better at exploiting the human host .
this will cause evolutionary change in the genetic composition of hiv within the infected individuals . at the same time , this suite of strains is also being transmitted to new hosts via sexual contact and potentially vector transmission .
the assumption of the above hypothesis is then that the strains that are better able to transmit to new hosts via vector transmission are not the ones best able to compete for resources within a host .
the simplest way to abstract these processes into a tractable model that still retains the fundamental processes at work is to make an assumption of superinfection .
specifically , we suppose that humans almost always harbor only a single strain , but occasionally new strains arise by mutation .
when such a mutation occurs , the mutant then either takes over the host or dies out instantaneously , resulting in a single strain infection once again ( levin and pimentel 1981 ; nowak and may 1994 ) . in keeping with our earlier notation we will use b to denote the vector - transmissible form , and a to denote the form best able to compete within a host and thus to transmit sexually . letting be the rate at which new mutations arise within an infected host , turning either an a pathogen into a b pathogen or vice versa , and using ij to denote the probability that an i mutant so produced will take over a host originally infected with type j , where i and j are either a or b , model ( 5 ) can be extended to yield ( day and proulx 2004 ; day and gandon 2006 ) with ra and rb again given by equations ( 6 ) .
the hypothesis under consideration supposes that within - host competition always favors the sexually transmitted form , and thus we take ab = 0 in equation ( 9 ) . in this case , there are then two possible evolutionary outcomes .
first , if rb ra > ba , then the frequency of vector transmission will ultimately evolve to the equilibrium value . on the other hand , if rb ra < ba , then vector transmission will never evolve .
in other words , if the significance of within - host evolution is large relative to the benefit of vector transmission , then vector transmission will never evolve .
this will be true whenever the mutation rate of the virus is high ( i.e. , large ) and when the selective advantage of sexual transmission in terms of within - host competition is large .
the first of these is certainly true of most retroviruses , although the second requirement is less well documented . nevertheless , this might provide a selective explanation for why no retrovirus appears to have evolved biological vector transmission .
how likely is it that hiv has not evolved vector transmission because of a lack of appropriate genetic variation ?
the most direct way to assess this possibility is to determine if genetic variants capable of vector transmission are currently present in the hiv population .
unfortunately , performing such an assay on all genotypes within the population would be next to impossible .
furthermore , if variants capable of vector transmission are selectively disadvantageous , then their frequency in the population might be extremely low .
nevertheless , there are some studies available that take this approach as far as is possible . a second approach to addressing this issue
if some of hivs close relatives have evolved vector - transmission , then the premise that genetic variation for this route of transmission in hiv does not exist would be significantly weakened .
below we review both types of evidence , for both mechanical and biological transmission . mechanical transmission :
a number of studies working directly with hiv have assayed its ability to be transmitted mechanically by arthropods .
such transmission is often difficult to assess under natural conditions , and therefore most studies have employed artificial experimental setups , using a variety of arthropods including mosquitoes , stable flies , and tabanids ( e.g. , horse flies and deer flies ) .
for example , hiv was found to be viable for up to 10 days in african soft ticks , and perhaps even up to 14 days ( humphrey - smith et al .
1989 found that hiv can remain infectious for up to 8 days in the gut of cimex hemipterus .
most research on this topic has stressed the need for large bloodmeal size , however , because hiv tends not to have a very high viral titre during infection in humans ( lifson 1988 ; webb et al .
this has led to a focus on arthropods , like ticks , whose bloodmeal sizes are often 70 times larger than that of mosquitoes ( humphrey - smith et al .
it has also been suggested that squashing mosquitoes while they are feeding , and subsequently scratching the area ( which might result in lacerations ) could increase the likelihood of transmission as well ( siemens 1987 ) .
other studies of closely related viruses suggest that , in principle , there is no obvious barrier to mechanical transmission of hiv by arthropods .
in fact , it has also been suggested that mechanical vector transmission might be the route through which hiv was initially transmitted to humans ( eigen et al .
at least three other retroviruses can be transmitted mechanically , including bovine leukemia virus , friend murine leukemia virus , and equine infectious anemia virus ( foil and issel 1991 ; humphrey - smith et al .
the latter ( equine infectious anemia virus ) is believed to be a close relative of hiv ( mcclure et al .
1988 ) , and epidemiological evidence suggests that vector transmission might play a significant role in its transmission ( foil and issel 1991 ) .
interestingly , however , all three of these retroviruses tend to reach viral titres in their hosts that are much higher than those typical of hiv ( foil and issel 1991 ) .
it has also been suggested that some hepatitis viruses can be transmitted mechanically by arthropods ( jupp et al .
1983 ) , although this has been controversial ( kuno 2004 ) . even if this does occur
, however , the viral levels of hiv in humans are thought to be about 10 to 100 times lower than that of some hepatitis viruses ( foil and issel 1991 ) , again implicating hivs low titre during infection of humans as the primary reason that mechanical transmission does not occur in this virus .
biological transmission : despite evidence that mechanical transmission of hiv by arthropods can occur , there is no evidence that biological transmission is possible .
( 1993 ) found that hiv can remain viable in ticks for up to 2 weeks , they failed to find any evidence that hiv can replicate in these vectors .
evidence has been reported of hiv - related nucleic acids being found in tsetse flies from central africa ( becker et al .
1986 ) , but this has been controversial , and epidemiological evidence is not indicative of vector transmission ( noireau et al .
furthermore , experiments using cell cultures from arthropods have demonstrated that hiv is not capable of replicating in these cells ( srinivasan et al .
in fact , no evidence exists to date that any retrovirus is capable of biological transmission by arthropods ( webb et al .
1989 ; foil and issel 1991 ; kuno 2004 ; kuno and chang 2005 ) .
it remains unclear exactly why such replication is not possible , but functional constraints on receptor use in arthropods versus mammals provides one proximate explanation ( van den heuvel et al .
alternatively , it remains possible that appropriate genetic variation can arise , but that selection simply does not favor the spread of biological transmission in hiv or other retroviruses .
the above empirical findings suggest that , in principle , hiv is capable of being transmitted mechanically . in practice , however , the typical hiv viral titre in the bloodstream of humans is too low for significant vector - borne transmission to occur ( lifson 1988 ; webb et al .
there is evidence of genetic variation for differences in viremia in hiv - infected patients ( kanki et al .
1999 ) and this therefore suggests that mechanical transmission should be evolutionarily feasible if it were selectively advantageous .
thus , we are forced to ask : why have genetic variants of hiv that induce higher viremia , and thus that transmit mechanically via arthropods , not increased in frequency ?
on the other hand , there is no evidence to suggest that biological transmission can occur , even in principle .
this might be because the relevant genetic variation for such transmission has not appeared ( or perhaps is not possible ) .
alternatively , perhaps such variation does occasionally arise , but that such strains are acted against by natural selection . in this section
we use some mathematical calculations to elucidate potential reasons why selection might not favor increased viremia and thus mechanical vector transmission in hiv .
mechanical transmission : to understand the form and strength of selection shaping the evolution of hiv viremia it is helpful to quantify the important properties of hiv epidemiology in terms of a mathematical model . at
present hiv infection is increasing in prevalence in the human population ( unaids 2006 ) .
the simplest description of this is exponential growth in the number of infected individuals ; where y(t ) is the number of hiv - positive individuals at time t , and r is the per capita growth rate .
although the rate of increase of hiv appears to be slowing in recent years , exponential growth nevertheless provides a useful benchmark for its initial spread in humans .
the per capita growth rate will depend on various properties of hiv , including its mode of transmission . to derive an expression for the per capita growth rate of hiv - positive individuals in terms of underlying epidemiological parameters , we first need to specify a model for the epidemiological dynamics .
this model will allow for both sexual transmission of hiv as well as insect transmission , and therefore it will also track the number of insects carrying hiv . using w(t ) for the number of insects carrying hiv at time t , and y(a , t ) as the number of hiv - positive people who were infected a years ago , we specify the dynamics as with boundary condition . in equations ( 2 )
, v is the population size of insects free of hiv , a is the insect - biting rate , b1 is the probability of an insect picking up hiv , given it feeds on an infected human , and is the per capita loss rate of infected insects ( which subsumes both insect mortality and the decay of hiv stores in or on the insect ) .
note that , although many insects display a characteristic time lag between feeding events , for simplicity we have ignored this .
also note that , for simplicity , equations ( 2 ) implicitly assume that the likelihood of an insect picking up hiv from an infected human is constant across all infection ages ( i.e. , it does not depend on a ) .
the parameter b2 is the probability that an insect - carrying hiv infects a human when feeding , and x is the number of hiv - negative people .
we assume that the number of susceptible insects and people are both constant over the timescale of interest because hiv infection is still increasing in prevalence in the human population ( unaids 2006 ) .
the parameter is the transmission rate through sexual contact of hiv , and is assumed to be independent of infection age .
our assumption is justified on the basis that we are concerned with average viremia throughout the entire infection , and viral loads during the acute infection phase are strongly correlated with viral loads during the subsequent chronic phase ( kelley et al .
results in appendix 1 also show that the main conclusions are not altered by relaxing this assumption .
lastly , (a ) is a function describing the mortality rate of hiv - positive people as a function of infection age . in particular
, we will suppose that (a ) has the form where is the time during the infection at which aids develops .
this model is analyzed in appendix 1 to show that the asymptotic per capita rate of increase , r , is defined implicitly , as a function of various epidemiological parameters , by the equation : some of the parameters in equation ( 4 ) will depend on the level of viremia in humans , and therefore viremia will affect the per capita rate of spread of hiv .
in particular , the amount of hiv picked up by an arthropod during a feeding , b1 , is expected to increase with viremia .
similarly , evidence suggests that sexual transmission rate , , also increases with viremia ( operskalski et al .
lastly , evidence also shows that high levels of viremia lead to a more rapid development of aids , and thus a lower value of in untreated patients ( mellors et al . 1997 ; levy 1998 ; raffanti et al .
, our question about the evolution of mechanical transmission of hiv can now be cast in population - genetic terms .
suppose the predominant strain of hiv is one that gives rise to a viremia too low for vector transmission ( i.e. , b1 0 ) .
also suppose that a mutant strain arises that produces a viremia high enough for mechanical vector transmission .
assuming that multiple infections do not occur ( an assumption that we relax below ) , the rate of change in frequency , p , of this mutant strain is ( day and gandon 2007 ) where ra is the per capita growth rate of the original strain and rb is the per capita growth rate of the mutant strain . specifically , ra and rb are implicitly defined , from equation ( 4 ) , as respectively
. equations ( 6 ) make the implicit assumption that the increased viremia of the mutant strain does not significantly increase its sexual transmission rate .
more precisely , sexual transmission must be a concave function of viremia with an optimal viral load that is lower than for vector transmission for the following argument to hold .
empirical evidence in support of this functional form for hiv exists ( quinn et al .
, a comparison of equations ( 6a ) and ( 6b ) reveals that rb will be smaller than ra ( i.e. , the mutant strain will decrease in frequency ) and hiv will be predominately sexually transmitted , when the following conditions hold : vector mortality , or the hiv decay rate , is high ( i.e. , large ) , vector population size is small ( i.e. , small v ) , vector biting rate is small ( i.e. , small a ) , or the hiv transfer rates to and from vectors , b1 and b2 , are small . indeed , rb is not only less than ra in such situations ( as in fig .
1 ) , but often negative as well , meaning that vector transmissible strains will not only decrease in frequency but in absolute numbers . therefore , some or all of the above conditions must hold if this analysis is to explain why hiv has not evolved mechanical vector transmission .
conversely , if the opposite conditions hold , then the mutant will increase in frequency , and both sexual and mechanical vector transmission will play a significant role in the disease s epidemiology ( fig .
sexual transmission rate ( solid line ) and vector transmission rate ( dashed line ) as a function of viremia , . the resulting per capita growth rate based on equation ( 4 ) is also plotted ( growth rate is negative where is falls below the horizontal axis , meaning that such strains can never increase in number ) .
b denote the sexually transmitted and vector transmitted genotypes considered in model ( 5 ) of the text .
parameter values : 0 = 1/65 , a = 1 , v = 100 , x = 5 , = 15 .
these considerations provide the conditions required for the spread of a mutant with mechanical vector transmission , but they do not tell us the time frame over which such spread occurs . for example , it would be useful to know by how much arthropods must increase the overall transmission rate of hiv if such strains are to have increased significantly in frequency during the period of time in which hiv has been evolving in humans . the calculations in appendix 2 demonstrate that the factor by which vectors must increase the overall transmission rate of hiv , in order for the relative frequency of the mutant , p/(1 p ) , to increase by a factor of k over a period of t years , is given by all parameters in equation ( 7 ) can be estimated except k , t , and b ( appendix 2 ) , yielding fig .
the factor by which vectors must increase the overall transmission rate of hiv in order for a vector transmissible virus to increase in relative frequency by a factor of k , as a function of the amount of time over which evolution occurs ( between 30 and 50 years for hiv in humans ) .
solid lines assume that the increased viremia caused by the vector - transmissible virus decreases the time until the development of aids from a = 8 to b = 5 years .
the parameter k has very little effect over several orders of magnitude , meaning that the benefit of vector transmission required for it to evolve over 3050 years is determined largely by the value of b .
first , note that changes in k ( the amount of increase that must occur for evolutionary change to be deemed
, we can view any of these curves as general requirements for significant evolution of vector transmission to have occurred during the past 30 to 50 years .
also , as expected , the curves are decreasing , reflecting the fact that a smaller benefit of vector transmission is required to generate significant evolution if evolution has longer to act .
more interestingly we can see that , if the increased viremia that allows for vector transmission also results in the development of aids after only 5 years as opposed to 8 years , then arthropods would need to cause a doubling of hiv transmission rate for appreciable evolution to have occurred . on the other hand , if increased viremia results in the development of aids after only 1 year , then arthropods would need to increase hiv transmission rate by 5- or 6-fold for appreciable evolution to have occurred .
unfortunately , there are currently no estimates available of these parameters , but these calculations nevertheless suggest that significant mechanical transmission could have evolved within the last 3050 years under biologically plausible conditions . therefore , the lack of vector transmission in hiv can not immediately be attributed to an insufficient evolutionary history of hiv in humans . biological transmission : although there is no evidence suggesting that the required genetic variation for biological transmission is possible in hiv ( or any other retrovirus ; foil and issel 1991 ; kuno 2004 ; kuno and chang 2005 ; webb et al .
1989 ) , it is nevertheless instructive to consider whether there might also be reasons associated with the nature and strength of selection for why such transmission has not evolved .
equation ( 5 ) can again be used in this context , with equation ( 4 ) again defining the per capita growth rate for different strains of hiv .
biological transmission need not require an increased viremia in humans , however , because the pathogen would replicate to transmissible levels once in the arthropod vector . as a result , strains that are capable of biological vector transmission need not result in the more rapid development of aids . without some associated cost , however
, biological vector transmission would clearly enhance the growth rate of hiv and thus would readily evolve .
thus , if an absence of such transmission is to be explained in terms of selection ( as opposed to an explanation based on a lack of genetic variation ) then there must be some associated cost .
there are at least two biologically plausible mechanisms through which such a cost might arise .
first , effective biological vector transmission might require evolutionary changes that reduce hivs capacity for sexual transmission . in this case
, the cost stems from an evolutionary trade - off between these two transmission routes .
the sexually transmitted form would have a growth rate defined by whereas the vector - transmitted form would have a growth rate defined by the parameter values in equations ( 8)
could readily be such that the growth rate of the vector - transmitted strain was less than that of the sexually transmitted strain .
there is , however , no a priori reason why this would be expected rather than the reverse .
therefore , it does not provide a very compelling answer as to why biological transmission has not evolved in hiv , particularly given that it is absent in all other retroviruses as well .
the second way in which a cost of biological transmission might arise is through a conflict between natural selection acting on transmission of hiv between hosts , and natural selection acting on the virus replicative capacity within a host .
it is well documented in hiv ( and other retroviruses ) that extensive genetic variation arises within an infected host via mutation .
if vector - transmissible strains suffer a cost in terms of their replicative ability within humans , then this within - host natural selection acting against vector transmission might be enough to prevent its spread .
modeling the evolutionary consequences of this in hiv is difficult because each infected human will harbor a suite of genetic variants , some of which will be better at exploiting the human host .
this will cause evolutionary change in the genetic composition of hiv within the infected individuals . at the same time , this suite of strains is also being transmitted to new hosts via sexual contact and potentially vector transmission .
the assumption of the above hypothesis is then that the strains that are better able to transmit to new hosts via vector transmission are not the ones best able to compete for resources within a host .
the simplest way to abstract these processes into a tractable model that still retains the fundamental processes at work is to make an assumption of superinfection .
specifically , we suppose that humans almost always harbor only a single strain , but occasionally new strains arise by mutation .
when such a mutation occurs , the mutant then either takes over the host or dies out instantaneously , resulting in a single strain infection once again ( levin and pimentel 1981 ; nowak and may 1994 ) . in keeping with our earlier notation we will use b to denote the vector - transmissible form , and a to denote the form best able to compete within a host and thus to transmit sexually . letting be the rate at which new mutations arise within an infected host , turning either an a pathogen into a b pathogen or vice versa , and using ij to denote the probability that an i mutant so produced will take over a host originally infected with type j , where i and j are either a or b , model ( 5 ) can be extended to yield ( day and proulx 2004 ; day and gandon 2006 ) with ra and rb again given by equations ( 6 ) . the hypothesis under consideration supposes that within - host competition always favors the sexually transmitted form , and thus we take ab = 0 in equation ( 9 ) . in this case , there are then two possible evolutionary outcomes .
first , if rb ra > ba , then the frequency of vector transmission will ultimately evolve to the equilibrium value . on the other hand ,
in other words , if the significance of within - host evolution is large relative to the benefit of vector transmission , then vector transmission will never evolve .
this will be true whenever the mutation rate of the virus is high ( i.e. , large ) and when the selective advantage of sexual transmission in terms of within - host competition is large .
the first of these is certainly true of most retroviruses , although the second requirement is less well documented . nevertheless , this might provide a selective explanation for why no retrovirus appears to have evolved biological vector transmission .
experiments and epidemiological data have unequivocally demonstrated , however , that such vector transmission does not occur at any significant level , and various aspects of hiv biology have been implicated as proximate reasons ( bockarie and paru 1996 ) .
these reasons do not offer an explanation for why vector transmission has not evolved , however , and as weiss ( 2001 ) points out , we ought to seriously consider whether such evolution might occur in the future ( for a summary , see table 1 ) .
main conclusions of the study existing data suggest that the lack of mechanical vector transmission in hiv is not due to genetic constraints . while ecological constraints , such as number of vectors and biting rates , may limit vector transmission in certain areas , these constraints
would likely not explain why hiv has not evolved this form of transmission in areas where vector - borne diseases ( e.g. malaria ) are endemic .
rather , there must presumably be a reason why such transmission is selectively disadvantageous in hiv .
effective mechanical vector transmission can be brought about only through the evolution of higher levels of viremia , and this also results in a more rapid onset of aids .
this reduces the duration over which such strains can be transmitted from an infected human , more than is made up for by the occurrence of vector transmission .
it also remains possible that insufficient time has elapsed for the evolution of vector transmission to occur , but our calculations suggest that this is not a very compelling possibility . on the other hand , existing data is largely consistent with the hypothesis that biological vector transmission has not evolved in hiv because of genetic constraints . at the same time
, it is not possible to rule out a selective explanation instead . in particular ,
if there is a genetic trade - off between efficient replication in humans and replication in arthropod vectors , then a conflict between selection favoring effective replication within humans , and selection favoring arthropod transmission between humans can readily prevent biological transmission from evolving .
this is particularly likely when the mutation rate of the virus is high , and thus might provide an explanation for the lack of biological vector transmission in all retroviruses .
our analysis might also be extended to include other forms of transmission , for instance needle transmission ( see bruneau et al .
several evolutionary consequences of this are possible depending both on the level of viremia required for such transmission to occur and the resulting transmission rate .
for instance , if needle transmission can be achieved with a lower viremia than sexual transmission , and if this leads to a sufficiently high transmission rate , less virulent strains could be favored .
conversely , if needle transmission requires a high viremia and leads to a sufficiently high transmission rate , more virulent strains would be favored .
the only situation in which enhanced needle use could lead to the evolution of vector - borne transmission would be if effective needle transmission requires a viremia close to that of vector - borne transmission , while leading to a much higher transmission rate than vector - borne transmission .
this way , strains with high viremia could be maintained in the population through needle transmission , and vector - borne transmission would then occur largely as a byproduct .
our conclusions in this article are necessarily speculative , but such speculation is a necessary part of the initial stages of any research .
one of our aims is to stimulate future research into the evolutionary biology of hiv transmission . from the results presented here ,
a number of different directions might be taken to ground these evolutionary ideas more firmly in empirical data .
one possibility would be to examine more closely mechanical vector transmission in immunodeficiency viruses of other species .
for example , more data on the epidemiological patterns of siv and its potential for alternative routes of transmission would be enormously useful .
since siv is believed to be at the evolutionary ancestor of hiv , it would be very interesting to know if the longer evolutionary history it has had with its host has resulted in different transmission patterns .
to the best of our knowledge , there are no empirical studies testing the potential for vector transmission of siv
. another fruitful approach might be to conduct artificial selection experiments with hiv in arthropod tissue culture .
experiments have demonstrated that hiv can not currently replicate significantly in arthropod cells , but no study to our knowledge has attempted to select for the evolution of hiv replication in such cells .
one could even imagine doing such experiments with both mammalian and arthropod cell cultures to determine of the evolutionary trade - off postulated here actually occurs .
ultimately , it will require innovative experiments and empirical studies to push the boundaries of our knowledge of hiv , and the use of evolutionary biology as a powerful tool for designing sensible intervention strategies .
these kinds of studies are beginning to appear for other aspects of hiv biology ( e.g. , see mller et al .
2006 for an interesting evolutionary analysis of hiv virulence ) but more work on transmission biology would be useful .
for example , if further empirical research validated the hypothesis presented here , that mechanical vector transmission has not evolved because of its associated mortality costs , this would then have important implications for how we attempt to stop the spread of hiv .
strategies such as condom use , while beneficial for reducing the extent of sexual transmission , could thereby enhance the relative benefit of vector transmission , potentially resulting in the evolution of this new route of transmission .
the use of antibiotics against bacterial pathogens has clearly brought home the fact that pathogens can readily evolve the means to circumvent our control measures , and there is no reason to expect things to be any different for other control measures .
the use of antiviral medication , on the other hand , not only reduces sexual transmission but also the level of viremia , and therefore would presumably not move the selective balance more towards vector transmission .
it is only by asking these kinds of questions , however , that we will have a chance at preventing adverse future outcomes .
finally , the question of biological vector transmission addressed here is really a special case of the more general question of the evolution of a pathogen s host range .
why do some pathogens have a relatively broad taxonomic host range while others are much more conservative ?
this continues to be an interesting and important question in the evolutionary ecology of parasites ( poulin 2007 ) and there are some theoretical results predicting when we might expect different outcomes ( gandon 2004 ) . from the standpoint of human diseases
this is also clearly an important question since emerging diseases , such as pandemic influenza , are precisely instances in which a pathogen evolves a different host range .
a better understanding of the evolutionary biology of parasite host ranges is an important goal for future research .
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abstractmany pathogens of humans are blood borne , including hiv , malaria , hepatitis b and c , west nile virus , dengue , and other viral hemorrhagic fevers .
although several of these pathogens are transmitted by blood - feeding arthropods , hiv is not .
a number of properties of hiv and its life cycle have been identified as proximate explanations for the absence of arthropod transmission , but little consideration has been given to why hiv has not evolved this form of transmission .
we consider the empirical evidence for arthropod transmission , and suggest that mechanical transmission has not evolved in hiv because such strains would induce a faster onset of aids during infection , which would thereby limit their ability to spread . on the other hand , it is not as clear why biological transmission has not occurred .
available data suggests that a lack of appropriate genetic variation in hiv is one explanation , but it is also possible that a conflict between natural selection occurring within and between infected individuals has prevented its evolution instead .
we discuss the potential significance of these ideas , and argue that taking such an evolutionary perspective broadens our understanding of infectious diseases and the potential consequences of public health interventions .
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Introduction
Why is HIV not vector-borne?
Genetic variation
Selection
Discussion
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copd is characterized by persistent airway obstruction , usually of a progressive nature and associated with an enhanced chronic inflammatory response in airways to noxious particles or gases.1 copd is associated with increased mortality and reduced life expectancy.2 of the 3.8 million mortalities caused by chronic respiratory system diseases in 2010 , 2.9 million were by copd.3 the first consensus report for the diagnosis , management , and prevention of copd was published in 2001 by the global initiative for chronic obstructive lung disease ( gold ) committee , and since then it has been updated several times .
the major objectives of gold are to increase the awareness of copd and to help the millions of people who suffer from this disease and die prematurely from it or its complications by increasing the standards of care .
the low adherence to guideline treatment recommendations has been reported in several studies.46 under or overprescription cause the inappropriate treatment of the disease.4,7 undertreatment of copd has also been reported in turkey.8 the bold study in adana , turkey showed that only 12.3% of the adults with copd used medication for their diseases in 2004.9 the chronic diseases and risk factors in turkey survey reported that 46.1% of the patients clinically diagnosed with copd used regular medication in 2011.10 to date , there has been no study on adherence to the gold guidelines for the treatment of copd among pulmonary physicians in turkey . at the time of the study
, the patients with copd were still treated according to the gold 2010 guideline recommendations .
therefore , stages and treatments in this study are also classified in accordance with the gold 2010.1 the aim of this study is to investigate the adherence rates to gold 2010 recommendations for stable copd treatment by pulmonary physicians with a national survey in outpatient clinics of turkish pulmonary units from different regions of the country .
this multi - center , cross - sectional study was conducted with the participation of the pulmonary outpatient clinics of eleven centers ( university hospitals , research and training hospitals , state hospitals , and private hospitals ) from eight cities ( adana , ankara , diyarbakir , erzurum , istanbul , izmir , kocaeli , and samsun ) across turkey .
sample size estimation has been calculated in order to have the representation of all gold stages , and sequential recruitment of 100 patients was anticipated per center .
the study was approved by the institutional ethics committee of ankara university school of medicine ( november 21 , 2010 ; # 24,596 ) prior to its initiation and conducted in accordance with the latest version of declaration of helsinki and international conference of harmonization / good clinical practice guideline .
the inclusion criteria included doctor s diagnosis of copd , age 40 years , available pulmonary function test ( pft ) results from within the past 2 years and copd treatment data from any time within 6 months of the test , and formal written consent .
patients with a concomitant chronic disease that leads to obstructive ( ie , asthma ) or restrictive ( ie , disseminated bronchiectasis ) changes on pft , with pft results discordant to gold diagnostic criteria ( symptoms of copd , and postbronchodilator spirometric evaluation confirming forced expiratory volume in 1 second ( fev1)/forced vital capacity [ fvc ] < 70% , exclusion of other obstructive lung diseases),1 and those with acute copd exacerbations were excluded from the study analysis . in nonsmoker patients ,
biomass exposure is considered as one of the major risk factors especially in rural areas , and they were not excluded from the study if they meet the gold spirometric diagnostic criteria .
patient recruitment has been performed according to the study inclusion and exclusion criteria in each study center .
patient data were collected from hospital records on standardized forms and following parameters were recorded : any chronic diseases accompanying copd , age at the time of copd diagnosis , spirometry results ( fev1 and fev1/fvc ) and copd treatment recommended within 6 months of this result , and demographic information such as sex , age , location of residence , occupation , level of education , and health insurance status .
overall , 1,125 patients have been recruited , and 719 patients meeting the gold diagnostic criteria ( fev1/fvc < 70% ) were included in this analysis .
appropriateness of treatment was established in accordance with the 2010 gold guideline recommendations below :
in stage i , add short - acting bronchodilator when needed.in stage ii , add regular treatment with one or more long - acting bronchodilator ( long - acting 2-agonist [ laba ] or long - acting muscarinic antagonist [ lama]).in stages iii and iv , add regular treatment with one or more long - acting bronchodilator and add inhaled corticosteroid ( ics ) for those experiencing frequent exacerbations ( gold 2010).1 in stage i , add short - acting bronchodilator when needed . in stage
ii , add regular treatment with one or more long - acting bronchodilator ( long - acting 2-agonist [ laba ] or long - acting muscarinic antagonist [ lama ] ) . in stages
iii and iv , add regular treatment with one or more long - acting bronchodilator and add inhaled corticosteroid ( ics ) for those experiencing frequent exacerbations ( gold 2010).1 all other treatment approaches ( under- or overtreatment ) were evaluated as inappropriate treatment .
data are reported as mean and sd ; median , minimum , and maximum ; or frequency and percentage .
monte carlo simulation was used for multigroup comparisons where chi - square test assumptions were not fulfilled .
bonferroni - corrected mann whitney u - test was used for subgroup comparisons within numeric variables .
seven - hundred and nineteen patients ( 614 males , 105 females ) with mean sd age of 62.99.7 years were included .
six - hundred and forty - two patients ( 89.3% ) had smoking history , and health insurance coverage rate was 95.3% .
of all the patients , 2.2% ( n=16 ) were in stage i , 33.1% ( n=238 ) in stage ii , 48.1% ( n=346 ) in stage iii , and 16.6% ( n=119 ) in stage iv .
mean sd fev1 value by stage was 88.6%9.3% in stage i , 60.4%8.1% in stage ii , 39.4%5.6% in stage iii , and 23.9%4.2% in stage iv .
there were significant differences between the disease stages in terms of age ( p=0.004 ) and duration of disease ( p<0.001 ) .
of the 547 ( 76% ) patients with available data on comorbidities , 412 ( 57.3% ) had at least one comorbidity .
the most common comorbidity was hypertension ( 60.9% ) , followed by cardiovascular diseases ( 43.9% ) , diabetes mellitus ( 22.6% ) and depression / anxiety ( 10.4% ) .
overall adherence to gold guideline treatment recommendations for different stages of copd was 59.5% in the study group .
appropriate and inappropriate treatment approaches in different stages are represented in table 2 . in stage
i , only one patient ( 6.25% ) was treated with appropriate regimen , and others were overtreated with regular long - acting bronchodilators or combination of bronchodilators and icss .
the two most common treatment options were long - acting muscarinic antagonist ( lama ) ( 37.5% ) and laba - ics - lama ( 37.5% ) . in stage ii ,
adherence to gold recommendations was 14.7% ( n=35 ) , and this group was also overtreated by a combination of bronchodilators and icss . in this stage , frequently preferred treatment options were laba - ics - lama ( 47.5% ) , laba - ics - lama - theophylline ( t ) ( 13% ) , and laba - ics ( 12.6% ) .
of all the patients in stage ii , 81.5% ( n=194 ) received treatment containing ics . in stage iii , adherence to guideline increased to 84.4% ( n=292 ) because of the wide use of bronchodilator - ics combinations .
the most preferred treatment combination was laba - ics - lama ( 45.1% ) , followed by laba - ics - lama - t ( 27.2% ) .
of all the patients in stage iii , 89.3% ( n=309 ) received treatment containing ics .
the undertreatment was due to inappropriate treatment with regular long-/short - acting bronchodilators without ics use , 1.7% ( n=6 ) of the patients were treated by ics alone without bronchodilator drug and 3.8% ( n=9 ) were not on any medication for copd . in stage
iv , the appropriate treatment rate was also high , as 83.2% ( n=99 ) received dual , triple , and quadruple therapies with bronchodilators and icss .
the most preferred regimens were laba - ics - lama and laba - ics - lama - t combinations ( both 31.1% ) .
of all the patients in stage iv , 87.3% ( n=104 ) received treatment containing ics .
the inappropriate treatment approaches were one or more long - acting bronchodilator administrations without ics , use of ics only ( 4.3% , n=5 ) , and lack of treatment ( 5.9% , n=7 ) . according to these findings , while 59.5% of all patients has been receiving appropriate treatment according to gold 2010 guideline , 40.5% was inappropriately treated .
inappropriate treatment rate were 93.8% in stage i , 85.3% in stage ii , 15.6% in stage iii , and 16.8% in stage iv ( table 2 ) .
the cause of inappropriate therapies was due to over - treatment ( 100% ) among stage i. in stage ii overtreatment ( 91.1% ) was the main reason for guideline discordant therapies , followed by undertreatment ( 8.9% ) , and lack of treatment ( 3.8% ) .
only 14.7% of the stage ii patients were treated appropriately . in severe and very severe patients ,
guideline concordant treatment rate was as high as 84.4% in stage iii and 83.2% in stage iv . in stage iii , 13.3% of patients were undertreated , and 2.3% of very severe copd patients did not receive any regular treatment .
while undertreatment rate was 10.9% among stage iv patients , 5.9% of stage iv patients had no treatment for copd .
the most administered treatment regimen was laba - ics - lama combination ( 43.4% , n=312 ) , followed by laba - ics - lama - t ( 22.5% , n=162 ) and laba - ics ( 10.9% , n=78 ) . in patients treated with regular bronchodilators , the most frequently used drug was laba ( n=613 ) , followed by lama ( n=575 ) , t ( n=197 ) , short - acting 2-agonist ( n=13 ) and short - acting muscarinic antagonistic drugs ( n=13 ) , respectively .
the distribution of patients on bronchodilator alone , treatment containing ics , and no maintenance drug by copd stage is shown in table 3 .
of all the patients , 614 ( 89% ) received treatment containing ics or ics alone .
the distribution of patients who received ics was 43.8% in stage i , 81.5% in stage ii , 89.2% in stage iii , and 86.5% in stage
iv . of the 25 ( 3.5% ) patients who were not on any medication ,
one patient was in stage i , nine in stage ii , eight in stage iii , and nine in stage iv . there were 327 ( 45.5% ) patients on short - acting bronchodilators used as rescue drugs in addition to maintenance therapy .
of all the patients , 56 ( 7.8% ) were on monotherapy , 125 ( 17.4% ) on dual , 350 ( 48.7% ) on triple , and 163 ( 22.6% ) on quadruple therapy .
long - term oxygen use rate was 22.4% ( n=112 patients ) in stages iii and iv patients .
seven - hundred and nineteen patients ( 614 males , 105 females ) with mean sd age of 62.99.7 years were included .
six - hundred and forty - two patients ( 89.3% ) had smoking history , and health insurance coverage rate was 95.3% .
of all the patients , 2.2% ( n=16 ) were in stage i , 33.1% ( n=238 ) in stage ii , 48.1% ( n=346 ) in stage iii , and 16.6% ( n=119 ) in stage iv .
mean sd fev1 value by stage was 88.6%9.3% in stage i , 60.4%8.1% in stage ii , 39.4%5.6% in stage iii , and 23.9%4.2% in stage iv .
there were significant differences between the disease stages in terms of age ( p=0.004 ) and duration of disease ( p<0.001 ) .
of the 547 ( 76% ) patients with available data on comorbidities , 412 ( 57.3% ) had at least one comorbidity .
the most common comorbidity was hypertension ( 60.9% ) , followed by cardiovascular diseases ( 43.9% ) , diabetes mellitus ( 22.6% ) and depression / anxiety ( 10.4% ) .
overall adherence to gold guideline treatment recommendations for different stages of copd was 59.5% in the study group .
appropriate and inappropriate treatment approaches in different stages are represented in table 2 . in stage
i , only one patient ( 6.25% ) was treated with appropriate regimen , and others were overtreated with regular long - acting bronchodilators or combination of bronchodilators and icss .
the two most common treatment options were long - acting muscarinic antagonist ( lama ) ( 37.5% ) and laba - ics - lama ( 37.5% ) . in stage ii ,
adherence to gold recommendations was 14.7% ( n=35 ) , and this group was also overtreated by a combination of bronchodilators and icss . in this stage , frequently preferred treatment options were laba - ics - lama ( 47.5% ) , laba - ics - lama - theophylline ( t ) ( 13% ) , and laba - ics ( 12.6% ) .
of all the patients in stage ii , 81.5% ( n=194 ) received treatment containing ics . in stage iii , adherence to guideline increased to 84.4% ( n=292 ) because of the wide use of bronchodilator - ics combinations .
the most preferred treatment combination was laba - ics - lama ( 45.1% ) , followed by laba - ics - lama - t ( 27.2% ) .
of all the patients in stage iii , 89.3% ( n=309 ) received treatment containing ics .
the undertreatment was due to inappropriate treatment with regular long-/short - acting bronchodilators without ics use , 1.7% ( n=6 ) of the patients were treated by ics alone without bronchodilator drug and 3.8% ( n=9 ) were not on any medication for copd . in stage
iv , the appropriate treatment rate was also high , as 83.2% ( n=99 ) received dual , triple , and quadruple therapies with bronchodilators and icss .
the most preferred regimens were laba - ics - lama and laba - ics - lama - t combinations ( both 31.1% ) .
of all the patients in stage iv , 87.3% ( n=104 ) received treatment containing ics .
the inappropriate treatment approaches were one or more long - acting bronchodilator administrations without ics , use of ics only ( 4.3% , n=5 ) , and lack of treatment ( 5.9% , n=7 ) . according to these findings , while 59.5% of all patients has been receiving appropriate treatment according to gold 2010 guideline , 40.5% was inappropriately treated .
inappropriate treatment rate were 93.8% in stage i , 85.3% in stage ii , 15.6% in stage iii , and 16.8% in stage iv ( table 2 ) .
the cause of inappropriate therapies was due to over - treatment ( 100% ) among stage i. in stage ii overtreatment ( 91.1% ) was the main reason for guideline discordant therapies , followed by undertreatment ( 8.9% ) , and lack of treatment ( 3.8% ) .
only 14.7% of the stage ii patients were treated appropriately . in severe and very severe patients ,
guideline concordant treatment rate was as high as 84.4% in stage iii and 83.2% in stage iv . in stage iii , 13.3% of patients were undertreated , and 2.3% of very severe copd patients did not receive any regular treatment .
while undertreatment rate was 10.9% among stage iv patients , 5.9% of stage iv patients had no treatment for copd .
the most administered treatment regimen was laba - ics - lama combination ( 43.4% , n=312 ) , followed by laba - ics - lama - t ( 22.5% , n=162 ) and laba - ics ( 10.9% , n=78 ) . in patients treated with regular bronchodilators , the most frequently used drug was laba ( n=613 ) , followed by lama ( n=575 ) , t ( n=197 ) , short - acting 2-agonist ( n=13 ) and short - acting muscarinic antagonistic drugs ( n=13 ) , respectively . the distribution of patients on bronchodilator alone , treatment containing ics , and no maintenance drug by copd stage is shown in table 3 .
of all the patients , 614 ( 89% ) received treatment containing ics or ics alone .
the distribution of patients who received ics was 43.8% in stage i , 81.5% in stage ii , 89.2% in stage iii , and 86.5% in stage iv . of the 25 ( 3.5% ) patients who were not on any medication , one patient was in stage i , nine in stage ii , eight in stage iii , and nine in stage iv . there were 327 ( 45.5% ) patients on short - acting bronchodilators used as rescue drugs in addition to maintenance therapy .
of all the patients , 56 ( 7.8% ) were on monotherapy , 125 ( 17.4% ) on dual , 350 ( 48.7% ) on triple , and 163 ( 22.6% ) on quadruple therapy .
long - term oxygen use rate was 22.4% ( n=112 patients ) in stages iii and iv patients .
this is the first large - scale real - life study in turkey that reflects the clinical practice of pulmonary specialists through an observational analysis of their adherence to treatment recommendations of gold 2010 guideline .
the survey was carried out in eleven pulmonary outpatient clinics spread over the national territory .
our findings show that only 59.5% of overall patients were treated in accordance with the guideline recommendations .
while inappropriate treatment regimens were more frequently prescribed for stages i ( 93.8% ) and ii ( 85.3% ) , overtreatment in early stages and high rate of ics administration ( 89% ) in all stages of disease resulted in improper therapeutic approach according to gold 2010 recommendations . in more severe disease stages ,
guideline - adherent therapeutic approaches were detected by wide use of ics addition to one or more long - acting bronchodilators .
most of the provider s guideline adherence studies have reported the current practices of general practitioners .
a few recent studies have reported similar rates of adherence to guideline recommendations for copd patients ( 37.9%54.7% ) between pulmonologists in other countries.4,5 stage i patients were overtreated with regular long - acting bronchodilators or combination of bronchodilators and icss , while guidelines recommend short - acting bronchodilators as initial therapy . in a study conducted among the pulmonary specialists in italy , corrado and rossi reported that 87.7% of the stage i patients received regular treatment containing long - acting bronchodilators , ics alone , or other treatment combinations of ics plus long - acting bronchodilators.4 in a retrospective database study in the united states , fitch et al reported that only 1% of the stage i patients received ics added to laba and lama , while 6% received ics plus single long - acting bronchodilators , and 76% did not receive any medication in stage i.11 shariff et al reported that 7.7% of the patients in stage i were overtreated and received inappropriate treatment according to the guidelines , in another study on guideline adherence among practitioners and pulmonary specialists.5 in our study , only 14.7% of the patients in stage ii received appropriate therapy , and this was mainly due to overtreatment which was combined long - acting bronchodilators and ics in 77.3% of the patients .
other reasons for nonadherent therapeutic approaches were single use of ics in 3.4% of the patients and lack of treatment in 3.8% of the patients . other studies found 26%49.2% adherence to gold recommendations in this stage .
overtreatment was 67.3% in one study,4 and lack of copd treatment has been reported in 54% of the patients in another report for stage ii.11 severe and very severe copd patients were treated by the combined use of one or more long - acting bronchodilators and ics which was compliant to gold recommendations .
this finding can be explained by the features of this group of patients who are more symptomatic and have more frequent exacerbations .
similar studies on guideline adherence in different countries also reported frequent use of dual and triple therapies in stages iii and iv.4,5,11 the ics was frequently used at all stages ( 43.8% in stage i , 81.5% in stage ii , 89.3% in stage iii , and 87.4% in stage iv ) mostly as a part of combination therapy .
this study demonstrates that the pulmonary specialists in turkey have a high tendency to use ics for the treatment of patients with copd . in a study by de miguel - diez et al in spain ,
34.5% of the patients were prescribed ics , alone or in combination , with a rate of inappropriate use of 18.2%.12 asche et al reported that 33% of the patients received combination treatment containing laba , lama , and/or ics consistent with the current gold guidelines.13 pulmonary specialists in our study mostly preferred to prescribe combination treatments containing ics .
this might be related to a number of factors :
inadequate time with patients , which leads to opting for combination therapies that can provide a broad - spectrum treatment.concerns about symptom control , exacerbation prevention alongside spirometric staging might influence the treatment choice of the specialist.reimbursement of all copd drugs in turkey , making them easily accessible for patients with health insurance.preferring to prescribe drug groups that are effective in the treatment of both asthma and copd , where a differential diagnosis is not available.increasing number of drugs that are provided in combinations and marketing strategies and publicity efforts that promote their use .
inadequate time with patients , which leads to opting for combination therapies that can provide a broad - spectrum treatment .
concerns about symptom control , exacerbation prevention alongside spirometric staging might influence the treatment choice of the specialist .
reimbursement of all copd drugs in turkey , making them easily accessible for patients with health insurance .
preferring to prescribe drug groups that are effective in the treatment of both asthma and copd , where a differential diagnosis is not available . increasing number of drugs that are provided in combinations and marketing strategies and publicity efforts that promote their use . in patients treated with regular bronchodilators ( alone or a part of a combination therapy ) ,
the most frequently used drug was laba ( n=613 ) , followed by lama ( n = 575 ) , t ( n=197 ) . among these results ,
t use was prescribed for 27.4% of stable copd patients which was remarkably higher than the finding of corrado and rossi that reported 2.2% of patients were using xanthines.4 gold remark on t is that it is effective in copd but , due to its potential toxicity , inhaled bronchodilators are preferred when available.1 in our study , this high rate of t prescription in addition to other inhaled bronchodilators may be explained by the choice of an oral bronchodilator drug even if it has potential toxicity and drug interactions in older patients .
another finding of this study was that the use of long - acting bronchodilators such as laba and lama alone remains considerably low , and they have been mostly preferred in stages i , ii , and iii .
the most commonly used bronchodilator in monotherapy was lama ( 4.2% ) , and this might be due to its single - dose use and duration of action of more than 24 hours.14,15 this study has several limitations .
individual patient data were retrospectively collected through patient records : number of acute exacerbations in the previous year , complete information on comorbidities , adherence to nonpharmacologic interventions were lacking .
gold guidelines were updated in 2011 to incorporate clinical presentation , including dyspnea as measured by copd assessment test and the modified british medical research council .
the updated version of the guidelines incorporates clinical presentation with spirometry findings ( gold 2011).16 this study was undertaken between december 2010 and november 2011 .
study protocol had been designed to evaluate gold 2010 guideline adherence . due to lack of knowledge on dyspnea level ( medical research council scale ) and exacerbation frequency during the last year
even this new gold classification ( updated in 2011 ) has been changed by considering sypmtoms and exacerbations in addition to spirometric staging , we have not enough data as to the exact superiorities over the old gold classification .
a very recent analysis showed that gold 2011 shifted the overall copd severity distribution to more severe categories .
there were nearly three times more copd patients in stage d than in former stage iv ( p<0.05 ) .
the predictive capacity for survival up to 10 years was significant for both systems ( p<0.01 ) and gold 2007 and 2011 did not differ significantly . in this study ,
the percent predicted fev1 thresholds of 85% , 55% , and 35% were found better to stage copd severity for mortality , which are similar to the ones used previously in the gold 2010 classification . increasing intensity of treatment of patients with copd according to their gold 2011 reclassification
is not known to improve health outcomes.17 the classification recommended by the gold committee since 2011 does not seem to be adopted universally throughout europe .
even if there is a real improvement over the previous classification due to taking into account clinical criteria , quality of life , and exacerbations , criticisms have arisen concerning the choice of certain pathways and therapeutic recommendations not based on prospective studies with a high level of evidence.18 at the national level , we do not have any published data on the adoption of the new gold 2011 classification . according to the previously mentioned obstacles of the new classification , and lack of the knowledge about the frequency of new gold 2011 classification use , instead of the gold 2010 .
we believe this data on the guideline adherence among pulmonologists may still reflect the current attitude toward the routine approach of the diagnosis and therapies of copd in turkey , and also is concordant with the real life situation around the world .
turkish thoracic society has published an updated report of copd prevention , diagnosis and treatment in 2014,19 and included in the global alliance against chronic respiratory disease ( gard ) with the ministry of health.20 the improvement of adherence to national and international copd guidelines may be achieved by continuing medical education supported by national thoracic societies . increased awareness of guidelines may influence the standardized care of copd patients , and will have prognostic influences .
promotion of the compliance to evidence - based medicine by health authorities may have also positive effects on the attitude change in the real - life applications .
this study showed that the pulmonary specialists in turkey have low rates of adherence to gold guidelines for treatment of stable copd patients .
major improper approaches were overtreatment in early stages and excessive use of inhaler corticosteroids in all stages of disease .
new strategies are needed to achieve a standardized approach for the treatment of copd , and thus to improve adherence to guideline recommendations , with particular focus on treatment indications and overtreatment in turkey .
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backgroundlow adherence to global initiative for chronic obstructive lung disease ( gold ) guideline recommendations has been reported worldwide .
there has been no study on the adherence to gold guidelines for copd treatment in turkey.objectivesto investigate the rates of adherence to gold 2010 guidelines for copd treatment among pulmonologists.designa multi - center , cross - sectional , observational study was carried out in eleven pulmonary outpatient clinics across turkey .
adherence to gold was evaluated through hospital records .
demographic and clinical data were recorded.resultsstudy included 719 patients ( mean age : 62.99.7 years ; males 85.4% ) of whom 16 was classified as gold stage i , 238 as ii , 346 as iii , and 119 as iv , and only 59.5% received appropriate treatment .
rates of guideline adherence varied across gold stages ( i , 6.3% ; ii , 14.7% ; iii , 84.4% ; and iv , 84% ) .
causes of inappropriate therapies were overtreatment ( stage i , 100% and stage ii , 91.1% ) , undertreatment ( stage iii , 3.3% and stage iv , 10.9% ) and lack of treatment ( stage ii , 3.8% ; stage iii , 2.3% ; and stage iv , 5.9% ) . the most preferred regimen ( 43.4% ) was long - acting 2-agonist - inhaled corticosteroid - long - acting muscarinic antagonist .
overall , 614 patients ( 89% ) received treatment containing inhaled corticosteroid.conclusionpulmonologists in turkey have low rates of adherence to gold guidelines in copd treatment .
inappropriateness of therapies was due to overtreatment in early stages and excessive use of inhaled corticosteroid ( ics ) in all disease stages .
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Introduction
Methods
Statistical analysis
Results
Patient characteristics
Adherence to GOLD 2010 guideline recommendations for treatment
Analysis of treatment approaches
Discussion
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o - glycans have been found to play an important role in protein stability and tertiary structure , stabilize protein conformation , modulate the activity of enzymes ( e.g. by reversible attachment of o - linked glcnac to cytoplasmic and nuclear proteins ) and signaling molecules , are essential for a number of recognition processes and are sorting determinants guiding the modified protein in the cell from the place of biosynthesis to its target location [ 13 ] .
unlike the n - glycans they may be linked to several different amino acid residues ( very often serine or threonine , but also hydroxylysine , tyrosine or hydroxyproline ) of the protein and their occurrence can not be predicted easily by a common consensus sequence .
extended galnac residues linked to serine or threonine residues ( mucin - type glycans ) are the most frequent and best investigated o - glycans in higher eukaryotes .
eight core subtype formations showing cell - tissue specific patterns related to their function have been described ( for a review see ) .
also glcnac--ser / thr without any elongation , a typical feature for nuclear and cytoskeletal proteins existing in a dynamic equilibrium with phosphorylation , and fuc--ser / thr as well as glc--ser , primarily found in epidermal growth factor domains , have been found in eukaryotes .
a linkage via mannose has been found to be the main o - glycan type in yeasts , but also some mammalian tissues carry this structural feature , which seems to be relevant in some muscular dystrophies , while o - xylosylation is the starting point for the biosynthesis of chondroitin and heparan sulfates , which have roles in development and morphogenesis .
the o - glycans of nematodes are rather complex with methylation as further modification [ 9 , 10 ] .
xenopus eggs have o - glycans with up to 10 monosaccharide constituents , often highly fucosylated , with a linkage to the protein via galnac [ 11 , 12 ] .
even parasites ( helminths and t. cruzi ) display small galnac linked o - glycans . in plants
o - glycans occur as arabinogalactans linked to hydroxyproline or serine ( gal--hyp / ser ) , or hydroxyproline is glycosylated with short arabinofuranosides ( ara--hyp , ara--hyp ) . here
we present for the first time an overview on o - glycans of some species of the mollusk branch of the evolutionary tree .
the glycosylation potential of snails is important in general , because some of them are intermediate hosts of pathogens ( biomphalaria glabrata and lymnaea stagnalis ) or a pest in agriculture
. a broad knowledge of their glycosylation abilities may show targets for pest control in the near future .
in the course of the years , n - glycan patterns of several snail species have been presented [ 1417 ] . methylated mannose and galactose residues were found to be frequent constituents . in the present study we determine the main o - glycan structures in snails and characterize a common core - trisaccharide .
cepaea hortensis , planorbarius corneus , arion lusitanicus , limax maximus and helix pomatia were collected by the authors under the supervision of dr .
manfred pintar ( department of integrative biology and biodiversity research , institute of zoology , university of natural resources and life sciences , vienna ) in the vicinity of vienna .
all chemicals purchased were of the highest quality available from sigma or fluka . while water living snails could be dissected right away , land living snails were extensively washed to remove the extraneous mucous components .
snails were dissected as previously described and proteins were purified according to with minor modifications : 1 g of wet tissue was homogenised in 10 ml of chaps - based lysis buffer ( 0.5 % ( w / v ) chaps , 150 mm nacl , 20 mm tris / hcl , 2.5 mm sodium pyrophosphate , 1 mm ethylene - glycol - bis(2-aminoethylether)-n , n , n,n-tetraacetic acid and 1 mm edta , ph 7.5 ) and incubated for 72 h at 4 c .
snail proteins were then reduced by the addition of 500 l of 10 mm dithiothreitol and incubation at 56 c for 45 min .
then 500 l of 55 mm iodoacetamide were added and incubated for 30 min at room temperature in the dark .
the aqueous samples were precipitated with a 4-fold amount of pre - chilled acetone at 20 c for at least 4 h and then centrifuged at 32000 g for 60 min .
the pellets were resuspended in 0.1 m sodium citrate buffer , ph 4.6 , and incubated with 2 units of -glucosidase from rice [ ec 3.2.1.20 ] at 37 c for 16 h. sugars and polysaccharides were eluted from cation exchange chromatography ( ag 50 w - x2 , biorad , ca ) with 2 % of acetic acid , while proteins were eluted by 1.0 m ammonium acetate , ph 9.0 .
the protein fraction was again precipitated by acetone followed by a similar precipitation by methanol .
the dry sample was dissolved in 0.1 m naoh containing 0.8 m nabh3 and incubated at 37 c for 68 h. the sample was carefully neutralised by the addition of 2 m acetic acid , dried in a vacuum centrifuge and washed at least twice with 30 % ( v / v ) methanol .
the glycans were directly extracted by solid phase extraction on a porous graphitized carbon cartridge ( supelclean envi - carb , 100 mg bed weight , 1 ml column volume or 500 mg bed weight , 6 ml column volume , sigma - aldrich , germany ) according to by using ammonium formate buffer / acetonitrile for elution and then on a reversed - phase c18 cartridge ( strata , c18-e , 55 m , 50 mg bed volume , 1 ml column volume , phenomenex , germany ) using 5 % ( v / v ) acetic acid as loading solvent and 25 % ( v / v ) isopropanol in 5 % ( v / v ) acetic acid for elution .
hplc separation was carried out on a porous graphitized carbon ( pgc ) column ( hypersil - keystone , hypercarb 5 150 3 mm , thermo scientific , austria ) at a flow rate of 0.6 ml / min using 0.3 % ( v / v ) formic acid adjusted to ph 3.1 with ammonium as solvent a and 95 % ( v / v ) acetonitrile in solvent a as solvent b. the elution protocol consisted of 2 min at 100 % ( v / v ) of solvent a followed by a gradient of 28 min till 25 % ( v / v ) of solvent b and a cleaning step of 4 min at 60 % ( v / v ) of solvent b. each fraction was tested by esi - ms for its glycan content .
single glycan structures were pooled and lyophilised . for monosaccharide analysis alditol acetates and for linkage analysis
gc analysis was carried out on a vf 5 ms capillary column ( 60 m , 0.25 mm inner diameter ; 0.1 mm film thickness ; varian , darmstadt , germany ) and electron impact ( ei ) mass spectrometry was performed in the positive ion mode using a polaris q instrument ( thermoquest - analytical systems ) . for analysis of small sample amounts with esi - ms an in - gel release method for glycans bound to coomassie
reduced oligosaccharides were analyzed by pgc - lc - esi - ms on a hypercarb column ( 0.32 150 mm , thermo fisher scientific , austria ) coupled to an ultimate 3000 ( dionex ) capillary hplc and a q - tof ultima ms ( waters ) as described previously [ 21 , 26 ] .
the interpretation of the ms data was performed with the help of the software tool glycoworkbench .
arion lusitanicus proteins were isolated and separated from contaminants through several purification steps in order to obtain sufficient amounts ( 12 g ) of pure glycans for further gc - ms analysis ( total monosaccharide and linkage analysis ) .
the preparation of o - glycans was carried out via -elimination followed by further separation steps . as a final procedure
the glycans were separated on a pgc ( porous graphitized carbon ) hplc - column by collecting 1-minute fractions which were screened by esi - ms to gain clean single structures . in the course of the -elimination releasing the glycan from the protein backbone , sodium borohydride was used .
in contrast , for reduction of monosaccharides before gc - ms analysis sodium borodeuteride was employed , which allowed further the discrimination between the sugar released by -elimination and those linked to other sugars previously . in fig
. 1 monosaccharide constituents of the core trisaccharide representing the main structure found in a. lusitanicus , with a pseudomolecular mass ( [ m+h ] ) of m / z 576.3 da , were determined by gc - ms of the corresponding alditol acetates and identified on the basis of retention times in gc and their characteristic electron impact ( ei ) mass spectra .
the results revealed the presence of a galnac - residue which was h - reduced , while all the other sugars were h reduced .
therefore , galnac can be considered as the protein bound sugar . the high amount of 4-o - me - gal indicated that this residue is the one which is mainly involved in the elongation of the glycan .
minor compounds were identified as contaminations by n - glycans ( glcnac and man ) or storage glycans ( glc and gal ) .
the lc - esi - ms / ms fragmentation pattern confirmed the occurrence of two methylated hexoses linked to the amino sugar ( fig .
1monosaccharide constituent analysis of the core trisaccharide of a. lusitanicus released by [ h ] reductive -elimination .
alditol acetates obtained after acid hydrolysis , reduction with sodium borodeuteride and peracetylation or hydrolysis and peracetylation of already existing alditols were identified by gc - ms .
peaks 14 arise from contaminating n - glycans and storage oligosaccharides that could not be completely removed ( 1 : man , h - reduced , 2 : glc , h - reduced , 3 : gal , h - reduced , 4 : glcnac , h - reduced)fig 2lc - es - ms / ms spectrum of the core trisaccharide of a. lusitanicus . registered ions represent proton adducts [ m+h ] monosaccharide constituent analysis of the core trisaccharide of a. lusitanicus released by [ h ] reductive -elimination .
alditol acetates obtained after acid hydrolysis , reduction with sodium borodeuteride and peracetylation or hydrolysis and peracetylation of already existing alditols were identified by gc - ms .
peaks 14 arise from contaminating n - glycans and storage oligosaccharides that could not be completely removed ( 1 : man , h - reduced , 2 : glc , h - reduced , 3 : gal , h - reduced , 4 : glcnac , h - reduced ) lc - es - ms / ms spectrum of the core trisaccharide of a. lusitanicus .
registered ions represent proton adducts [ m+h ] moreover , gc - ms analysis of partially methylated alditol acetates was carried out for further confirmation of the compounds and also for linkage determination .
methylated sugar standards purified by preparative gc were used to elucidate the type of methylated hexose ( man or gal ) based on retention time .
the selected ion chromatogram of the monosaccharide derivatives obtained from the o - glycan core structure identified 2,3,4,6-tetra - o - methyl - galacitol at 20.66 min and 1,4,5-tri - o - methyl galnac - ol at 33.33 min ( fig
. 3a ) which could be verified by ei - ms data ( figs .
3b and c ) . to determine the position of the naturally occurring methyl group on the galactose
this allows in the ei - ms fragmentation pattern the discrimination between the naturally occurring and introduced methylgroups in the fragments by a mass difference of 3 da . that way the occurrence of terminal 4-o - methylated galactose
4 ) . combining these data , the main o - glycan structure of a. lusitanicus was elucidated as a trisaccharide containing two terminal 4-o - methylated galactoses which are 3- and 6-linked to an inner galnac residue ( fig .
5).fig 3linkage analysis of the core trisaccharide - alditol from a. lusitanicus ( a ) selected ion chromatogram ( m / z 145 and m / z 318 ) of partially methylated alditol acetates obtained after permethylation with [ h ] methyliodide , hydrolysis , reduction with sodium borohydride and peracetylation ; ( b ) ei - ms spectrum and fragmentation pattern of 1,4,5-tri - o - methyl - galnac - ol and ( c ) 2,3,4,6-tetra - o - methyl - galacitol .
characteristic primary selected secondary fragment ions are assignedfig 4ei - ms spectra and fragmentation patterns of ( a ) 1,4,5-tri - o - deuteromethyl - galnac - ol and ( b ) terminal 4-o - methyl-2,3,6-tri - o - deuteromethyl - galacitol obtained after permethylation of the core trisaccharide alditol from a. lusitanicus with [ h ] methyliodide .
the structure plot is generated in the notation of the consortium for functional glycomics ( http://www.functionalglycomics.org ) using the visual editor of glycoworkbench .
this software application is developed and available as part of the eurocarbdb project ( http://www.eurocarb.db.org/applications/ms-tools ) .
square = galnac , circles = gal , me = methyl group linkage analysis of the core trisaccharide - alditol from a. lusitanicus ( a ) selected ion chromatogram ( m / z 145 and m / z 318 ) of partially methylated alditol acetates obtained after permethylation with [ h ] methyliodide , hydrolysis , reduction with sodium borohydride and peracetylation ; ( b ) ei - ms spectrum and fragmentation pattern of 1,4,5-tri - o - methyl - galnac - ol and ( c ) 2,3,4,6-tetra - o - methyl - galacitol .
characteristic primary selected secondary fragment ions are assigned ei - ms spectra and fragmentation patterns of ( a ) 1,4,5-tri - o - deuteromethyl - galnac - ol and ( b ) terminal 4-o - methyl-2,3,6-tri - o - deuteromethyl - galacitol obtained after permethylation of the core trisaccharide alditol from a. lusitanicus with [ h ] methyliodide .
the structure plot is generated in the notation of the consortium for functional glycomics ( http://www.functionalglycomics.org ) using the visual editor of glycoworkbench .
this software application is developed and available as part of the eurocarbdb project ( http://www.eurocarb.db.org/applications/ms-tools ) .
square = galnac , circles = gal , me = methyl group a small amount of an isoform with the same molecular weight , but slightly later retention time in the pgc ( porous graphitized carbon ) selected ion chromatogram ( fig .
6c ) , was identified analogously by gc - ms and ei - ms as trisaccharide containing a galnac residue substituted by one 4-o - methylated galactose and one 3-o - methylated mannose ( data not shown).fig 6selected ion chromatograms of partially methylated structures obtained by lc - esi - ms .
a core trisaccharide carrying in addition one methylated and one unmethylated hexose ( m / z 914.4 [ m+h ] ) ; b core trisaccharide with one additional hexose ( m / z 738.3 [ m+h ] ) ; c core trisaccharide and its isoform ( m / z 576.3 [ m+h ] ) .
square = amino sugar , circle = hexose , me = methyl group selected ion chromatograms of partially methylated structures obtained by lc - esi - ms . a core trisaccharide carrying in addition one methylated and one unmethylated hexose ( m / z 914.4 [ m+h ] ) ; b core trisaccharide with one additional hexose ( m / z 738.3 [ m+h ] ) ; c core trisaccharide and its isoform ( m / z 576.3 [ m+h ] ) .
square = amino sugar , circle = hexose , me = methyl group besides the trisaccharide core several other glycan species were found during purification on pgc - column .
a glycan consisting of the methylated core trisaccharide and one further hexose ( m / z 738.3 [ m+h ] ) was found .
this additional hexose was an unmethylated gal residue linked alternatively to one of the two arms of the trisaccharide explaining the two isoforms detected by pgc selected ion chromatogram ( fig .
the fourth structure ( m / z 914.4 [ m+h ] ) which was obtained in sufficient amounts for gc - ms consisted of the core trisaccharide elongated by an unmethylated hexose on one arm and a methylated hexose on the other one ( fig .
the presence of significant amounts of man and 3-o - me - man indicated that these two residues were responsible for the elongation .
gc - ms analysis using different derivatisation protocols requested a high amount of material and high quality of purification . especially for snail tissues this was a challenge and
therefore this method could not be carried out for a large number of samples in due time .
an in - gel -elimination after sds - page followed by a short clean - up ( pgc - cartridge ) procedure was sufficient .
the shortened protocol also ensured that no weakly bound modifications , such as sulfate or sialic acids for example , got lost . combining our data on snail monosaccharide analysis , data from previous gc - ms methylation analysis and the fragmentation results of lc - esi - ms / ms analysis of selected snails we could determine the main structures of snail o - glycans and classify them according to their modification of the established core structure into six groups : ( i ) glycans smaller than the core , missing one or both methyl groups or missing one hexose ; ( ii ) the trisaccharide core containing galnac and two 4-o - methylated hexose residues ; ( iii ) the core with additional methylated hexoses ; ( iv ) the core with one or two additional unmethylated hexoses and sometimes one more methylated hexose ; ( v ) glycans containing fucose ; ( vi ) glycans containing one hexnac and up to six unmethylated hexoses .
for an overview on the structures see table 1.table 1overview of o - glycan distribution in selected snail species given in [ % ] of total o - glycans .
compositions are given in terms of hexose ( hex ) , methylated hexose ( mehex ) , n - acetylhexosamine ( hexnac ) and deoxyhexose ( fucose ; dhex)glycan compositiongroupobserved mass [ m+h]calculated mass [ m+h]arion lusitanicusachatina fulicacepaea hortensisplanorbarius corenusbiomphalaria glabratahelix pomatialimax maximusclea helenahexhexnac(i ) - glycans smaller than the core386.2386.1707000000mehexhexnac400.2400.1850310200hex2hexnac548.2548.22traces000traces000hexmehexhexnac562.3562.2390012182600(mehex)2hexnac(ii ) - core576.3576.2554692612292910089(mehex)3hexnac(iii ) - core with additional methyl hexoses752.3752.325212302700(mehex)4hexnac928.4928.39traces0000000(mehex)5hexnac1104.41104.46traces0000000hex(mehex)2hexnac(iv ) core with additional hexoses and methyl hexoses738.3738.30141617164512011hex2(mehex)2hexnac900.4900.36203720100hex(mehex)3hexnac914.4914.3725505400(mehex)2hexnacdhex(v ) - fucosylated structures722.3722.31220traces1traces00(mehex)3hexnacdhex898.4898.3860000000(mehex)4hexnacdhex1074.41074.44traces0000000hex3hexnac(vi ) hexnac and hexoses710.3710.2700061traces00hex4hexnac872.3872.32000460000hex5hexnac1034.31034.3800030000hex6hexnac1197.01196.43000traces0000 overview of o - glycan distribution in selected snail species given in [ % ] of total o - glycans .
compositions are given in terms of hexose ( hex ) , methylated hexose ( mehex ) , n - acetylhexosamine ( hexnac ) and deoxyhexose ( fucose ; dhex ) analyzing snails in detail , it was obvious that the o - glycan trisaccharide core is an important decoration of snail proteins in all investigated species . in five of the analyzed snails ( a. lusitanicus , a. fulica , h. pomatia , l. maximus , c. helena ) this glycan was the most abundant type of o - glycan .
b. glabrata and c. hortensis displayed as their main glycan the trisaccharide elongated by one or two unmethylated hexoses , respectively .
only p. corneus was an exception possessing a large amount of hex4hexnac glycan . whereas the addition of one hexose to the core was relatively frequent , larger structures were minor compounds or were even not present in several species .
the only detected amino sugar was galnac which provided in all cases the connection to the protein .
gal and man were present in unmethylated or mono - methylated ( 3-o - me - gal , 4-o - me - gal , 3-o - me - man ) versions .
fucose , which had been identified as such in previous works [ 23 , 28 ] , occurred only in low amounts and was detected linked to galnac as well as to terminal sugar residues by ms / ms fragmentation .
during the last decades our knowledge on glycosylation processes - structure of the glycans and the corresponding biochemical pathways including the responsible enzymes - increased enormously , especially for glycans of mammalian origin .
the glycosylation capabilities of other species were only under investigation if their glycans were for any reason connected with human life ( e.g. some recognition processes of pathogens or allergy on food or plant glycans ) or if they were potent candidates for cell culture systems for the expression of therapeutics ( some insect , yeast and plant cells ) .
in the meantime more and more invertebrate systems are investigated because of their potential usefulness for biotechnological reasons or to get some deeper insights into the processing pathways of unusual glycan structures .
previous studies on various invertebrates showed that glycosylation has a lot more varieties than we expected as long as only mammals were investigated , but the data are scattered .
still the main focus is laid on n - glycans but the interest in o - glycans is growing .
a number of difficulties arose during snail glycan analysis due to the tough tissue , which was not easy to homogenize and the rigid mucus , which had to be discarded diligently .
the removal of storage carbohydrates , which might influence monosaccharide analysis and the separation of n- and o - glycans were other struggles to resolve . while the first was done by -glucosidase digestion from rice , the latter was an even more difficult task .
n - glycosidases ( pngase a and f ) did not remove the n - glycans completely and even mild -elimination conditions seemed to be able to release , at least in part , residual n - glycans which resulted in n - glycan impurities in o - glycan fractions .
furthermore some n - glycans of snails and digested storage glycans are so small ( 45 sugar residues [ 16 , 17 ] ) that they may co - migrate on hplc with o - glycans
. therefore only a limited number of glycans could be prepared in terms of sufficient amount and purity for permethylation followed by maldi - tof - ms and gc - ms linkage analysis .
however , in combination with lc - esi - ms of native glycans which is capable of a high throughput of samples the o - glycan pattern of 8 snail species ( land snails with shell : achatina fulica , cepaea hortensis and helix pomatia ; slugs : arion lusitanicus and limax maximus ; water snails : biomphalaria glabrata , clea helena , and planorbarius corneus ) could be identified .
four monosaccharide constituents galnac , gal , man and fuc are the building blocks of the structures .
the only further modification is a methylation of the hexoses , resulting in 3-o - me - gal , 4-o - me - gal and 3-o - me - man , which has already been shown for snail n - glycans [ 1417 , 23 ] .
each o - glycan contains only one amino sugar , which is the protein linked galnac .
the linkage amino acid was not determined in detail , but due to the easy release by -elimination the common galnac - ser / thr motif is very likely .
the connection to other amino acids , for example tyrosine , is usually more stable . in snail o - glycans the inner galnac
is frequently elongated by two 4-o - me - gal residues in ( 13)- and ( 16)-linkage . elongations of this trisaccharide core by one or two hexoses with or without methyl group appeared in most snail species , whereas further elongation or fucosylation are rare events .
insects , one neighbor of mollusks in the phylogenetic tree , display also internal galnac elongated by one gal in their o - glycan structures , but further elongations and/or branching are frequently done by another amino sugar ( galnac or glcnac ) .
the main o - glycans of toxocara canis are trisaccharides consisting of galnac , gal or 4-o - gal and 2-o - fuc .
so far nothing is known about biological functions of snail glycans . for sure these glycans
are similarly involved in recognition processes as they are in all other species , but especially the significance of the high degree of methylation of hexoses of n- as well as of o - glycans is waiting for elucidation . here
we enlarge the current knowledge on glycosylation abilities of lower animals by presenting the first o - glycan analysis of snails , representatives of the mollusk phylum .
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the glycosylation abilities of snails deserve attention , because snail species serve as intermediate hosts in the developmental cycles of some human and cattle parasites . in analogy to many other host - pathogen relations , the glycosylation of snail proteins may likewise contribute to these host - parasite interactions . here
we present an overview on the o - glycan structures of 8 different snails ( land and water snails , with or without shell ) : arion lusitanicus , achatina fulica , biomphalaria glabrata , cepaea hortensis , clea helena , helix pomatia , limax maximus and planorbarius corneus .
the o - glycans were released from the purified snail proteins by -elimination .
further analysis was carried out by liquid chromatography coupled to electrospray ionization mass spectrometry and for the main structures by gas chromatography / mass spectrometry .
snail o - glycans are built from the four monosaccharide constituents : n - acetylgalactosamine , galactose , mannose and fucose .
an additional modification is a methylation of the hexoses .
the common trisaccharide core structure was determined in arion lusitanicus to be n - acetylgalactosamine linked to the protein elongated by two 4-o - methylated galactose residues .
further elongations by methylated and unmethylated galactose and mannose residues and/or fucose are present .
the typical snail o - glycan structures are different to those so far described .
similar to snail n - glycan structures they display methylated hexose residues .
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Introduction
Material and methods
Results
Discussion and conclusion
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assessment of myocardial viability in patients with coronary artery disease is of great clinical importance , as dysfunctional but viable myocardium has the ability to regain contractility after coronary revascularization with subsequent improvements in cardiac function and prognosis . the perfusable tissue index ( pti )
can be used as a marker of myocardial viability , having been validated in patients with ischaemic heart disease [ 27 ] .
however , pti has never been used in clinical practice , primarily due to the complex imaging protocol consisting of dynamic [ o]h2o and [ o]co scans , together with a transmission scan , and the lack of high - quality , clinically usable parametric images .
recently , however , a method was developed to derive the pti from a single [ o]h2o pet / ct scan .
given the current potential of clinical [ o]h2o - based perfusion imaging and the rapid growth in the availability of cardiac pet / ct systems , pti viability measurements could become incorporated into clinical practice [ 9 , 10 ] .
parametric pti images derived from [ o]h2o pet / ct scans , however , have not yet been validated .
the purpose of this study was to compare these novel parametric pti images with late gadolinium - enhanced cardiovascular magnetic resonance ( lge - cmr ) imaging , an established method for quantifying scar size and a marker of viability .
the study population comprised 46 patients with documented or suspected coronary artery disease who had been studied using both pet / ct and cmr within a 2-month period .
all patients were in a stable clinical condition and no ischaemic events or revascularizations had occurred during the period between the two examinations .
patients with contraindications for pet / ct or cmr ( e.g. pacemaker , claustrophobia , atrial fibrillation ) were excluded .
the study was approved by the institutional medical ethics review committee , and all participants gave written informed consent prior to inclusion .
[ o]h2o scans were acquired using a gemini tf-64 ( philips healthcare , best , the netherlands ) pet / ct scanner .
[ o]h2o ( 370 mbq ) was injected as a 5-ml bolus ( 0.8 ml s ) followed by a 35-ml saline flush ( 2 ml s ) , simultaneously starting a 6-min list mode emission scan .
this scan was followed immediately by a respiration - averaged slow low - dose ( ld ) ct scan ( 55 mas , rotation time 1.5 s , pitch 0.825 , collimation 16 0.625 , acquiring 20 cm in 37 s ) during normal breathing .
all scans were checked for misalignment between the ld ct scan and the [ o]h2o scan ; in none of the patients were corrections needed .
dynamic [ o]h2o images were reconstructed into 22 frames ( 1 10 , 8 5 , 4 10 , 2 15 , 3 20 , 2 30 , 2 60 s ) using the three - dimensional row action maximum likelihood algorithm and applying all appropriate corrections for the scanner , normalization , dead time , decay , scatter , randoms and attenuation based on the corresponding ld ct scan . using capp software ( siemens / cti , knoxville , tn ) , regions of interest ( rois ) of 1 cm diameter were placed over the ascending aorta in at least ten transaxial image planes of the frame showing the first pass of the injected bolus .
these rois were combined into one volume of interest ( voi ) for the ascending aorta .
a second set of rois were placed over the right ventricle ( rv ) cavity in at least five transaxial planes , with roi boundaries at least 1 cm from the rv wall to avoid spill - in from myocardial activity .
both vois were then projected onto all dynamic [ o]h2o images , thereby generating arterial ( ca(t ) ) and rv ( crv(t ) ) time activity curves .
parametric pti images were calculated as previously described . in brief , parametric images of myocardial blood flow ( mbf ) , perfusable tissue fraction ( ptf ) , and arterial and venous blood volume fractions were calculated using a basis function implementation of the standard single - tissue compartment model for [ o]h2o [ 1114 ] .
parametric images of arterial and venous blood volume fractions were subtracted from normalized ct transmission images , resulting in parametric anatomic tissue fraction ( atf ) images . finally , parametric pti images were calculated as the ratio of ptf and atf images ( fig . 1 ) .
finally , 16 myocardial vois were defined manually on parametric ptf images , according to the 16 segments model of the american heart association , after which this voi template was projected onto the parametric pti images.fig .
( b ) atf ( g ml ) , ( c ) ptf ( g ml ) , and ( d ) pti in a patient without coronary artery disease example of short axis images of ( a ) blood volume , ( b ) atf ( g ml ) , ( c ) ptf ( g ml ) , and ( d ) pti in a patient without coronary artery disease cmr studies were performed on a 1.5-t whole - body scanner ( magnetom sonata or avanto ; siemens , erlangen , germany ) , using a six - channel phased - array body coil . after survey scans ,
a retrotriggered , balanced steady - state free precession gradient - echo sequence was used for cine imaging .
image parameters included : slice thickness 5 mm , slice gap 5 mm , temporal resolution < 50 ms , repetition time 3.2 ms , echo time 1.54 ms , flip angle 60 , typical image resolution 1.3 1.6 5.0 mm .
the cardiac cycle consisted of 20 phases . after obtaining four- , three- and two - chamber view cines ,
stacks of 10 to 12 short - axis slices were acquired to fully cover the left ventricle ( lv ) .
contrast images were acquired 10 to 15 min after administration of 0.2 mmol kg gadolinium - dtpa in the same views as in the cine images , using a two - dimensional segmented inversion - recovery prepared gradient echo sequence ( te 4.4 ms , tr 9.8 ms , inversion time 250 to 300 ms , typical voxel size 1.3 1.6 5.0 mm ) .
images were analysed off - line using the software package mass ( mr analytical software system ; medis , leiden , the netherlands ) .
the endocardial and epicardial borders of the lv were outlined manually in both end - diastolic and end - systolic phases of all short - axis images .
end - diastolic volume , end - systolic volume and ejection fraction were computed using these analyses .
the amount of fibrosis was calculated using the full - width at half - maximum method , which defines fibrosis as myocardial tissue with a signal intensity 50 % of the maximum hyperenhancement intensity . if no enhancement was found in a slice , the maximum signal of the nearest slice with enhancement was used .
all areas of enhancement were quantified by computer - assisted planimetry on each of the short - axis images and the segmental extent of enhancement was expressed as a percentage of the segmental area .
cmr images were analysed according to the same 16-segment model as used for the parametric pet images .
finally , myocardial segments were graded as viable or nonviable using the previously defined cut - off value of 50 % lge per segment [ 15 , 17 ] .
continuous variables are presented as means sd , and categorical data are summarized as frequencies and percentages .
multiple datasets were compared using analysis of variance ( anova ) , and specific differences were identified using student s t - test with bonferroni inequality adjustment .
receiver operating characteristic curves were generated for ptf , pti and mbf for the prediction of myocardial viability assessed by lge cmr .
the area under the curve ( auc ) was considered a measure of accuracy to discriminate between viable and nonviable myocardium .
the study population comprised 46 patients with documented or suspected coronary artery disease who had been studied using both pet / ct and cmr within a 2-month period .
all patients were in a stable clinical condition and no ischaemic events or revascularizations had occurred during the period between the two examinations .
patients with contraindications for pet / ct or cmr ( e.g. pacemaker , claustrophobia , atrial fibrillation ) were excluded .
the study was approved by the institutional medical ethics review committee , and all participants gave written informed consent prior to inclusion .
[ o]h2o scans were acquired using a gemini tf-64 ( philips healthcare , best , the netherlands ) pet / ct scanner .
[ o]h2o ( 370 mbq ) was injected as a 5-ml bolus ( 0.8 ml s ) followed by a 35-ml saline flush ( 2 ml s ) , simultaneously starting a 6-min list mode emission scan .
this scan was followed immediately by a respiration - averaged slow low - dose ( ld ) ct scan ( 55 mas , rotation time 1.5 s , pitch 0.825 , collimation 16 0.625 , acquiring 20 cm in 37 s ) during normal breathing .
all scans were checked for misalignment between the ld ct scan and the [ o]h2o scan ; in none of the patients were corrections needed .
dynamic [ o]h2o images were reconstructed into 22 frames ( 1 10 , 8 5 , 4 10 , 2 15 , 3 20 , 2 30 , 2 60 s ) using the three - dimensional row action maximum likelihood algorithm and applying all appropriate corrections for the scanner , normalization , dead time , decay , scatter , randoms and attenuation based on the corresponding ld ct scan . using capp software ( siemens / cti , knoxville , tn ) , regions of interest ( rois ) of 1 cm diameter were placed over the ascending aorta in at least ten transaxial image planes of the frame showing the first pass of the injected bolus .
these rois were combined into one volume of interest ( voi ) for the ascending aorta .
a second set of rois were placed over the right ventricle ( rv ) cavity in at least five transaxial planes , with roi boundaries at least 1 cm from the rv wall to avoid spill - in from myocardial activity .
both vois were then projected onto all dynamic [ o]h2o images , thereby generating arterial ( ca(t ) ) and rv ( crv(t ) ) time activity curves .
parametric pti images were calculated as previously described . in brief , parametric images of myocardial blood flow ( mbf ) , perfusable tissue fraction ( ptf ) , and arterial and venous blood volume fractions were calculated using a basis function implementation of the standard single - tissue compartment model for [ o]h2o [ 1114 ] .
parametric images of arterial and venous blood volume fractions were subtracted from normalized ct transmission images , resulting in parametric anatomic tissue fraction ( atf ) images . finally , parametric pti images were calculated as the ratio of ptf and atf images ( fig . 1 ) .
finally , 16 myocardial vois were defined manually on parametric ptf images , according to the 16 segments model of the american heart association , after which this voi template was projected onto the parametric pti images.fig .
( b ) atf ( g ml ) , ( c ) ptf ( g ml ) , and ( d ) pti in a patient without coronary artery disease example of short axis images of ( a ) blood volume , ( b ) atf ( g ml ) , ( c ) ptf ( g ml ) , and ( d ) pti in a patient without coronary artery disease
cmr studies were performed on a 1.5-t whole - body scanner ( magnetom sonata or avanto ; siemens , erlangen , germany ) , using a six - channel phased - array body coil . after survey scans ,
a retrotriggered , balanced steady - state free precession gradient - echo sequence was used for cine imaging .
image parameters included : slice thickness 5 mm , slice gap 5 mm , temporal resolution < 50 ms , repetition time 3.2 ms , echo time 1.54 ms , flip angle 60 , typical image resolution 1.3 1.6 5.0 mm .
the cardiac cycle consisted of 20 phases . after obtaining four- , three- and two - chamber view cines ,
stacks of 10 to 12 short - axis slices were acquired to fully cover the left ventricle ( lv ) .
contrast images were acquired 10 to 15 min after administration of 0.2 mmol kg gadolinium - dtpa in the same views as in the cine images , using a two - dimensional segmented inversion - recovery prepared gradient echo sequence ( te 4.4 ms , tr 9.8 ms , inversion time 250 to 300 ms , typical voxel size 1.3 1.6 5.0 mm ) .
images were analysed off - line using the software package mass ( mr analytical software system ; medis , leiden , the netherlands ) .
the endocardial and epicardial borders of the lv were outlined manually in both end - diastolic and end - systolic phases of all short - axis images .
end - diastolic volume , end - systolic volume and ejection fraction were computed using these analyses .
the amount of fibrosis was calculated using the full - width at half - maximum method , which defines fibrosis as myocardial tissue with a signal intensity 50 % of the maximum hyperenhancement intensity .
if no enhancement was found in a slice , the maximum signal of the nearest slice with enhancement was used .
all areas of enhancement were quantified by computer - assisted planimetry on each of the short - axis images and the segmental extent of enhancement was expressed as a percentage of the segmental area .
cmr images were analysed according to the same 16-segment model as used for the parametric pet images .
finally , myocardial segments were graded as viable or nonviable using the previously defined cut - off value of 50 % lge per segment [ 15 , 17 ] .
continuous variables are presented as means sd , and categorical data are summarized as frequencies and percentages .
multiple datasets were compared using analysis of variance ( anova ) , and specific differences were identified using student s t - test with bonferroni inequality adjustment .
receiver operating characteristic curves were generated for ptf , pti and mbf for the prediction of myocardial viability assessed by lge cmr .
the area under the curve ( auc ) was considered a measure of accuracy to discriminate between viable and nonviable myocardium . a p value of < 0.05 was considered statistically significant .
lge was seen in the cmr images of 34 patients ( 74 % ) . of the 736 myocardial segments ,
364 ( 49 % ) showed some degree of lge.table 1patient characteristics ( n = 46)characteristicvalueage ( years)65 10gender ( male)36 ( 78 % ) previous myocardial infarction34 ( 74 % ) lv end - diastolic volume ( ml)226 65lv end - systolic volume ( ml)135 71lv ejection fraction ( % ) 42 16lv mass ( g)132 39 patient characteristics ( n = 46 ) pet / ct and cmr data are summarized in table 2 . there was a gradual decrease in ptf , pti and mbf values with increasing degree of lge on cmr images ( p < 0.001 by anova ) .
atf values remained relatively constant , except for a significant decrease in the ( near ) transmurally enhanced segments ( p < 0.001 by anova).table 2segmental pet / ct and lge dataextent of lge ( % ) control
( n = 372)025 ( n = 190)2550 ( n = 83)5075 ( n = 54)75 ( n = 37)p value ( anova)atf0.76 0.090.77 0.090.75 0.110.73 0.080.68 0.11**<0.001ptf ( g ml)0.72 0.080.72 0.090.70 0.090.60 0.08**0.51 0.12***<0.001pti0.91 0.080.89 0.090.89 0.090.77 0.10**0.70
0.16***<0.001mbf ( ml g min)1.02 0.300.91 0.26 * 0.85 0.30 * 0.83 0.24 * 0.67 0.30**<0.001*p < 0.05 vs. control ; * * p < 0.05 vs. control , 025 % lge and 2550 % lge ; * * * p < 0.05 vs. control , 025 % lge , 2550 % lge and 5075 % lge . segmental pet / ct and lge data * p < 0.05 vs. control ; * * p < 0.05 vs. control , 025 % lge and 2550 % lge ; * * * p < 0.05 vs. control , 025 % lge , 2550 % lge and 5075 % lge .
figure 2 shows a concordant pattern between parametric ptf , pti and lge cmr images in a patient with ischaemic cardiomyopathy after an anterior myocardial infarction.fig .
2long axis images ( a d ) and polar maps ( e h ) in a patient with an anterior myocardial infarction : a , e cmr with lge ( arrow , % transmurality ) ; b , f ptf ( g ml ) ; c , g pti ; d , h mbf ( ml g min)fig .
3long axis images in a patient with a nontransmural anterior myocardial infarction : a cmr with lge ; b pti long axis images ( a d ) and polar maps ( e h ) in a patient with an anterior myocardial infarction : a , e cmr with lge ( arrow , % transmurality ) ; b , f ptf ( g ml ) ; c , g pti ; d , h mbf ( ml g min ) long axis images in a patient with a nontransmural anterior myocardial infarction : a cmr with lge ; b pti using cmr as a reference , 91 of 364 ( 25 % ) segments showing some degree of lge were judged to be nonviable ( lge > 50 % ) . as shown in fig . 4 ,
the values of ptf and pti for predicting myocardial viability in all 736 segments were comparable ( auc 0.87 , ci 0.830.90 , and 0.86 , ci 0.820.91 , respectively , p = 0.541 ) .
mbf was able to predict myocardial viability with less accurate ( auc 0.69 , ci 0.630.75 , p < 0.001 ) .
optimal cut - off values of ptf , pti and mbf for predicting ( near ) transmural lge on cmr were 0.69 g ml , 0.80 , and 0.78 ml min g with sensitivities of 69 % , 91 % and 72 % , and specificities of 87 % , 73 % and 56 % , respectively .
figure 3 shows an example of a patient with a nontransmural scar and a corresponding relatively high pti.fig .
4receiver operator characteristics curves for the abilities of ptf , pti and mbf to differentiate between viable and nonviable segments based on late gadolinium enhancement receiver operator characteristics curves for the abilities of ptf , pti and mbf to differentiate between viable and nonviable segments based on late gadolinium enhancement
lge was seen in the cmr images of 34 patients ( 74 % ) . of the 736 myocardial segments ,
364 ( 49 % ) showed some degree of lge.table 1patient characteristics ( n = 46)characteristicvalueage ( years)65 10gender ( male)36 ( 78 % ) previous myocardial infarction34 ( 74 % ) lv end - diastolic volume ( ml)226 65lv end - systolic volume ( ml)135 71lv ejection fraction ( % ) 42 16lv mass ( g)132 39 patient characteristics ( n = 46 )
pet / ct and cmr data are summarized in table 2 . there was a gradual decrease in ptf , pti and mbf values with increasing degree of lge on cmr images ( p < 0.001 by anova ) .
atf values remained relatively constant , except for a significant decrease in the ( near ) transmurally enhanced segments ( p < 0.001 by anova).table 2segmental pet / ct and lge dataextent of lge ( % ) control
( n = 372)025 ( n = 190)2550 ( n = 83)5075 ( n = 54)75 ( n = 37)p value ( anova)atf0.76 0.090.77 0.090.75 0.110.73 0.080.68 0.11**<0.001ptf ( g ml)0.72 0.080.72 0.090.70 0.090.60 0.08**0.51 0.12***<0.001pti0.91 0.080.89 0.090.89 0.090.77 0.10**0.70 0.16***<0.001mbf ( ml g min)1.02 0.300.91 0.26 * 0.85 0.30 * 0.83 0.24 * 0.67 0.30**<0.001*p < 0.05 vs. control ; * * p < 0.05 vs. control , 025 % lge and 2550 % lge ; * * * p < 0.05 vs. control , 025 % lge , 2550 % lge and 5075 % lge .
segmental pet / ct and lge data * p < 0.05 vs. control ; * * p < 0.05 vs. control , 025 % lge and 2550 % lge ; * * * p < 0.05 vs. control , 025 % lge , 2550 % lge and 5075 % lge .
figure 2 shows a concordant pattern between parametric ptf , pti and lge cmr images in a patient with ischaemic cardiomyopathy after an anterior myocardial infarction.fig .
2long axis images ( a d ) and polar maps ( e h ) in a patient with an anterior myocardial infarction : a , e cmr with lge ( arrow , % transmurality ) ; b , f ptf ( g ml ) ; c , g pti ; d , h mbf ( ml g min)fig .
3long axis images in a patient with a nontransmural anterior myocardial infarction : a cmr with lge ; b pti long axis images ( a d ) and polar maps ( e h ) in a patient with an anterior myocardial infarction : a , e cmr with lge ( arrow , % transmurality ) ; b , f ptf ( g ml ) ; c , g pti ; d , h mbf ( ml g min ) long axis images in a patient with a nontransmural anterior myocardial infarction : a cmr with lge ; b pti using cmr as a reference , 91 of 364 ( 25 % ) segments showing some degree of lge were judged to be nonviable ( lge > 50 % ) . as shown in fig . 4 ,
the values of ptf and pti for predicting myocardial viability in all 736 segments were comparable ( auc 0.87 , ci 0.830.90 , and 0.86 , ci 0.820.91 , respectively , p = 0.541 ) .
mbf was able to predict myocardial viability with less accurate ( auc 0.69 , ci 0.630.75 , p < 0.001 ) .
optimal cut - off values of ptf , pti and mbf for predicting ( near ) transmural lge on cmr were 0.69 g ml , 0.80 , and 0.78 ml min g with sensitivities of 69 % , 91 % and 72 % , and specificities of 87 % , 73 % and 56 % , respectively .
figure 3 shows an example of a patient with a nontransmural scar and a corresponding relatively high pti.fig .
4receiver operator characteristics curves for the abilities of ptf , pti and mbf to differentiate between viable and nonviable segments based on late gadolinium enhancement receiver operator characteristics curves for the abilities of ptf , pti and mbf to differentiate between viable and nonviable segments based on late gadolinium enhancement
the present study was conducted to validate the use of a parametric myocardial viability imaging technique using [ o]h2o pet / ct in patients with ischaemic heart disease .
viability assessed using parametric ptf and pti imaging was in good agreement with that assessed using lge cmr .
furthermore , these images and myocardial perfusion imaging are obtained simultaneously allowing both myocardial viability and ischaemia to be evaluated in a single scanning session .
[ o]h2o is generally considered to be the gold standard for absolute quantification of myocardial perfusion in vivo [ 10 , 18 , 19 ] .
apart from mbf , [ o]h2o also provides estimates of the extent of myocardium within a roi that is able to exchange water rapidly , i.e. ptf .
subsequently , ptf can be corrected for partial volume effects by dividing it by its anatomical counterpart atf , resulting in pti .
it has been shown that both ptf and pti can identify myocardial scarring and thus can act as markers of viability . only recently ,
however , a method has been developed that enables generation parametric atf , ptf and pti images from a single [ o]h2o pet / ct scan .
in addition , the traditional ( long ) transmission scan can be replaced by a ( rapid ) low - dose ct scan , thereby shortening the total scanning time substantially .
now that the method has been shown to produce high - quality parametric images , its implementation into clinical practice needs investigation . using the described parametric imaging approach
, the present study demonstrated that atf was relatively constant independent of tissue characteristics of the myocardium . only in ( near )
transmurally infarcted segments was atf significantly reduced , most likely due to wall thinning , as atf is prone to partial volume effects . in contrast , ptf and pti progressively decreased with increasing extent of scarring , as shown by lge cmr .
these results are in line with those of previous studies in which reductions in both ptf and pti were observed in nonviable scarred myocardium .
pti in control segments appeared to be somewhat lower than the expected value of unity for normal segments ( 0.91 0.08 ) .
this phenomenon has previously been observed in cardiomyopathy in both animal experiments and human studies [ 6 , 20 ] .
slight misalignment between pet and cmr segments may have occurred to additionally account for this reduction in pti .
furthermore , the presence of interstitial fibrosis in dilated cardiomyopathy may also explain this reduction in
normal segments that remains undetected on lge cmr [ 21 , 22 ] . taking lge as a reference , the optimal cut - off values for discriminating between viable and nonviable myocardium
although this would reduce the data processing time , it would not affect the scanning protocol , as for both parameters a dynamic [ o]h2o pet scan in combination with a low - dose ct scan are required .
in addition , the sensitivity of pti exceeded that of ptf ( 91 % and 68 % , respectively ) , whereas for specificity the opposite pattern was observed ( 73 % and 78 % , respectively ) .
although further study is need to determine the cause of this discrepancy , as a potential clinical marker of viability pti may be favoured over ptf to reduce false - negative findings in patients who might benefit from revascularization .
it is of interest to note that , compared to ptf and pti , mbf performed relatively poorly in distinguishing viable from nonviable tissue , rendering it less suitable for viability imaging .
previous studies have indicated that the optimal cut - off value for pti is in the range 0.70.9 .
lge was used as a surrogate end - point of myocardial viability instead of functional recovery of dysfunctional myocardium after revascularization .
lge has been shown to be a good , but not perfect , marker of myocardial viability [ 15 , 17 ] .
therefore , the results should be interpreted with caution , and more studies are warranted to establish the value of parametric pti as a viability marker .
ptf and pti , obtained from a single [ o]h2o pet / ct scan , can be used as markers of myocardial viability in patients with coronary artery disease .
ptf and pti , obtained from a single [ o]h2o pet / ct scan , can be used as markers of myocardial viability in patients with coronary artery disease .
this article is distributed under the terms of the creative commons attribution license which permits any use , distribution , and reproduction in any medium , provided the original author(s ) and the source are credited .
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purposethe perfusable tissue index ( pti ) is a marker of myocardial viability .
recent technological advances have made it possible to generate parametric pti images from a single [ 15o]h2o pet / ct scan .
the purpose of this study was to validate these parametric pti images.methodsthe study population comprised 46 patients with documented or suspected coronary artery disease who were studied with [ 15o]h2o pet and late gadolinium - enhanced ( lge ) cardiac magnetic resonance imaging ( cmr).resultsof the 736 myocardial segments included , 364 showed some degree of lge .
pti and perfusable tissue fraction ( ptf ) diminished with increasing lge .
the areas under the curve of the pti and ptf , used to predict ( near ) transmural lge on cmr , were 0.86 and 0.87 , respectively .
optimal sensitivity and specificity were 91 % and 73 % for pti and 69 % and 87 % for ptf , respectively.conclusionpti and ptf assessed with a single [ 15o]h2o scan can be utilized as markers of myocardial viability in patients with coronary artery disease .
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Introduction
Materials and methods
Study population
PET/CT image acquisition
PET/CT image analysis
CMR image acquisition
CMR image analysis
Statistical analysis
Results
Baseline characteristics
PET/CT and CMR parameters
Predictive values for viability
Discussion
Conclusion
Open Access
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while advances in free trade and globalization increase the movement and accelerate the accumulation of invasive species ( lockwood et al .
2005 ) , it is still unclear how introduced populations can successfully establish . as elton ( 1958 ) pointed out , for every successful invasion that occurs , there are enormously more invasions that never happen , or fail quite soon or even after a good many years ( page 109 ) .
this modern biological paradox can not readily be reconciled , especially in the characteristic case where the founder population is small , as such populations are definitely in a precarious position ( mayr 1965 ; page 42 ) .
introductions of populations at low density and/or small size are often faced with inverse density dependent effects , attributed to demographic stochasticity or reduced cooperative interactions ( courchamp et al .
allee ( 1931 ) first proposed that under these conditions , populations may suffer a decrease in the per - capita rate of increase , from here on referred to as the allee effect . upon arrival in a novel environment , individuals need to overcome a series of challenges in order to reduce the population 's risk of extinction .
the time period in which this occurs is generally considered the initial establishment phase , and is thought to be a common feature and general pattern of invasion and the process of growth and expansion ( shigesada and kawasaki 1997 ; sakai et al .
the occurrence of a lag phase that precedes a noticeable increase in population growth and density can result from ecological and/or evolutionary phenomena ( sakai et al .
small populations that undergo logistic growth slowly increase through the initial phase of the exponential curve , leading to the perception of a time lag .
where this time lag is more pronounced , populations may be recovering from inverse density dependent effects ( i.e. , allee effects ) .
individuals may suffer a reduction in fitness at low densities for many reasons ( reviewed by courchamp et al .
even when the initial population is large , rapid dispersal required for expansion could be suicidal as the population density decreases , thereby enhancing inverse density dependent effects ( lewis and kareiva 1993 ; drake et al .
component allee effect where the population size and density affects the mean overall fitness or some component of individual fitness , respectively ( stephens et al . 1999 ; gascoigne et al .
it is often difficult to decipher the exact mechanism that manifests allee effects ( and it is not always the case that component allee effects lead to demographic allee effects ) .
nevertheless , we focus our attention on population level demographic allee effects with the underlying assumption that a component allee effect led to the demographic allee effect . essentially , it is the case that introduced individuals may be maladapted to small population sizes where their survival and reproductive ability are significantly impacted , and these impacts on individual fitness combine to produce an overall decrease in abundance ( i.e. , demographic allee effects ) . as allee effects impact individual fitness , the underlying traits that influence these effects (
i.e. , component allee effects ) may be subject to natural selection ( courchamp et al . 2008 ) . whereas propagule pressure is an emerging explanation for the establishment of invasive species ( lockwood et al . 2005 ) , it relies on an obvious relationship between the number and size of introduction events and the probability of success , as safety in numbers helps combat allee effects and stochastic extinction . in the event that propagule size is not large enough to overcome inverse density dependent effects , a population
traits that may be responsible for reproductive success and survival at small population densities and sizes include mate finding cues ( pheromones and vocal / visual signals ) , dispersal / aggregation behavior , habitat preferences , mating synchronicity , and gamete morphology and performance ( see courchamp et al .
direct evidence for the evolution of these traits as functional adaptations to allee effects is limited , but we can infer an adaptive evolutionary origin of these traits from studies of sexual selection ( courchamp et al . 2008 ; gascoigne et al .
an evolutionary response to intensive selection pressure imposed by density dependent survival and reproduction relies on genetic variants for adaptive evolution .
according to neutral quantitative genetic theory , a loss of genetic variation is expected from population bottlenecks and founder effects ( nei et al .
however it is not neutral variation that matters , but rather evolvability depends on the variation relevant to selection .
maintenance or even increases of this ( additive ) genetic variation has been theoretically and empirically observed following a bottleneck or in small founder populations ( bryant et al .
1986 ; goodnight 1988 ; willis and orr 1993 ; cheverud and routman 1996 ; briggs and goldman 2006 ; turelli and barton 2006 ) .
additionally , many recent studies suggest that there is actually no significant reduction in genetic diversity in most successful invaders ( lee 2002 ; allendorf and lundquist 2003 ; roman and darling 2007 and references therein ) , and that evolution can occur on contemporary timescales ( reznick and ghalambor 2001 ; carroll et al . 2007 ; kinnison and hairston 2007 ) .
our purpose here is to explore the feasibility of small populations that may adaptively respond to overcome allee effects in order to establish , given any amount of genetic variation . in this paper
, we present evidence for the enhanced potential for growth and spread of a small introduced population of organisms faced with allee effects when adaptation occurs .
we model the growth and spread of this population according to a reaction - diffusion equation , and allow evolution to influence inverse density dependent effects through a genetic subsystem that provides the opportunity for successful invasion when otherwise ( under the current , ecological paradigm ) the population would go extinct .
the deterministic model that we explore in this paper broadly describes population dynamics with density - mediated growth ( i.e. , an allee effect ) and diffusive dispersal .
this type of demographic model has been used as a compact and tractable representation of invasion ( e.g. , skellam 1951 ; lewis and kareiva 1993 ) .
specifically , it has been applied to systems such as introductions of nonindigenous freshwater and marine species through ballast water discharge ( drake et al .
2005 ; drury et al . 2007 ) . using this approach , drake et al .
( 2005 ) report acceptable volumes of discharge for various organisms ( with differing reproductive rates ) for a range of invasion risk tolerances . here
, we consider populations that are introduced below the invasion risk threshold , but nonetheless succeed if evolutionary dynamics are considered in conjunction with the ecological system .
the growth and spread of an introduced population of organisms is represented by a reaction - diffusion equation described by ( lewis and kareiva 1993 ) : which determines the rate of change in the local population density relative to the carrying capacity [ u which denotes u(x , t ) ] over time , at a point in space .
this equation models the growth of the population ( the first term on the right hand side of eqn .
1 ) at a spatial location that is subject to an allee effect in addition to migration ( which depends on the second term on the rhs of eqn .
1 ) . the diffusion coefficient ( d ) scales the rate of population spread , in this case across a one - dimensional habitat x. the reproductive rate is regulated by r , and a ( which is a function of space and time , derived below ) is the local critical density or allee threshold that determines if population growth is positive or negative ( fig .
growth dynamics of the model population ( a ) , and the solution of eqn .
( 1 ) without diffusion ( b ) with reproductive rate , r = 0.6 , and allee threshold , a = 0.3 .
trajectories for population size ( b ) are given for initial densities from 0 to 1 in increments of 0.05 .
there are many variations of single - species models of population dynamics that exhibit allee effects ( see table 1 of boukal and berec 2002 ) , however the growth function of eqn .
the behavior of this verhulst ( 1838 ) logistic model modified to include a nonlinear cubic term ( based on the fitzhugh - nagumo equations ; fitzhugh 1960 ; nagumo et al .
1962 ) , is bistable , with equilibria at u = 0 ( extinction ) , u = a ( unstable threshold ) , and u = 1 ( carrying capacity ) .
figure 1a shows this behavior in terms of the growth of the population ( change in population density with respect to time ) versus the population density . at densities below the critical threshold ( a )
there is negative population growth declining to extinction ( from here on the population is considered extinct below a cutoff density of 0.0001 , as a declining population trajectory will only asymptotically approach zero in a deterministic model ; gomulkiewicz and holt 1995 ) ; otherwise the population will reach carrying capacity .
1b with the graph of the solution of the growth function ( population size versus time ) at various initial densities . when diffusion is added to this model of population growth ,
there are two critical elements that emerge based on the solution to the partial differential equation ( pde ) .
the first is the wave speed , which is determined by the allee threshold ( a ) . as we are considering a strong allee effect in this model ( i.e. , 0 < a < 1 , where the population below this threshold exhibits negative growth versus reduced positive growth from a weak allee effect )
, there exists a unique wave speed of the invasion front that is a result of being
pushed from the inside out , as opposed to being pulled by the leading edge ( lewis and kareiva 1993 )
. this velocity can be derived through the solution of the pde ( 1 ) : ( lewis and kareiva 1993 ; murray 1993 ) .
this result suggests that in order for a wave to maintain a positive velocity of advance , the magnitude of the allee threshold ( a ) must be less than half of the maximum value of the population density relative to the carrying capacity .
in addition to the velocity of the wave front , the region occupied by the invading population must exceed a certain critical size for positive growth to occur ( skellam 1951 ; kierstead and slobodkin 1953 ) .
this phenomenon is clearly explained by okubo ( 1980 ) by noting that whereas reproduction takes place within a region or patch , diffusion takes place at the boundaries resulting in a loss of organisms , thus reducing the density within the patch .
this tradeoff in the ratio of inner region volume to outer surface area will either allow a population to grow or decline with an inverse relationship of diffusivity to rate of growth .
this relationship gives a minimum region within which reproduction can not compensate for loss due to diffusion , especially when allee effects influence population growth .
thus , lewis and kareiva ( 1993 ) derive a minimum size condition ( i.e. , the radius of the initial beachhead ) based on the wave speed that is required for the population to establish and radially expand .
we address this critical size threshold qualitatively , as the analytical solution ( i.e. , ; lewis and kareiva 1993 ) is for two - dimensional spread , while we work with a simpler one - dimensional model .
the minimum critical radius is proportional to the ratio of diffusivity ( i.e. , diffusion coefficient , d ) to the reproductive rate ( controlled by r ) .
the inclusion of diffusion in the model provides a spatially explicit understanding of how all of the components interact to affect invasion / establishment success .
the diffusion process has been extensively analyzed in invasion processes ( e.g. , fisher 1937 ; skellam 1951 ; okubo 1980 ) . in order to incorporate evolutionary factors that may influence invasion success ,
this genetic subsystem is coupled to the ecological model to explore the effects of selection and genetic variance on traits that may increase a population 's likelihood of survival .
specifically , we allow the allee threshold to become a dynamic parameter that is considered to be a fitness related trait ( e.g. , a trait impacting the component allee effect ) . from here on , except in the absence of evolution , referring to the allee threshold implies that that value is the initial value , as it changes over time .
this quantitative trait influences an organism 's ability to survive and reproduce in a small population .
the results reveal the possibility that an introduced population that would fail to persist in the ecological context of this model has the potential to succeed through evolutionary means . including evolution within the context of ecological invasions can serve to provide more robust predictions for management strategies .
therefore , it is important to investigate the possibility of evolution in the analysis of invasions .
the framework that is used to link evolutionary change with ecological processes involves developing a relationship between the fast , ecological and slow , evolutionary timescales in order to make these rates comparable ( kondrashov and khibnik 1996 ) . in the coupled evolutionary ecology model , the reaction - diffusion eqn .
( 1 ) describes the change in the population density over time and is tied into a genetic subsystem that allows the organismal response to population density to evolve in terms of the selection gradient and genetic variance .
as the population dynamics vary across space , the genetic subsystem describes the rate of change of the trait mean ( i.e. , the allee parameter ) at each location x by : ( pease et al .
1989 ; garca - ramos and kirkpatrick 1997 ; kirkpatrick and barton 1997 ; hare et al .
the first term on the right hand side reflects the force of local directional selection , where the selection gradient for frequency - independent selection is the rate of change of the mean malthusian fitness function ( i.e. , per - capita growth rate : ) with respect to the trait , a ( lande 1976 ; falconer 1989 ) .
thus , , where we assume that individual fitness approaches the population mean fitness , as most individuals are close to the average phenotype ( webb 2003 ) .
this suggests that the genetic variance ( ) is small ( and constant in this model ) .
this small parameter for the genetic variance can be used to couple the fast ecological timescale , t , with the slow evolutionary timescale , = t ( kondrashov and khibnik 1996 ; webb 2003 ) . combing these two components of genetic variance and selection ,
quantifies the effect of natural selection on the local mean value of the quantitative trait ( the allee parameter ; lande 1976 ; falconer 1989 ) . in order to account for the influence of migration on the trait 's local mean , the latter two terms in eqn .
the middle term takes into account asymmetrical gene flow caused by the variation of density across space ( pease et al .
1989 ; garca - ramos and kirkpatrick 1997 ; kirkpatrick and barton 1997 ; hare et al .
this captures the influence of the mean trait value ( i.e. , genetic contribution ) from more abundant populations to less abundant neighboring locations due to the spatial gradient , as more individuals migrate from areas with relatively high population densities .
the last term mirrors the diffusion term from the ecological model , and describes the homogenizing effect of random dispersal .
we solved the spatially explicit system numerically using matlab 7.0 ( 2004 , the mathworks , natick , ma , usa ) using a finite difference method to incorporate diffusion and gene flow ( adapted from garvie 2007 ) . by iterating eqns ( 1 ) and ( 2 ) forward in time
, the population density and allee threshold at each location are updated with diffusion following growth and selection , respectively , while incorporating the spatial gradient .
the simulated populations , with and without evolution , behaved as we expected from the model eqns ( 1 ) and ( 2 ) , and adequately approximate / represent the critical conditions that govern this dynamical system .
the growth and spread of an introduced population of organisms is represented by a reaction - diffusion equation described by ( lewis and kareiva 1993 ) : which determines the rate of change in the local population density relative to the carrying capacity [ u which denotes u(x , t ) ] over time , at a point in space .
this equation models the growth of the population ( the first term on the right hand side of eqn .
1 ) at a spatial location that is subject to an allee effect in addition to migration ( which depends on the second term on the rhs of eqn .
1 ) . the diffusion coefficient ( d ) scales the rate of population spread , in this case across a one - dimensional habitat x. the reproductive rate is regulated by r , and a ( which is a function of space and time , derived below ) is the local critical density or allee threshold that determines if population growth is positive or negative ( fig .
growth dynamics of the model population ( a ) , and the solution of eqn .
( 1 ) without diffusion ( b ) with reproductive rate , r = 0.6 , and allee threshold , a = 0.3 .
trajectories for population size ( b ) are given for initial densities from 0 to 1 in increments of 0.05 .
there are many variations of single - species models of population dynamics that exhibit allee effects ( see table 1 of boukal and berec 2002 ) , however the growth function of eqn .
the behavior of this verhulst ( 1838 ) logistic model modified to include a nonlinear cubic term ( based on the fitzhugh - nagumo equations ; fitzhugh 1960 ; nagumo et al .
1962 ) , is bistable , with equilibria at u = 0 ( extinction ) , u = a ( unstable threshold ) , and u = 1 ( carrying capacity ) .
figure 1a shows this behavior in terms of the growth of the population ( change in population density with respect to time ) versus the population density . at densities below the critical threshold ( a )
there is negative population growth declining to extinction ( from here on the population is considered extinct below a cutoff density of 0.0001 , as a declining population trajectory will only asymptotically approach zero in a deterministic model ; gomulkiewicz and holt 1995 ) ; otherwise the population will reach carrying capacity .
1b with the graph of the solution of the growth function ( population size versus time ) at various initial densities . when diffusion is added to this model of population growth ,
there are two critical elements that emerge based on the solution to the partial differential equation ( pde ) .
the first is the wave speed , which is determined by the allee threshold ( a ) . as we are considering a strong allee effect in this model ( i.e. , 0 < a < 1 , where the population below this threshold exhibits negative growth
versus reduced positive growth from a weak allee effect ) , there exists a unique wave speed of the invasion front that is a result of being
pushed from the inside out , as opposed to being pulled by the leading edge ( lewis and kareiva 1993 )
. this velocity can be derived through the solution of the pde ( 1 ) : ( lewis and kareiva 1993 ; murray 1993 ) .
this result suggests that in order for a wave to maintain a positive velocity of advance , the magnitude of the allee threshold ( a ) must be less than half of the maximum value of the population density relative to the carrying capacity .
in addition to the velocity of the wave front , the region occupied by the invading population must exceed a certain critical size for positive growth to occur ( skellam 1951 ; kierstead and slobodkin 1953 ) .
this phenomenon is clearly explained by okubo ( 1980 ) by noting that whereas reproduction takes place within a region or patch , diffusion takes place at the boundaries resulting in a loss of organisms , thus reducing the density within the patch .
this tradeoff in the ratio of inner region volume to outer surface area will either allow a population to grow or decline with an inverse relationship of diffusivity to rate of growth .
this relationship gives a minimum region within which reproduction can not compensate for loss due to diffusion , especially when allee effects influence population growth .
thus , lewis and kareiva ( 1993 ) derive a minimum size condition ( i.e. , the radius of the initial beachhead ) based on the wave speed that is required for the population to establish and radially expand .
we address this critical size threshold qualitatively , as the analytical solution ( i.e. , ; lewis and kareiva 1993 ) is for two - dimensional spread , while we work with a simpler one - dimensional model .
the minimum critical radius is proportional to the ratio of diffusivity ( i.e. , diffusion coefficient , d ) to the reproductive rate ( controlled by r ) .
the inclusion of diffusion in the model provides a spatially explicit understanding of how all of the components interact to affect invasion / establishment success .
the diffusion process has been extensively analyzed in invasion processes ( e.g. , fisher 1937 ; skellam 1951 ; okubo 1980 ) .
in order to incorporate evolutionary factors that may influence invasion success , we develop a quantitative genetic subsystem .
this genetic subsystem is coupled to the ecological model to explore the effects of selection and genetic variance on traits that may increase a population 's likelihood of survival .
specifically , we allow the allee threshold to become a dynamic parameter that is considered to be a fitness related trait ( e.g. , a trait impacting the component allee effect ) . from here on , except in the absence of evolution , referring to the allee threshold implies that that value is the initial value , as it changes over time .
this quantitative trait influences an organism 's ability to survive and reproduce in a small population .
the results reveal the possibility that an introduced population that would fail to persist in the ecological context of this model has the potential to succeed through evolutionary means . including evolution within the context of ecological invasions can serve to provide more robust predictions for management strategies .
therefore , it is important to investigate the possibility of evolution in the analysis of invasions .
the framework that is used to link evolutionary change with ecological processes involves developing a relationship between the fast , ecological and slow , evolutionary timescales in order to make these rates comparable ( kondrashov and khibnik 1996 ) . in the coupled evolutionary ecology model , the reaction - diffusion eqn .
( 1 ) describes the change in the population density over time and is tied into a genetic subsystem that allows the organismal response to population density to evolve in terms of the selection gradient and genetic variance . as the population dynamics vary across space , the genetic subsystem describes the rate of change of the trait mean ( i.e. , the allee parameter ) at each location x by : ( pease et al .
1989 ; garca - ramos and kirkpatrick 1997 ; kirkpatrick and barton 1997 ; hare et al .
the first term on the right hand side reflects the force of local directional selection , where the selection gradient for frequency - independent selection is the rate of change of the mean malthusian fitness function ( i.e. , per - capita growth rate : ) with respect to the trait , a ( lande 1976 ; falconer 1989 ) .
thus , , where we assume that individual fitness approaches the population mean fitness , as most individuals are close to the average phenotype ( webb 2003 ) .
this suggests that the genetic variance ( ) is small ( and constant in this model ) .
this small parameter for the genetic variance can be used to couple the fast ecological timescale , t , with the slow evolutionary timescale , = t ( kondrashov and khibnik 1996 ; webb 2003 ) . combing these two components of genetic variance and selection ,
quantifies the effect of natural selection on the local mean value of the quantitative trait ( the allee parameter ; lande 1976 ; falconer 1989 ) . in order to account for the influence of migration on the trait 's local mean , the latter two terms in eqn .
the middle term takes into account asymmetrical gene flow caused by the variation of density across space ( pease et al .
1989 ; garca - ramos and kirkpatrick 1997 ; kirkpatrick and barton 1997 ; hare et al .
this captures the influence of the mean trait value ( i.e. , genetic contribution ) from more abundant populations to less abundant neighboring locations due to the spatial gradient , as more individuals migrate from areas with relatively high population densities .
the last term mirrors the diffusion term from the ecological model , and describes the homogenizing effect of random dispersal .
we solved the spatially explicit system numerically using matlab 7.0 ( 2004 , the mathworks , natick , ma , usa ) using a finite difference method to incorporate diffusion and gene flow ( adapted from garvie 2007 ) . by iterating eqns ( 1 ) and ( 2 ) forward in time
, the population density and allee threshold at each location are updated with diffusion following growth and selection , respectively , while incorporating the spatial gradient .
the simulated populations , with and without evolution , behaved as we expected from the model eqns ( 1 ) and ( 2 ) , and adequately approximate / represent the critical conditions that govern this dynamical system .
the dynamics of the evolutionary ecology model can be interpreted using the idea of fast and slow timescales ( kondrashov and khibnik 1996 ; webb 2003 ) .
earlier , we assumed that the genetic variation ( ) was small ( to use mean fitness as a proxy for individual fitness ) , which can subsequently be taken advantage of for our analysis of the coupled evolutionary ecological dynamics . when = 0 , the situation without evolution , the genetic subsystem is frozen and the population moves towards a stable equilibrium of the ecological subsystem ( carrying capacity or extinction ) depending on its initial density ( greater than or less than the allee threshold respectively ; fig .
when > 0 but small , the allee threshold evolves relatively slowly and influences the ecological system . whenever the population is below its carrying capacity ( u = 1 for each spatial coordinate x when space is explicit ) , eqn .
( 2 ) is negative , and decreases the mean allee threshold ( a ) , as the intensity of selection is density dependent .
thus , fitness increases as allee effects are suppressed , and selection drives the allee threshold towards zero .
if the population density is greater than the allee threshold , but still below the carrying capacity , it will progress towards carrying capacity more rapidly than it would without evolution as a decreases ; as the rate at which the population density changes ( eqn .
1 ) is proportional to the difference between u and a. the ecological dynamics are reversed when the population density is below the allee threshold as the population declines towards extinction , but more slowly than it does without evolution . when u < a , eqn .
( 1 ) is negative , and the population density approaches extinction more rapidly with a constant ( as the difference between u and a increases ) , than it does with evolution as a decreases ( revealing a more pronounced time lag to extinction ) . during this time lag ,
as the population slowly declines , the opportunity for evolution to overcome inverse density dependent effects occurs .
if the rate of evolution is fast enough , the allee threshold can fall below the population density , causing the rate of change of population density to become positive ( where u > a ) and the population grows and can successfully invade .
the chance that evolution can rescue the population from extinction depends on the relative rates of genetic change in the quantitative trait ( i.e. , allee threshold ) and of population decline ( gomulkiewicz and holt 1995 ) ; hence the amount of genetic variance greatly impacts the ability to adapt and survive .
a nonspatial example of this process , referred to as evolutionary rescue ( gomulkiewicz and holt 1995 ) , is shown in fig .
2 , where a population is introduced below the allee threshold . without evolution , the population declines to extinction ( fig . 2a , solid line ) as the allee threshold remains constant ( fig . 2b , solid line ) . when the population can evolve ( fig .
2 , dotted line ) , it declines at first until it can overcome the magnitude of inverse density dependence , and is then able to successfully establish . as it is difficult to measure the allee effect empirically ( tobin et al .
2007 ) , we use an extreme value that exaggerates density dependent effects in order to investigate the worst case scenario ( a = 0.3 , where the population exhibits deterministic decline when its density is less than 30% of its carrying capacity ) . when evolution is included , we used a small value for the genetic variance , = 0.02 , in order to remain consistent with fast - slow dynamics , unless otherwise indicated .
comparison of an invading population introduced at a density below the allee threshold , a = 0.3 ( u = 0.25 , r = 0.6 ) .
the solid line represents the nonspatial system ( d = 0 ) described by eqns ( 1 ) and ( 2 ) without evolution ( = 0 ) which results in extinction ( a ) and a constant allee threshold ( b ) .
the dotted line indicates population growth ( a ) when evolution ( = 0.02 ) acts to reduce the allee threshold ( b ) . in general , there is a range of parameter space that permits persistence for a population below the allee threshold in the nonspatial model with evolution ( instead of simple decline to extinction ) .
as genetic variance increases , we are essentially relaxing the assumption of fast - slow timescales and allow evolution to occur more rapidly .
3 where initial population densities below the allee threshold require a minimum amount of genetic variance in order to avoid extinction . in this case
, the rate of reproduction , r , also influences the potential for evolutionary rescue , as it impacts both population growth and rate of evolution ( eqns 1 and 2 , respectively ) .
as we relax the assumption of fast - slow timescales , the behavior remains qualitatively the same as that described analytically under a strict fast - slow timescale assumption .
parameter combinations of reproductive rate : r ; genetic variance : , and initial population density : u , that result in extinction or evolutionary rescue . in this nonspatial scenario ,
initial population densities greater than the allee threshold ( a = 0.3 ) always succeed , thus the focus is on the parameter space that allows for evolutionary rescue ( i.e. , where the population growth changes from negative to positive ) . as the reproductive rate increases from 0.1 to 1
, there is less genetic variance needed for a population to evolve to overcome inverse density dependence as increased reproduction will contribute to suppressing allee effects .
nonetheless , the additional complexities result in qualitatively similar behavior to the nonspatial model . in this case , not only will evolution influence population growth , it affects the wave speed and the critical size threshold , rmin .
as the population overcomes allee effects with a decreasing allee threshold , the wave speed accelerates and the critical patch size becomes smaller .
thus , in addition to the initial density of the introduced population and the genetic variance , the initial radius or patch size of the initial invasion area , the ratio of diffusion to reproduction , and gene flow will factor into successful establishment and give rise to a wider range of interactions between the ecology and evolution of this system .
( 1 ) ( without the evolutionary subsystem ) in one - dimensional space , with an initial population density below the allee threshold , declines to extinction ( fig .
this is contrasted by the results when the evolutionary subsystem is included . with the initial population density below the allee threshold , fig .
the same type of rescue occurs for a population that starts near carrying capacity , but occupies an initial spatial size below that which is necessary for a population to successfully establish .
figure 5a shows a rapidly declining population that goes extinct . under the same circumstance , but where evolution of the allee threshold occurs , fig .
5b shows the population density at first beginning to shrink and then growing and expanding .
in addition to the time evolution of population density across space in figs 4 and 5 , the evolution of the mean trait value across space illustrates how gene flow and the density dependent selection gradient influences its rate of change and distribution ( figs 4c and 5c ) . as the intensity of selection is density dependent ( and we assume constant genetic variance ) , locations with smaller populations can evolve the trait value more rapidly compared to other areas where allee effects may not be as strong and experience weaker selection .
the trait distribution over time , figs 4c and 5c , therefore reflect the population density distribution , but are also influenced by the trait values of the migrants . as individuals disperse out to new locations and push the boundaries of the species range , their trait values are averaged to determine the demographic allee threshold for that spatial coordinate . this demographic allee threshold combines with their local population density to influence individual fitness and population growth ( where the distance between the density and mean trait value is the initial degree of maladaptation ) .
diffusive dispersal of an introduced population at an initial density ( bold dashed line ) below the initial allee threshold , a = 0.3 ( u = 0.25 , r = 1 , d = 0.1 ) across a linear , one dimensional habitat .
the population collapses over time to extinction ( a ) where there is no evolution ( = 0 ) , and succeeds ( b ) after an initial decline with evolution ( = 0.02 ) .
( c ) the evolution of the mean value of the allee threshold across space ( where the initial distribution is given by the bold dashed line ) . the population density distribution and corresponding trait values ( i.e. , allee threshold ) are plotted at equal time increments ( every 20 of 1200 model iterations ) .
population density of a diffusion dispersed population across one dimensional space . the initial population density ( bold dashed line )
is near carrying capacity ( u = 0.95 , a = 0.3 , r = 1 , d = 0.5 ) , but introduced below the minimum radius of area determined to be critical for invasion success .
( a ) is collapsing to extinction without evolution ( = 0 ) , whereas ( b ) shows success of an invader with evolution ( = 0.02 ) after initial decline .
( c ) the evolution of the mean value of the allee threshold across space ( where the initial distribution is given by the bold dashed line ) .
the population density distribution and corresponding trait values ( i.e. , allee threshold ) are plotted at equal time increments ( every 20 of 1200 model iterations ) .
we explored when evolutionary rescue occurred across a range of parameter values for the spatially explicit model . according to drake et al .
( 2005 ) , variability among locations and over time makes it unreasonable to determine precise estimates for the diffusion coefficient , d. we therefore explored a range of values , and present those that best illustrate breadth of behavior .
the parameter that controls the reproductive rate , r , was also varied substantially , but as the spatial dynamics depend on the ratio of diffusion to rate of reproduction ( resulting in a measure of length ) ; we fixed r and varied d , unless otherwise noted .
this was justified as the results of the spatial simulations are qualitatively identical for equivalent ratios .
the effects of the critical patch size , initial population density , ratio of diffusion to growth and genetic variation on evolutionary rescue and population dynamics are shown in fig .
the ratio of the diffusion coefficient ( d ) to the reproductive rate ( r ) determines whether the population will expand or collapse according to the initial radius of the introduced population .
the areas under the curves denote combinations of genetic variance and initial population density that result in extinction .
areas above the curves are combinations of genetic and/or demographic conditions that produce inevitable persistence .
the parameter space between the vertical dashed lines refers to the different ways population survival is influenced . to the left of the initial allee threshold
, the initial population density will either go extinct due to density dependent effects ( below the d / r curve ) , or given enough genetic variation , will be evolutionarily rescued ( above the d / r curve ) .
the area to the right of the initial allee threshold [ and between the dashed lines in ( b ) and ( c ) ] is the case where the initial population density is greater than the allee threshold but due to the initial spatial size and the ratio of diffusion to reproduction , the population may go extinct without sufficient genetic variance ( below the d / r curve ) , otherwise it will evolve to overcome the critical patch size effect . for initial population densities
thus , the area between the dashed lines in ( b ) and ( c ) truly delineates evolutionary rescue when d / r = 1 .
the rightmost vertical line moves slightly to the left to the point of intersection of the d / r curve and the x - axis for other values of d / r .
graphs ( a ) , ( b ) , and ( c ) represent different radii of the linear habitat that the introduced population initially occupies .
when the size of the initial population is too small ( i.e. , a radius of 1 ) , a population at carrying capacity ( i.e. , u = 1 ) will go extinct without evolution due to the relative effect of diffusion to reproduction ( fig .
if evolution occurs rapidly enough ( i.e. , > 0.02 ) , the population can overcome inverse density dependent effects and compensate for the loss due to diffusion and rebound from low densities . when the initial radius of the population is increased ( fig .
6b , c ) , the chance of survival and establishment ( growth and expansion ) of populations above or below the allee threshold increases with initial density and genetic variance .
therefore , the initial radius of the population can significantly impact the likelihood of evolutionary rescue for populations with the same amount of genetic variance .
this is demonstrated further in fig . 7a , where the rate of recovery ( i.e. , the inverse of the time lag before growth becomes positive and the population reaches carrying capacity ) for a population near carrying capacity depends on its initial size / radius and genetic variance . where size and variance are small
, rescue never occurs . as these parameters increase , the rate of recovery gradually becomes faster until it essentially plateaus ( although with greater variance and initial radius , the rate of recovery may slow slightly if the initial spatial extent is large enough for the population to experience early growth before diffusion causes decline prior to recovery ) .
if the population occupies a large enough spatial extent , it will succeed without evolution ( where the genetic variance is zero ) , however the lag time may be more pronounced depending on the ratio of diffusion to reproduction through the tradeoff between growth and spread ( e.g. , if spread is relatively fast compared to reproduction , d / r = 1 ) . the population density may thus initially decline across space until reproduction can sufficiently overcome the loss due to diffusion , and the population can grow to carrying capacity .
similar to the nonspatial case , a population ( greater than the allee threshold ) above the spatial threshold will grow to carrying capacity more rapidly with evolution than without .
rate of recovery in terms of the inverse of the time lag before population growth becomes positive , where one timestep equals 24 iterations of the model . in ( a )
, the initial population density is near carrying capacity ( u = 0.95 , a = 0.3 , d / r = 1 ) , and the initial radius and genetic variance , , varies .
where the rate of recovery is zero , the population goes extinct as it initially occupies an area smaller than the critical patch size ( in this case , a radius of 1.4 ) or does not have sufficient genetic variance to evolve quickly enough to be rescued prior to extinction . increasing the genetic variance and initial radius will decrease this time lag until the population no longer experiences any negative growth ( in this case , for initial radii 3.8 and 0.036 ; for initial radii > 2.7 , the rate of recovery slows slightly due to early growth followed by a transient decline that precedes ultimate recovery ) .
when the initial population density varies ( indicating the initial degree of maladaptation where a = 0.3 ) , ( b ) shows the rate of recovery with the initial radius fixed ( as in fig .
5a where the radius = 1 and d / r = 1 ) . in this case
, extinction will occur without evolution not only for an initial density below the initial allee threshold , but for any density as the initial radius is below the critical patch size .
hence , a nonzero rate implies evolutionary rescue and a zero rate means extinction . as shown in fig .
7b , when evolutionary rescue is possible , the initial level of maladaptation ( a0u0 ) and the genetic variance ( ) also determine the rate at which evolutionary rescue proceeds .
figure 7b uses parameters ( i.e. , radius and ratio of d to r ) for a population that would decline and go extinct without evolution regardless of the initial density .
hence , it is clear that the amount of time required for a population to begin growing depends on its initial level of maladaptation ( to both the critical density and spatial thresholds ) and/or genetic variance . as the rate at which this rescue occurs depends on the amount of genetic variance ( eqn .
2 ) , it may take an extremely long time ( as 0 , the rate of recovery 0 ) for the allee threshold to fall below the population density . in this circumstance , as the population density becomes very close to zero , the rate of change of the allee threshold is greater than that of the population density ( as u 0 , u/t 0 and a/t -2ra )
. thus , theoretically , rescue would always occur ( gomulkiewicz and holt 1995 ) . however , to maintain biological realism , when solving this system numerically , we always considered the population extinct when the maximum density ( across space , when diffusion is included ) becomes reasonably close to zero ( i.e. , u = 0.0001 ; we chose this protocol instead of the total population across space due to the diffusion dynamics based on the gaussian dispersal kernel and the pushed wave front behavior ) .
overall , the numerical results qualitatively hold for a wide range of dimensional parameter values and initial conditions with and without diffusion and in one- and two - dimensional space .
results for two - dimensional space are not shown as they are qualitatively similar to the simpler , one dimensional model .
from these results , it is apparent that adaptations that enable organisms to overcome the negative effects of low densities can allow the population to rebound from a trajectory toward extinction to grow to reach carrying capacity .
current management strategies ( e.g. , reducing population density or size ) are based on ecological theory ( e.g. drake et al . 2005 ) , but this evolutionary ecology model suggests that adaptive evolution can enable successful establishment and that ecological considerations alone may not be sufficient . under the assumptions of an allee effect and diffusive dispersal , the idea of ecological size thresholds fits well with the ecological evidence that a large founding population is a primary cause of successful establishment ( lockwood et al .
however , by incorporating evolution , we see that the situation is not quite this simple because ecological size thresholds and genetic variance can interact to determine successful establishment . as
the ratio of diffusion to reproduction decreases , the spatial constraint on population growth becomes weaker , and less genetic variance is needed to rescue populations with densities below the allee threshold . as the initial spatial radius of introduction increases ,
furthermore , the rate of this rescue depends on the initial genetic load or maladaptation ( i.e. , how far the population density is from the allee threshold ) , as well as the amount of genetic variance .
because bottlenecks during founding events do not always result in highly reduced genetic variability , even small founding populations may have sufficient genetic variation to evolve to overcome allee effects and establish , contrary to solely ecologically based predictions .
additionally , we can draw several general insights about how dispersal impacts selection and evolution of allee effects in an invasion context .
as species are transported from their native environment into novel habitats or simply disperse on their own , it is clear that the genetic composition of the local population can influence the rate of evolution and adaptation to the new local conditions . given enough genetic diversity , local populations can adapt to their local environment , but dispersal may hinder survival across ecological clines as dispersers tend to be maladapted to the new local environment . essentially , local population persistence depends on the race between the rate of evolution and the degree of maladaptation ( gomulkiewicz and holt 1995 ) . in this case
, gene flow will play a major role in determining the outcome . as kirkpatrick and barton ( 1997 ) and garca - ramos and kirkpatrick ( 1997 )
demonstrate , individuals moving from one selection regime from the center of their species range to the periphery can introduce enough maladaptation that the new area becomes a sink environment . on the other hand , holt et al .
( 2003 , 2004 ) show that immigration can have a positive influence on adaptation to sink environments , in some circumstances .
resolving the disparity between these perspectives requires understanding what is contributing to the severity of maladaptation and the population 's ability to overcome it . in our model ,
dispersal impacts survival ecologically through the critical patch size , and genetically , as individuals may move from areas where they are well adapted ( i.e. , the population density is greater than the allee threshold or mean trait value ) to sink regions , where they are maladapted .
as individuals disperse across space , they may be contributing positively in an ecological sense to the quality of their new local environment ( by increasing the local population density ) .
however , dispersers are more likely to come from higher density areas where allee effects , and hence selection , are locally weak .
these dispersers potentially introduce more maladaptation to their new location , because they increase the average phenotype ( allee threshold ) in the new location where density is likely to be lower .
interestingly , the evolutionary impacts of migration in this model do not dramatically influence the dynamics .
changes to the local mean phenotype through local selection and simple mixing ( i.e. , diffusion ) actually slightly hastens the evolutionary rescue effect over a model that considers only the impact of local selection .
as the selection intensity is density dependent and proportional to u 1 for each point in space , the peripheral individuals faced with stronger selection with lower trait values have a small positive influence on the more dense neighboring populations .
the gradient term accounts for asymmetric gene flow due to differential migration from areas of relatively high population densities .
however , this term does not alter the overall evolutionary dynamics based on local selection any more than adding the diffusion term , as the negative effects of gene flow and the local rate of evolution ( which is relatively fast , based on the selection intensity ) essentially cancel each other out . in this context ,
similar to that of holt et al . ( 2003 , 2004 ) , the immigrants simply contribute to the local population density , which helps prevent extinction long enough for evolutionary rescue to occur locally ( i.e. , positive population growth ; note that whereas holt et al .
( 2003 ) attribute the main effect of immigration to the contribution of variation , this is not the case in our model , as we assume constant genetic variance ) .
( 2004 ) where immigration has a demographic effect on increasing fitness that can essentially outweigh the
the primary determinant of invasion success depends on positive population growth at the center of the introduced range .
this result comes from the allee effect [ and the solution to the pde ( 1 ) ] by forcing a pushed travelling wave front ( lewis and kareiva 1993 ) , where the wave speed causes population expansion , contraction , or propagation failure ( i.e. , pinning ; keitt et al
. 2001 ) . intuitively , aggregation - like behavior emerges based on the strength of the allee effects .
individuals that disperse too far from the whole are likely to die before they can pull others in their vicinity . in this
regard , growth occurs from the inside out , where the population seemingly spills out and overflows to expand its range .
consequently , in this study , and for biological invasions that exhibit similar dynamics , it is more important to focus on the center of the invader 's range and whether the initial beachhead can survive ( through evolutionary rescue ) , than the fate of peripheral populations at the wave front when determining the importance of evolution on invasion success .
the overall dynamics are qualitatively similar in the parameter space that allows for evolutionary rescue to occur .
even though gene flow and spatial structure do not dramatically influence the establishment of an introduced population , additional invasion criteria need to be considered .
when analyzing the model behavior in a spatially explicit context , there is an ecological tradeoff between growth and spread that affects establishment and the rate of recovery . as previously mentioned , reproduction needs to compensate for the loss due to diffusion . including evolution and suppressing allee effects
, actually contributes to the acceleration of the wave front ( i.e. , enhancing dispersal speed ) . a population then can more rapidly disperse as it evolves , and may become more of an invasion threat as long as this range expansion does not reduce their density too quickly . whereas this increasing wave speed can lead to a slightly longer lag phase prior to positive population growth , the population will likely be inevitably rescued because this effect primarily influences the dynamics at the periphery and is offset by the reduction in the critical invasion area ( rmin ) .
although there is no range contraction ( as there is always a positive wave velocity with unbounded expansion due to the parameter values and absence of environmental heterogeneity or range limitations ; filin et al .
2008 ) , as the critical patch size ( rmin ) becomes smaller with the decreasing allee threshold , rescue occurs more readily at the range center as the critical patch size threshold criteria weakens and the behavior approaches that of the nonspatial model .
this may seem like an oversimplification of the global dynamics ; however these conclusions are valid in the context of this investigation which focuses on the establishment phase rather than subsequent range expansion and spread . recognizing that evolution can significantly affect the establishment success of invasive species is becoming more widely accepted , influencing the ways in which invasion biologists conduct their research ( see the other articles in this issue ) .
specifically , adaptations that diminish allee effects and evolutionary responses to density dependence are beginning to emerge as viable explanations for sustaining vulnerable populations at low density and size ( gascoigne et al .
as it is difficult to conclusively support this claim empirically ( as the origin of the adaptation or the associated cost may be unknown ; courchamp et al . 2008 ; gascoigne et al .
2009 ) , mathematical models that incorporate evolution and compare the effects of various strategies ( e.g. , mitigating component allee effects ) can help decipher the mechanisms that both limit and facilitate population growth .
two such models that incorporate adaptations to component mate - finding allee effects compare the efficiency and survival of populations at various densities that attract mates with or without a sexual pheromone ( jonsson et al .
another study suggests that broadcast spawners that evolve their gamete morphology and performance under sperm limitation ( at low density ) bear a cost of decreased fitness at high density due to hybridization and competition ( levitan 2002 ) .
in these cases , particular strategies are shown to influence population viability in addition to an associated tradeoff , whereas our investigation provides broad , albeit simplistic , results dealing with generalized demographic allee effects and evolution . in order to understand how the results of this simplistic model extend to more realistic and complex evolutionary scenarios , spatially explicit
, individually - based stochastic simulation of the introduced populations should be developed to investigate more closely the mechanisms that allow these population level dynamics to emerge .
in particular , tracking the mean value of a component allee effect is sufficient to illustrate how evolution can overcome inverse density dependence and result in invasion .
however , this approach may not be sufficient to make the specific quantitative predictions necessary for management of invasive species .
this is due to the simplifying assumption of constant genetic variance based on mutation - selection balance ( lande 1976 ) .
complex simulations could relax this assumption and permit genetic variation to change via mutation , selection , and drift , in tandem with the demographic processes in a heterogeneous environment , and explicitly investigate the costs associated with avoiding allee effects .
hence , future models should incorporate how propagule pressure ( size and frequency of introduction events ) impacts genetic variation and how more realistic genetic architectures contribute to the evolutionary trajectory of invasive species .
although there is still much more work to be done to elucidate the factors that determine establishment success of founder populations , this theoretical approach has the promise to provide evidence in support of our working hypothesis that adaptive evolution can mitigate allee effects and be an important driver of biological invasions .
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the mechanisms that facilitate success of an invasive species include both ecological and evolutionary processes . investigating the evolutionary dynamics of founder populations can enhance our understanding of patterns of invasiveness and provide insight into management strategies for controlling further establishment of introduced populations .
our aim is to analyze the evolutionary consequences of ecological processes ( i.e. , propagule pressure and threshold density effects ) that impact successful colonization .
we address our questions using a spatially - explicit modeling approach that incorporates dispersal , density dependent population growth , and selection .
our results show that adaptive evolution may occur in small or sparse populations , providing a means of mitigating or avoiding inverse density dependent effects ( i.e. , allee effects ) .
the rate at which this adaptation occurs is proportional to the amount of genetic variance and is a crucial component in assessing whether natural selection can rescue a population from extinction .
we provide theoretical evidence for the importance of recognizing evolution in predicting and explaining successful biological invasions .
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Introduction
Model description
The ecological system
The evolutionary subsystem
Results
Discussion
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inspection of the database of clusters of orthologous groups of proteins ( cogs ) revealed only one family of such proteins that is represented in most of the sequenced bacterial , archaeal and eukaryotic genomes .
the prototype of this family is the rhomboid ( rho ) protein from drosophila melanogaster , a developmental regulator involved in epidermal growth factor ( egf)-dependent signaling pathways [ 2 - 4 ] .
not only were homologs of rhomboid detected in prokaryotes and eukaryotes , but the pattern of sequence conservation in this family appeared uncharacteristic of nonenzymatic membrane proteins , such as transporters .
specifically , several polar amino - acid residues are conserved in nearly all members of the rhomboid family , suggesting the possibility of an enzymatic activity . as three of these conserved residues were histidines , it has been hypothesized that rhomboid - family proteins could function as metal - dependent membrane proteases .
recently , however , it has been shown that rho cleaves a transmembrane helix ( tmh ) in the membrane - bound precursor of the tgf-like growth factor spitz , enabling the released spitz to activate the egf receptor , and that a conserved serine and a conserved histidine in rho are essential for this cleavage .
thus , it appears that rhomboid - family proteins are a distinct group of intramembrane serine proteases .
altogether , the genome of drosophila encodes seven rho paralogs ( now designated rho1 - 7 , with the original rhomboid becoming rho-1 ) , at least three of which are involved in distinct egf - dependent pathways , apparently through proteolytic activation of diverse ligands of the egf receptor .
the newly discovered intramembrane proteolytic activity of rho places the rhomboid family within the framework of regulated intramembrane proteolysis ( rip ) , a new paradigm of signal transduction , which appears to be prominent in all forms of life . under rip , signaling proteins undergo site - specific proteolysis within tmh , resulting in the release of active fragments , which are the actual effectors in signal tranduction cascades . until recently
, the only characterized cases of rip in eukaryotes involved presenilin-1 , an aspartyl protease , which cleaves a transmembrane helix in type-1 membrane proteins such as amyloid -precursor protein ( app ) , notch and ire1 , and the metalloprotease s2p , which cleaves a tmh in a type-2 transmembrane protein , the sterol - dependent transcription factor srebp .
notably , s2p has highly conserved bacterial homologs , and the protease domain of presenilins also might be homologous to bacterial and archaeal type iv prepilin peptidases , although , in this case , the sequence similarity is low . in the case of the rhomboid family ,
the existence of homologs of rho in most prokaryotes is particularly remarkable because animal rho proteins are involved in signaling pathways that are not found outside metazoa , which seems to make functional conservation in prokaryotes a remote possibility .
the only prokaryotic protein of the rhomboid family that has been characterized experimentally in considerable detail is aara from the bacterium providencia stuartii .
this protein is involved in the export of a quorum - sensing peptide , a function that , in physiological terms , resembles that of rho , although the signaling molecules , other than rho and aara , are obviously unrelated . in a striking recent development
, two independent research groups have shown that several bacterial rhomboid - family proteins , including aara , can cleave the egf receptor ligands ( spitz , keren and gurken ) that are normally cleaved by rho paralogs .
the cleavage depended on the conserved serine and histidine residues and , moreover , transgenic flies that expressed aara developed a phenotype indistinguishable from that induced by overexpression of rho , whereas rho could substitute for aara in providencia stuartii .
these unexpected findings demonstrated the conservation of a rip mechanism producing extracellular signals in eukaryotes and prokaryotes .
eukaryotic rhomboid family proteins seem to show considerable functional variability ; in particular , cross - talk might exist between different rip pathways . a distinct representative of the rhomboid family has been shown to physically interact with presinilins 1 and 2 , and was accordingly named presenilins - associated rhomboid - like protein ( parl ) .
the yeast ortholog of parl has been suggested to participate in the processing of cytochrome c peroxidase precursor during its import into the mitochondrion .
the near ubiquity of the rhomboid family among bacteria , archaea and eukaryotes , along with the remarkable functional conservation , suggests that a signaling mechanism mediated by rhomboids might have functioned already in the last common ancestor of all extant life forms , with subsequent loss in several lineages . to address this possibility
although the sequence similarity between eukaryotic and prokaryotic rhomboid family proteins is relatively low ( around 10 - 15% identity in the conserved region ) , the entire superfamily could be retrieved from the protein sequence databases within three iterations of the psi - blast program with a high statistical significance and without any false positives .
the conserved core of the rhomboid family consists of six conserved tmhs ( figure 1 ) .
the predicted catalytic serine is located in tmh5 , whereas the predicted catalytic histidine is in tmh7 ; tmh3 contains two additional histidines and an asparagine , which are conserved in the great majority of the rhomboid - family proteins ( figure 1 ) .
the roles of these conserved residues are not known , but , given the remarkable evolutionary conservation , it seems likely that they also contribute to catalysis ; indeed , it has been shown that the conserved asparagine is required for the cleavage of spitz by rho .
when examining the multiple alignment of the rhomboid superfamily proteins , we noticed that several eukaryotic members appear to be inactivated proteases , as indicated by the loss of the predicted catalytic serine or histidine ( figure 1 , and data not shown ) ; these inactivated forms could be regulators of active rhomboid proteases .
several other proteins lack one or more of the conserved residues in tmh3 ; it remains unclear whether or not these are active proteases . bacterial and archaeal members of the rhomboid superfamily contain six tmh , whereas the eukaryotic members typically have an additional seventh tmh , which may be attached to the core either from the amino terminus or from the carboxyl terminus as discussed below .
the phyletic distribution pattern of the rhomboid family shows that this intramembrane protease is extremely common in all three kingdoms of life , but is not necessarily essential for cell function .
rhomboids are missing in the microsporidian encephalitozoon cuniculi , a eukaryotic intracellular parasite with a highly degraded genome , the archaea methanothermobacter thermoautotrophicus and thermoplasma volcanium , and several bacterial species , primarily parasites with small genomes but also species with moderately sized genomes , such as xylella fastidiosum ( see cog0705 at ) . in two instances ,
a representative of the rhomboid family is present in only one of a pair of relatively close genomes ( present in t. acidophilum but missing in t. volcanium ; present in the spirochete treponema pallidum but missing in the related bacterium borrelia burgdorferi ) , which suggests relatively recent , repeated losses of this gene .
most of the prokaryotic species have a single gene coding for a rhomboid - family protein , although some have two or three paralogs ( see cog0705 ) ; in contrast , eukaryotes show expansion of the rhomboid family , with seven members in drosophila , and as many as 13 in arabidopsis .
the multiple alignment of the 6-tmh core of the rhomboid family ( figure 1 ) was employed to construct a phylogenetic tree using the least - squares algorithm with subsequent optimization using the maximum likelihood ( ml ) method ( see materials and methods ) . only the conserved regions including the tmh and
short adjacent stretches shown in figure 1 were used as the input for tree building , whereas the poorly conserved intervening regions were omitted to avoid noise from potentially misaligned residues ( except for the bayesian analysis , which used the complete alignment ; see materials and methods ) .
the phylogenetic tree of the rhomboid family presents a complex and unexpected picture ( figure 2 ) .
neither the eukaryotic nor the archaeal subsets of the family appear to form monophyletic clades .
instead , the eukaryotic rhomboids are split between two major subfamilies , which are positioned in the midst of different prokaryotic branches ( figure 2 ) .
the first subfamily , which includes six of the seven drosophila rhomboids , clusters with a distinct prokaryotic assemblage , consisting primarily of gram - positive bacteria as well as a subset of archaea ; this clade is strongly supported by bootstrap analysis ( figure 2 ) .
the proteins in this group of eukaryotic rhomboids , which we designated the rho subfamily , typically have an extra tmh added carboxy - terminally to the 6-tmh core ; some of these proteins also contain ef - hand calcium - binding domains amino - terminally of the core ( figure 2 ) .
the second eukaryotic subfamily , which we designated the parl subfamily , after parl , the human ortholog of drosophila rho7 , resides within a large , heterogeneous prokaryotic cluster ( figure 2 ) . within this subfamily , parl and its orthologs from other animals and from fungi
have distinct domain architecture , with an extra tmh added to the amino terminus of the core , whereas the rest have only the core ( a carboxy - terminal tmh and a ubiquitin - associated domain are appended in one arabidopsis protein ; figure 2 ) .
thus , the existence of two distinct subfamilies of eukaryotic rhomboids is supported by features of domain architectures that appear to comprise shared derived characters . within these two major eukaryotic subfamilies ,
several lineage - specific expansions of paralogs are noticeable , in insects , mammals and plants ( figure 2 ) .
archaeal rhomboids are scattered over the phylogenetic tree , with two major clusters and , in addition , three isolated proteins joining different bacterial branches ( figure 2 ) .
there is no indication of an affinity between any of the archaeal and eukaryotic rhomboids .
although many of the bacterial rhomboids form phylogenetically coherent clusters corresponding to the established bacterial lineages , there are also several clusters that have an odd composition , such as the grouping of proteobacterial and gram - positive species ; some of these clusters are well supported by bootstrap ( see clusters 1 - 4 in figure 2 ) .
this concern is particularly serious for highly divergent families of membrane proteins , such as the rhomboids , in which parallel amino - acid substitutions are likely .
therefore we investigated the phylogeny of the rhomboid family in greater detail using several independent phylogenetic methods and the corresponding statistical tests .
first , we assessed the robustness of the topology of the tree shown in figure 2 using the kishino - hasegawa ( kh ) test whereby the clade of interest is forced into various positions on the tree and the likelihoods of the resulting topologies are estimated .
specifically , the kh test was used to evaluate two alternative topologies , in which the rho and parl subfamilies formed a clade , and two topologies , in which the rho subfamily formed a clade with archaeal rhomboids ( figure 2 and table 1 ) .
each of these alternative topologies had a significantly lower likelihood than the original topology shown in figure 2 ( see table 1 ) .
in addition , a tree of the rhomboid family was constructed using the bayesian inference method , which has recently become a practical alternative to the more traditional methods of phylogenetic analysis .
the tree produced using the mrbayes package showed the same major clades as the tree in figure 2 ( data not shown ) ; moreover , clustering of the rho and parl subfamilies of eukaryotic rhomboids with the respective prokaryotic clades was supported by high posterior probabilities ( figure 2 ) .
we also attempted to construct a phylogenetic tree of the rhomboid family by using the maximum parsimony method .
the resulting tree contained the same major clades as the trees constructed using ml and mrbayes ; however , the number of parsimony - informative sites was insufficient to obtain high bootstrap support with this approach ( data not shown ) .
the alternative phylogenies reflected two distinct hypotheses : first , clustering of the rho and parl subfamilies of eukaryotic rhomboids with the prokaryotic rhomboid families as suggested by the tree topology in figure 2 ; and second , monophyly of the eukaryotic rhomboids ( figure 3 ) .
the phylogenies corresponding to these alternative hypotheses were compared to the best phylogeny using three statistical tests ( table 2 ) .
the hypothesis 1 tree was not significantly different from the best tree under any of these tests whereas the hypothesis 2 tree was significantly ( p < 0.05 ) worse than the best tree according to each of the tests ( table 2 ) .
the concordance of the results obtained with several independent methods for phylogenetic tree construction and statistical analysis specifically aimed at testing the alternative hypothesis of monophyletic origin of eukaryotic rhomboids shows strong support for the major aspects of the tree topology in figure 2 and , in particular , for the polyphyly of eukaryotic rhomboids .
the phylogenetic tree of the rhomboid family shown in figure 2 and supported by the additional tests described above follows neither the ' standard model ' scenario , with the major split between the archaeo - eukaryotic and bacterial lineages nor the ' mitochondrial ' scenario , which postulates acquisition of a gene by eukaryotes from the pro - mitochondrial endosymbiont .
neither can this tree be explained by postulating a small number of lineage - specific gene losses .
the parsimonious interpretation of the rhomboid family tree seems to be that the evolutionary history of this family had been replete with horizontal gene transfer ( hgt ) and lineage - specific gene loss events . in particular , in spite of the presence of rhomboids in the majority of modern life forms from all three primary superkingdoms , phylogenetic analysis suggests that this family has not been inherited from the last universal common ancestor ( luca ) .
instead , the tree topology seems to indicate that this family emerged in some bacterial lineage and afterwards had been widely disseminated by hgt , and then lost in some lineages .
in particular , at least two hgt events seem to have contributed to the origin of eukaryotic rhomboids , one of them yielding the rho subfamily and the other one the parl subfamily , with a possible additional hgt in plants ( figures 2,3 ) . given the broad phyletic representation of both subfamilies of eukaryotic rhomboids , both the rho subfamily and the parl subfamily
must have been acquired through hgt at an early stage of eukaryotic evolution , definitely before the divergence of the major crown - group lineages .
this early epoch in eukaryotic evolution is thought to have been dominated by hgt from multiple bacterial symbionts .
an alternative to this multiple - hgt scenario is that luca already had multiple , paralogous rhomboids , which evolved by a series of ancient gene duplications , and the odd topology of the phylogenetic tree is due primarily to differential loss of these ancient paralogs .
although this can not be ruled out formally , this hypothesis implies the existence of an elaborate signaling system in luca and , accordingly , suggests that luca was a complex organism , which might have had as many genes as modern bacteria .
theoretical analysis of evolutionary scenarios constructed on the basis of the phyletic patterns of cogs by applying
the parsimony principle shows that the complexity of the inferred gene set of luca critically depends on the relative rates of gene loss and hgt at the early stages of evolution .
a complex luca with around 2,000 genes is predicted only when one assumes that the rate of gene loss is an order of magnitude greater than the rate of hgt .
however , explicit reconstruction of the gene set of luca under the assumption of equal rates of gene loss and hgt leads to a hypothetical genome that consists of only around 600 genes but appears to be ' compatible with life ' , that is , it includes genes responsible for most , if not all , essential cellular functions .
we currently believe that this is the most realistic , albeit inevitably imprecise , reconstruction of luca 's gene set . with respect to the rhomboid family and other families
whose phylogenetic trees show similar patterns , this makes the multiple - hgt interpretation the scenario of choice . further theoretical
, comparative - genomic and experimental analyses aimed at determining relative rates of gene loss and hgt will help in a more objective assessment of the validity of this argument .
the multiple - hgt interpretation of the evolutionary history of the rhomboid family , while supported by the above argument , seems , at least at first glance , distinctly counter - intuitive , given that this family is nearly ubiquitous among extant life forms .
indeed , when attempts are made to construct parsimonious evolutionary scenarios on the basis of phyletic patterns alone [ 31 - 33 ] , there is no chance that such a widespread family is not assigned to luca .
it should be realized , however , that these approaches are inherently probabilistic , and extensive hgt can fool them . for the rhomboid family , the multiple - hgt mode of evolution seems to be particularly plausible .
it seems likely that the ultimate ancestor of the rhomboid family evolved from a nonenzymatic integral membrane protein , probably a transporter that might have been involved in an early primitive form of export of signaling peptides in bacteria .
the protease active center might have evolved in such a transporter by chance emergence of the suitable catalytic amino acids within two or three of the tmhs ( figure 4 ) .
this would enable the transition from simple transport to the rip mode of controlled export of signaling molecules .
emergence of rip could have conferred a major selective advantage on the respective bacteria and might have resulted in an evolutionary sweep whereby the gene carrying this trait was repeatedly fixed , rather than eliminated , after hgt . in terms of the evolution of sequence itself ,
the requirements for the conservation of the protease activity apparently ' locked ' the rhomboid family in a regime of relatively slow evolution , which ensures significant sequence similarity between all family members ( figure 1 ) .
the scenario of origin from non - catalytic transporters might potentially apply to other integral membrane enzymes , including intramembrane proteases involved in rip , such as presenilins and their homologs and the archaeo - eukaryotic signal peptide peptidase .
although the sequence similarity between eukaryotic and prokaryotic rhomboid family proteins is relatively low ( around 10 - 15% identity in the conserved region ) , the entire superfamily could be retrieved from the protein sequence databases within three iterations of the psi - blast program with a high statistical significance and without any false positives .
the conserved core of the rhomboid family consists of six conserved tmhs ( figure 1 ) .
the predicted catalytic serine is located in tmh5 , whereas the predicted catalytic histidine is in tmh7 ; tmh3 contains two additional histidines and an asparagine , which are conserved in the great majority of the rhomboid - family proteins ( figure 1 ) .
the roles of these conserved residues are not known , but , given the remarkable evolutionary conservation , it seems likely that they also contribute to catalysis ; indeed , it has been shown that the conserved asparagine is required for the cleavage of spitz by rho .
when examining the multiple alignment of the rhomboid superfamily proteins , we noticed that several eukaryotic members appear to be inactivated proteases , as indicated by the loss of the predicted catalytic serine or histidine ( figure 1 , and data not shown ) ; these inactivated forms could be regulators of active rhomboid proteases .
several other proteins lack one or more of the conserved residues in tmh3 ; it remains unclear whether or not these are active proteases . bacterial and archaeal members of the rhomboid superfamily contain six tmh , whereas the eukaryotic members typically have an additional seventh tmh , which may be attached to the core either from the amino terminus or from the carboxyl terminus as discussed below .
the phyletic distribution pattern of the rhomboid family shows that this intramembrane protease is extremely common in all three kingdoms of life , but is not necessarily essential for cell function .
rhomboids are missing in the microsporidian encephalitozoon cuniculi , a eukaryotic intracellular parasite with a highly degraded genome , the archaea methanothermobacter thermoautotrophicus and thermoplasma volcanium , and several bacterial species , primarily parasites with small genomes but also species with moderately sized genomes , such as xylella fastidiosum ( see cog0705 at ) . in two instances ,
a representative of the rhomboid family is present in only one of a pair of relatively close genomes ( present in t. acidophilum but missing in t. volcanium ; present in the spirochete treponema pallidum but missing in the related bacterium borrelia burgdorferi ) , which suggests relatively recent , repeated losses of this gene .
most of the prokaryotic species have a single gene coding for a rhomboid - family protein , although some have two or three paralogs ( see cog0705 ) ; in contrast , eukaryotes show expansion of the rhomboid family , with seven members in drosophila , and as many as 13 in arabidopsis .
the multiple alignment of the 6-tmh core of the rhomboid family ( figure 1 ) was employed to construct a phylogenetic tree using the least - squares algorithm with subsequent optimization using the maximum likelihood ( ml ) method ( see materials and methods ) .
only the conserved regions including the tmh and short adjacent stretches shown in figure 1 were used as the input for tree building , whereas the poorly conserved intervening regions were omitted to avoid noise from potentially misaligned residues ( except for the bayesian analysis , which used the complete alignment ; see materials and methods ) .
the phylogenetic tree of the rhomboid family presents a complex and unexpected picture ( figure 2 ) .
neither the eukaryotic nor the archaeal subsets of the family appear to form monophyletic clades . instead
, the eukaryotic rhomboids are split between two major subfamilies , which are positioned in the midst of different prokaryotic branches ( figure 2 ) .
the first subfamily , which includes six of the seven drosophila rhomboids , clusters with a distinct prokaryotic assemblage , consisting primarily of gram - positive bacteria as well as a subset of archaea ; this clade is strongly supported by bootstrap analysis ( figure 2 ) .
the proteins in this group of eukaryotic rhomboids , which we designated the rho subfamily , typically have an extra tmh added carboxy - terminally to the 6-tmh core ; some of these proteins also contain ef - hand calcium - binding domains amino - terminally of the core ( figure 2 ) .
the second eukaryotic subfamily , which we designated the parl subfamily , after parl , the human ortholog of drosophila rho7 , resides within a large , heterogeneous prokaryotic cluster ( figure 2 ) . within this subfamily , parl and its orthologs from other animals and from fungi
have distinct domain architecture , with an extra tmh added to the amino terminus of the core , whereas the rest have only the core ( a carboxy - terminal tmh and a ubiquitin - associated domain are appended in one arabidopsis protein ; figure 2 ) .
thus , the existence of two distinct subfamilies of eukaryotic rhomboids is supported by features of domain architectures that appear to comprise shared derived characters . within these two major eukaryotic subfamilies ,
several lineage - specific expansions of paralogs are noticeable , in insects , mammals and plants ( figure 2 ) .
archaeal rhomboids are scattered over the phylogenetic tree , with two major clusters and , in addition , three isolated proteins joining different bacterial branches ( figure 2 ) .
there is no indication of an affinity between any of the archaeal and eukaryotic rhomboids .
although many of the bacterial rhomboids form phylogenetically coherent clusters corresponding to the established bacterial lineages , there are also several clusters that have an odd composition , such as the grouping of proteobacterial and gram - positive species ; some of these clusters are well supported by bootstrap ( see clusters 1 - 4 in figure 2 ) .
this concern is particularly serious for highly divergent families of membrane proteins , such as the rhomboids , in which parallel amino - acid substitutions are likely .
therefore we investigated the phylogeny of the rhomboid family in greater detail using several independent phylogenetic methods and the corresponding statistical tests .
first , we assessed the robustness of the topology of the tree shown in figure 2 using the kishino - hasegawa ( kh ) test whereby the clade of interest is forced into various positions on the tree and the likelihoods of the resulting topologies are estimated .
specifically , the kh test was used to evaluate two alternative topologies , in which the rho and parl subfamilies formed a clade , and two topologies , in which the rho subfamily formed a clade with archaeal rhomboids ( figure 2 and table 1 ) .
each of these alternative topologies had a significantly lower likelihood than the original topology shown in figure 2 ( see table 1 ) .
in addition , a tree of the rhomboid family was constructed using the bayesian inference method , which has recently become a practical alternative to the more traditional methods of phylogenetic analysis .
the tree produced using the mrbayes package showed the same major clades as the tree in figure 2 ( data not shown ) ; moreover , clustering of the rho and parl subfamilies of eukaryotic rhomboids with the respective prokaryotic clades was supported by high posterior probabilities ( figure 2 ) .
we also attempted to construct a phylogenetic tree of the rhomboid family by using the maximum parsimony method .
the resulting tree contained the same major clades as the trees constructed using ml and mrbayes ; however , the number of parsimony - informative sites was insufficient to obtain high bootstrap support with this approach ( data not shown ) .
the alternative phylogenies reflected two distinct hypotheses : first , clustering of the rho and parl subfamilies of eukaryotic rhomboids with the prokaryotic rhomboid families as suggested by the tree topology in figure 2 ; and second , monophyly of the eukaryotic rhomboids ( figure 3 ) .
the phylogenies corresponding to these alternative hypotheses were compared to the best phylogeny using three statistical tests ( table 2 ) .
the hypothesis 1 tree was not significantly different from the best tree under any of these tests whereas the hypothesis 2 tree was significantly ( p < 0.05 ) worse than the best tree according to each of the tests ( table 2 ) .
the concordance of the results obtained with several independent methods for phylogenetic tree construction and statistical analysis specifically aimed at testing the alternative hypothesis of monophyletic origin of eukaryotic rhomboids shows strong support for the major aspects of the tree topology in figure 2 and , in particular , for the polyphyly of eukaryotic rhomboids .
the phylogenetic tree of the rhomboid family shown in figure 2 and supported by the additional tests described above follows neither the ' standard model ' scenario , with the major split between the archaeo - eukaryotic and bacterial lineages nor the ' mitochondrial ' scenario , which postulates acquisition of a gene by eukaryotes from the pro - mitochondrial endosymbiont .
neither can this tree be explained by postulating a small number of lineage - specific gene losses .
the parsimonious interpretation of the rhomboid family tree seems to be that the evolutionary history of this family had been replete with horizontal gene transfer ( hgt ) and lineage - specific gene loss events .
in particular , in spite of the presence of rhomboids in the majority of modern life forms from all three primary superkingdoms , phylogenetic analysis suggests that this family has not been inherited from the last universal common ancestor ( luca ) .
instead , the tree topology seems to indicate that this family emerged in some bacterial lineage and afterwards had been widely disseminated by hgt , and then lost in some lineages .
in particular , at least two hgt events seem to have contributed to the origin of eukaryotic rhomboids , one of them yielding the rho subfamily and the other one the parl subfamily , with a possible additional hgt in plants ( figures 2,3 ) . given the broad phyletic representation of both subfamilies of eukaryotic rhomboids , both the rho subfamily and the parl subfamily
must have been acquired through hgt at an early stage of eukaryotic evolution , definitely before the divergence of the major crown - group lineages .
this early epoch in eukaryotic evolution is thought to have been dominated by hgt from multiple bacterial symbionts .
an alternative to this multiple - hgt scenario is that luca already had multiple , paralogous rhomboids , which evolved by a series of ancient gene duplications , and the odd topology of the phylogenetic tree is due primarily to differential loss of these ancient paralogs .
although this can not be ruled out formally , this hypothesis implies the existence of an elaborate signaling system in luca and , accordingly , suggests that luca was a complex organism , which might have had as many genes as modern bacteria .
theoretical analysis of evolutionary scenarios constructed on the basis of the phyletic patterns of cogs by applying the parsimony principle shows that the complexity of the inferred gene set of luca critically depends on the relative rates of gene loss and hgt at the early stages of evolution . a complex luca with around 2,000 genes
is predicted only when one assumes that the rate of gene loss is an order of magnitude greater than the rate of hgt .
however , explicit reconstruction of the gene set of luca under the assumption of equal rates of gene loss and hgt leads to a hypothetical genome that consists of only around 600 genes but appears to be ' compatible with life ' , that is , it includes genes responsible for most , if not all , essential cellular functions .
we currently believe that this is the most realistic , albeit inevitably imprecise , reconstruction of luca 's gene set . with respect to the rhomboid family and other families
whose phylogenetic trees show similar patterns , this makes the multiple - hgt interpretation the scenario of choice . further theoretical
, comparative - genomic and experimental analyses aimed at determining relative rates of gene loss and hgt will help in a more objective assessment of the validity of this argument .
the multiple - hgt interpretation of the evolutionary history of the rhomboid family , while supported by the above argument , seems , at least at first glance , distinctly counter - intuitive , given that this family is nearly ubiquitous among extant life forms . indeed ,
when attempts are made to construct parsimonious evolutionary scenarios on the basis of phyletic patterns alone [ 31 - 33 ] , there is no chance that such a widespread family is not assigned to luca .
it should be realized , however , that these approaches are inherently probabilistic , and extensive hgt can fool them . for the rhomboid family , the multiple - hgt mode of evolution seems to be particularly plausible .
it seems likely that the ultimate ancestor of the rhomboid family evolved from a nonenzymatic integral membrane protein , probably a transporter that might have been involved in an early primitive form of export of signaling peptides in bacteria .
the protease active center might have evolved in such a transporter by chance emergence of the suitable catalytic amino acids within two or three of the tmhs ( figure 4 ) .
this would enable the transition from simple transport to the rip mode of controlled export of signaling molecules .
emergence of rip could have conferred a major selective advantage on the respective bacteria and might have resulted in an evolutionary sweep whereby the gene carrying this trait was repeatedly fixed , rather than eliminated , after hgt . in terms of the evolution of sequence itself ,
the requirements for the conservation of the protease activity apparently ' locked ' the rhomboid family in a regime of relatively slow evolution , which ensures significant sequence similarity between all family members ( figure 1 ) .
the scenario of origin from non - catalytic transporters might potentially apply to other integral membrane enzymes , including intramembrane proteases involved in rip , such as presenilins and their homologs and the archaeo - eukaryotic signal peptide peptidase .
the rhomboid family might be the most widespread and conserved group of integral membrane proteins . in and by itself , this would suggest that this family is part of the gene repertoire of luca .
however , phylogenetic analysis suggests a different scenario , one of emergence in a bacterial lineage with subsequent multiple , independent hgt events and gene losses . although caution is due in the evolutionary interpretation of phylogenetic trees for large families , particularly when membrane proteins with a relatively small number of conserved positions , such as the rhomboids ,
are involved , the multiple - hgt scenario seemed to be supported by several methods of tree analysis and statistical tests .
eukaryotes probably acquired their two major rhomboid subfamilies , rho and parl , as the result of two independent , early hgt events .
these events , which might have introduced rip as a means of intercellular communication , could have been pivotal in the evolution of eukaryotic multicellularity along the lines discussed previously with regard to the apparent bacterial origin of key components of eukaryotic programmed cell death machinery .
subsequent evolution of rhomboids in eukaryotes proceeded by lineage - specific expansion of paralogs followed by diversification through the addition of an extra tmh in different positions relative to the catalytic core , some limited domain accretion ( see figure 2 ) and sequence divergence .
phylogenetic analysis of the rhomboid family described here carries a general message for studies aimed at the reconstruction of ancestral life forms , particularly luca .
although most of the ( nearly ) ubiquitous protein families probably do derive from luca , explicit phylogenetic analysis is required to ascertain this in each case .
the nonredundant ( nr ) protein sequence database at the national center for biotechnology information ( nih , bethesda ) was searched iteratively using the psi - blast program with multiple starting queries .
psi - blast was normally run with expectation ( e ) value of 0.01 as the cut - off for inclusion of sequences into the position - specific scoring matrix .
multiple alignments of protein sequences were constructed using the clustalw program and manually adjusted on the basis of the examination of psi - blast search outputs and the superposition of the predicted tmhs , which were identified using the programs tmpred and tmap .
phylogenetic trees were built using the least - squares method implemented in the fitch program of the phylip package , with subsequent local rearrangement using the protml program of the molphy package to obtain the maximum likelihood tree .
the reliability of the tree topology was assessed using the rell ( resampling of estimated log - likelihoods ) bootstrap method of molphy , with 10,000 replications .
alternative placements of selected clades in maximum - likelihood trees were compared by using the rearrangement optimization method ( kishino - hasegawa test ) as implemented in the protml program [ 43 - 45 ] .
maximum parsimony trees were constructed using the heuristic search option of paup * . in addition , trees were constructed by bayesian inference using the markov chain monte carlo method as implemented in the mrbayes package .
the complete alignment information , including columns with gaps , was used for the mrbayes analysis .
constraint trees were imported into paup * and subjected to neighbor - joining search to generate the phylogenies corresponding to alternative hypotheses .
these phylogenies were compared using the kh , templeton ( wilcoxon signed - ranks ) and winning - sites ( sign ) tests implemented in paup*.
l.p . is supported by a grant from the natural sciences and engineering research council of canada .
the alignment includes the majority of the detected rhomboid family proteins ; some closely related sequences were omitted . only the six conserved ( predicted ) transmembrane helices ( tmh ) and short surrounding regions are shown .
the boundaries of the predicted tmh are indicated by gray shading and overline and they are numbered 1 - 6 . the number of amino - acid residues in the omitted terminal and internal regions are indicated .
the consensus shows amino - acid residues present in at least 90% of the aligned sequences ; h stands for hydrophobic residues ( a , c , i , l , v , m , f , y , w in the single - letter amino - acid code ) and s for small residues ( g , a , s , d , n , v ) .
the proposed catalytic serine ( tmh4 ) and histidine ( tmh6 ) as well as conserved residues in tmh2 with possible ancillary roles in catalysis are highlighted in color .
the proteins are identified with the gene identification ( gi ) number from the nonredundant database and an abbreviated species name .
bacterial species are color - coded green , eukaryotic species blue and archaeal species yellow .
species name abbreviations : aerpe , aeropyrum pernix ; agrtu , agrobacterium tumefaciens ; anoga , anopheles gambiae ; arath , arabidopsis thaliana ; arcfu , archaeoglobus fulgidus ; bacsu , bacillus subtilis ; brume , brucella melitensis ; caeel , caenorhabditis elegans ; caucr , caulobacter crescentus ; chlte , chlorobium tepidum ; cloac , clostridium acetobutilicum ; corgl , corynebacterium glutamicum ; deira , deinococcus radiodurans ; dicdi , dictyostelium discoideum ; drome , drosophila melanogaster ; escco , escherichia coli ; haein , haemophilus influenzae ; halsp , halobacterium sp . ;
homsa , homo sapiens ; lacla , lactococcus lactis ; lisin , listeria innocua ; metja , methanoccocus jannaschii ; metka , methanopyrus kandleri ; metma , methanosarcina mazei ; meslo , mesorhizobium loti ; mycle , mycobacterium leprae ; myctu , mycobacterium tuberculosis ; neucr , neurospora crassa ; nossp , nostoc sp .
; prost , providencia stuartii ; pyrab , pyrococcus abyssi ; pyrae , pyrobaculum aerophilum ; ralso , ralstonia solanaraceum ; sacce , saccharomyces cerevisiae ; schpo , schizosaccharomyces pombe ; sinme , sinorhizobium meliloti ; strco , streptomyces coelicolor ; strpn , streptococcus pneumoniae ; sulso , sulfolobus solfataricus ; sulto , sulfolobus tokodaii ; synsp , synechocystis sp . ; theac , thermoplasma acidophilum ; thema , thermotoga maritima ; thete , thermus thermophilus ; vibch , vibrio cholerae ; xanca , xanthomonas campestris ; xylfa , xylella fastidiosa .
the sequences and their regions used to construct the tree are exactly those shown in figure 1 . the color coding and abbreviations are as in figure 1 .
the two major eukaryotic subfamilies are denoted as rho and parl ( see text ) and four clusters containing unexpected ( from a phylogenetic viewpoint ) sets of species are denoted 1 - 4 .
the clades that were investigated in the kh test are denoted a through d. although the tree is shown in a pseudorooted form for convenience , this is an unrooted tree .
internal nodes with at least 70% rell bootstrap supported are denoted by black circles and nodes with a 50 - 70% support by blue circles .
the posterior probabilities reported by the mrbayes program are indicated for some key internal branches .
the rho and parl subfamilies are denoted ; the remaining clusters include prokaryotic rhomboids designated as in figure 2 ( with ' a ' added to the gi number ) . within each cluster ,
the trees are unrooted , although shown in a pseudorooted form . a hypothetical scenario for the origin and dissemination of the rhomboid family proteases .
the figure schematically shows the proposed three stages of evolution of the rhomboid family . in ( a ) , the progenitor of the rhomboid family functions as a transporter for a regulatory peptide in some bacterial lineage . in ( b ) , the catalytic site of the intramembrane protease evolves , allowing the switch to rip as the mechanism of the regulatory peptide release . in ( c ) , the emergence of rip is followed by a burst of hgt .
the transmembrane helices of rhomboid are designated as in figure 1 ; their topology in the membrane is based on that proposed in .
the catalytic histidine and serine are shown and connected by a dotted line to indicate the proposed charge - relay system of the protease ; possible ancillary catalytic residues are not shown .
log - likelihood analysis of possible placements of selected branches of maximum likelihood trees for the proteins analyzed * a - d , clades that were subjected to local rearrangements in the tree as indicated in figure 2 and discussed in the text .
bootstrap probability of the given tree calculated using the rell method ( resampling of estimated log - likelihoods ) .
statistical comparisons of the best neighbor - joining tree with the hypothesis 1 and hypothesis 2 trees * probability of getting a more extreme test statistic under the null hypothesis of no difference between the two trees ( two - tailed test ) .
|
the near - universal presence of the rhomboid family in bacteria , archaea and eukaryotes appears to suggest that this protein is part of the heritage of the last universal common ancestor , phylogenetic tree analysis indicates a likely bacterial origin with subsequent dissemination by horizontal gene transfer .
|
Background
Results and discussion
Sequence and structural features and phyletic distribution of the rhomboid family
Phylogeny and evolutionary history of the rhomboid family
Conclusions
Materials and methods
Acknowledgements
Figures and Tables
|
the potential of rnai to silence any gene has made it an attractive therapeutic modality .
however , the main obstacle to rnai in the clinic is delivery . to be effective
, sirnas must be transported through the body , bind , and be taken up by target cells where they must traverse the plasma membrane and gain access to the cytosolic compartment , where the rnai machinery resides . to be useful ,
ease of formulation and administration , overall cost , and any associated toxicities are essential considerations .
dcs are heterogeneous , with subsets defined phenotypically , functionally and by location ( reviewed in ref .
2 ) . a delivery vehicle that knocks down expression of specific genes in a distinct dc population(s ) would be a valuable tool for targeting diverse diseases including cancers , infectious diseases , autoimmunity and as a vaccine component .
therefore , a platform that delivers sirnas to specific dc subsets in situ would be useful for inhibiting or activating immune responses .
lipid nanoparticles ( lnps ) are one of the most advanced platforms for sirna delivery to hepatocytes , and are under clinical evaluation for conditions that require hepatic gene silencing .
these lnps typically contain ionizable cationic lipids ( pka ~6.5 ) that bind nucleic acids via electrostatic interactions at low ph , but are charge neutral at ph 7.4 . as a well - perfused organ
furthermore , lnp uptake by hepatocytes is mediated by association with serum apoe leading to efficient uptake via low - density lipoprotein receptors in the liver .
following cellular uptake of the lnp , the ionization of the lipid within acidic endosomes is thought to promote sirna escape to the cytosol .
importantly , these lnps are associated with minimal toxicity , including little induction of proinflammatory cytokines following administration of physiologically relevant doses .
in contrast to the liver , sirna delivery to extra - hepatic cells is challenging . in particular , immune cells such as dcs are relatively resistant to sirna uptake in vitro and in vivo .
although designed for hepatic gene silencing , the efficacy of these lnps and their derivatives has been assessed for rnai - mediated gene silencing in macrophages ( mos ) and dcs in vitro and in vivo .
lnp uptake and modest gene silencing was achieved at high doses of lnps ( ~ 5mg / kg ) .
together with a complementary study , this work demonstrates the feasibility of gene silencing in immune cells using lnp technology . clearly
, however , lnp formulations must be modified to achieve optimal gene silencing in immune cells .
enhanced uptake of lnps by primary hepatocytes is mediated by apolipoprotein e binding to the neutral lnps .
this results in recognition by receptors including the low - density lipoprotein receptor and scavenger receptors largely expressed on hepatocytes .
the mechanism of uptake strongly suggests that retargeting of these particles to other receptors or other tissues is possible .
indeed , akinc et al . demonstrated that lnps modified with n - acetylgalactosamine ( galnac ) were retargeted to the asialoglycoprotein receptor ( asgpr ) expressed by hepatocytes in vivo .
for delivery to nonhepatic cells , we have shown in vitro that anti - transferrin receptor aptamers can be used to redirect similar lnps .
more recently , liang et al . described a similar approach using aptamer - coated lnps to target osteoblasts in vivo .
for uptake by immune cells , a recent study coated lnps with a full - length anti - cd4 antibody for sirna delivery to primary cd4 t cells .
lnp uptake and gene silencing was observed in cd4 t cells in various organs including the spleen , lymph nodes , and blood .
these studies demonstrate the potential for attaching exogenous ligands to lnps for delivery to hepatic , and importantly nonhepatic primary cells in vivo . drawing from these works
, we reasoned that this lnp platform could be a useful foundation for the delivery of sirnas to dcs to modulate immune responses . to achieve this
, we modified lnps using a single chain antibody ( scfv ) specific for the dc receptor dec205 , a c - type lectin expressed at high levels on cd8 dcs .
dec205 dcs mediate cross - presentation of antigen resulting in modulation of cd8 t - cell responses . therefore , inhibition of gene expression by dec205 dcs is a potentially powerful approach for regulating cd8 t - cell activation . using this approach , we assessed the ability of dec - lnps ( lnps coated with scfv specific for murine dec205 , containing sirnas specific for costimulatory molecules ) to inhibit immune responses .
injection of dec - lnps resulted in preferential uptake of the dec - lnps by splenic dec205 dcs .
furthermore , when coadministered with adjuvant , lnps containing sirna targeting cd40 , cd80 , and cd86 reduced expression of these costimulatory molecules to levels similar to those observed in immature dcs .
most importantly , dcs isolated from mice injected with a low dose ( ~0.6 mg / kg ) of these dec - lnps , were able to suppress a robust mixed lymphocyte reaction ( mlr ) , demonstrating the functional efficacy of this approach .
interestingly , when we performed experiments using 2 ' fluoro ( 2'f ) modified sirna , a formulation reported to be nonimmunostimulatory and nonimmunogenic , significant immune activation of the targeted dcs was detected . this effect was not observed in assays performed with human peripheral blood mononuclear cells ( pbmcs ) or monocyte - derived dcs ( modcs ) and could be mitigated through selective 2-o - methyl ( 2ome ) modification of the sirna .
overall , our study demonstrates the functionality of lnps modified for receptor targeting for sirna delivery to dcs .
additionally our results suggest that specific cell targeting can mediate immunological responses to sirna formulations .
while care needs to be taken in designing and evaluating targeted approaches using lnps , we found that 2ome modification of the sirna was sufficient to negate immune activation .
our approach enhances the efficacy of sirna - mediated gene silencing by ~10-fold when compared to nontargeted delivery .
therefore , this targeted strategy should prove effective for regulating multiple immune - mediated diseases .
lnps containing sirnas specific either for cd80 , cd86 , cd40 , or a control ( nontarget ) sequence were synthesized by extrusion using a mixture of 1,2-distearoyl - sn - glycero-3-phosphocholine ( dspc):dlindma : dspe - peg : cholesterol at a ratio of 15:40:5:40 ( based on refs .
murine anti - dec205 scfv with a c - terminal cysteine was attached to the lipid dspe - peg by a maleimide group ( figure 1a , b ) . following synthesis , we subjected lnps to quality control to ensure consistency between batches .
dynamic light scattering was used to determine diameter and polydispersity of lnps , and cryo - electron microscopy confirmed lnp size and their unilamellar structure ( figure 1c , d ) .
the efficiency of sirna incorporation and scfv binding to dspe - peg was also assessed ( figure 1c , e ) . to show specificity of binding to dec205 ,
scfv - lnps containing dy547-labeled sirna were cultured with either parental ( dec205 ) or dec205 cho cells . as seen in figure 2a , at 4 c dec205 cells bind dec - lnps approximately fourfold better compared with nontargeted lnps ( nt - lnps : lnps not coated with any scfv ) , or lnps coated with an isotype scfv ( iso - lnps ; cho - dec205 panel ) . when similar assays were performed at 37 c to allow cell uptake , cell staining increased by at least fivefold .
little uptake was observed when lnps ( targeted or nontargeted ) were incubated with parental cho cells ( cho panel ) .
similar results were obtained when bone marrow - derived dcs ( bmdcs ) , derived from wild - type mice ( b6 ) were incubated with dec - lnps .
we note that as dec205 is expressed at lower levels on bmdcs compared with cho - dec205 , less dec - lnp binding and uptake was observed in bmdcs ( ~2-fold enhancement compared with nt- or iso - lnps ; b6 bmdc panel ) . to further confirm target - specific uptake we performed experiments using bmdcs derived from dec205 mice , and no specific cell binding or uptake
the dec205 antibody is internalized via receptor - mediated endocytosis , and is targeted to late endosomes and lysosomes .
confocal microscopy confirmed that similar to dec205 antibody , dec - lnps were targeted to lamp-1 late endosomes or lysosomes ( figure 2b ) .
having demonstrated dec205-mediated uptake of dec - lnps in vitro , we investigated their localization in vivo .
b6 mice were injected systemically ( intravenous , i.v . ) with fluorescently labeled scfv - lnps .
as the dec205 receptor is endocytosed when it binds its ligand , we could not use this molecule to identify dec205 dcs .
therefore , we used cd8 , a protein that is coexpressed on dec205 dcs , as a surrogate marker for tracking these cells .
more than 50% of cd11ccd8 cells took up dec - lnps , and uptake was largely restricted to the cd8 dc population , with little uptake observed by cd8 dcs ( figure 3a , b ) .
specificity was demonstrated by comparison of uptake of dec - lnps and iso - lnps .
significant uptake was only observed in dec205 dcs following dec - lnp injection , and other splenic immune cells took up little dec- or iso - lnps ( figure 3a , c ) . when dec - lnps were injected into dec205 mice ,
no uptake was detected ( figure 3d ) . to show that dec - lnps were competent for specific gene silencing , we generated dec - lnps containing a sirna specific for cd80 ( having identified the most effective sirna sequence ; see supplementary figure s1 ) and injected into b6 mice .
cd11cdec205 cells took up similar amounts of lnps following injection with dec - lnp containing either cd80- or control - sirna ( lnp uptake panel , mean fluorescence intensity ( mfi ) : dec - lnp - control = 443 versus dec - lnp - sicd80 = 464 ) . in mice injected with dec - lnp encapsulating cd80-specific sirna , cd80 protein expression approached basal levels , and was reduced by ~2-fold when compared with dec - lnp containing control - sirna ( figure 4a ) .
a 75% reduction in cd80 mrna was observed in dec205 dcs that had taken up dec - lnps containing cd80-specific sirna ( compared with dec - lnps containing control sirnas ; figure 4b ) .
we confirmed that gene knockdown was rnai - mediated using 5 race to detect sirna - directed mrna cleavage products ( figure 4c ) .
sequencing of the polymerase chain reaction ( pcr ) fragment verified that it was derived from cd80 mrna and that the cut site corresponded with nucleotide positions 1011 of the sirna guide strand ( not shown ) .
interestingly , while the data in figure 4 demonstrate the ability of our dec - lnps to effectively knockdown cd80 expression in dcs in vivo , we observed significant dc activation following injection of dec - lnps containing control sirnas ( figure 4a , right panel ) .
this observation was surprising considering these experiments were performed using sirnas containing 2f modified rna , previously reported to have reduced immunostimulatory activity . to better assess this effect , we synthesized a series of luciferase - specific control sirnas ( luc ) containing 2oh rna ( unmodified ) , 2f pyrimidines or selected 2ome modifications ( see methods for modification strategy ; based on refs .
lnps were generated and their ability to stimulate immune responses using multiple assays was examined .
first , b6 mice were injected with dec - lnps that contained either unmodified , 2f or 2ome modified luc - specific sirnas in the absence of any adjuvant .
one day later , splenic dcs were analyzed for the expression of costimulatory receptors ( supplementary figure s2a ) .
as expected , dcs isolated from mice that had received unmodified sirnas demonstrated significant upregulation of cd40 , cd80 , and cd86 .
consistent with our previous results ( figure 4a ) , animals treated with dec - lnps containing 2f modified sirnas also upregulated these costimulatory molecules . importantly , and similar to work by other groups , we found that 2ome modified sirnas induced minimal immune activation . to attempt to circumvent the need for a time and resource intensive in vivo assay , we wanted to determine whether an in vitro assay could be used to detect immunostimulatory sirnas .
however , when we cultured b6-derived bmdcs with dec - lnps ( containing cd80-specific sirnas ) no difference between 2oh modified sirna , 2f or 2ome modified sirna was detected ( supplementary figure s2b ) .
we also used nt - lnps to assess the effects of various sirna formulations on human pbmcs and modcs using standard preclinical assays ( supplementary figure s2c ) .
pbmcs or modcs were cultured for 24 hours at which time cells were assessed for induction of apoptosis and culture supernatants were tested for the presence of proinflammatory cytokines ( see methods ) .
apoptosis was not observed under any conditions tested ( data not shown ) . as expected , pbmcs cultured with the highest concentration of lnps containing unmodified sirnas elicited the secretion of several cytokines .
2f and 2ome luc sirnas induced production of one cytokine ( il8 ) , also at the highest concentration tested .
no cytokine production was observed following culture of modcs with lnps encapsulating unmodified , 2f or 2ome sirnas .
taken together , these experiments suggest that the assays tested show marked differences in sensitivity . surprisingly , bmdcs and modcs were the least receptive for detecting immunostimulatory sirnas .
conversely , dec - lnp injection was useful for the identification of sirnas with immune - activating potential . from these results
, we used sirnas containing selective 2ome substitutions for all subsequent studies . to target autoimmune diseases , reducing the expression of several costimulatory molecules
therefore , we next determined the ability of dec - lnps that encapsulated 2ome modified cd86 specific sirnas , to reduce gene expression following i.v . administration ( optimal sirna identified as previously ; supplementary figure s1 ) .
as the sirnas were 2ome modified , mice were coinjected with lps to stimulate expression of costimulatory molecules .
analysis of splenic dec205 dcs 1 day following dec - lnp injection showed reduction of cd86 protein to near steady - state levels ( figure 5a ) .
total rna was isolated from dec205 dcs that had taken up dec - lnps encapsulating either cd86 or control sirnas .
an approximately 70% reduction in cd86 mrna levels was observed from dcs isolated from mice that received dec - lnps containing cd86 sirna in comparison to dcs derived from control sirna treated mice . to verify knockdown occurred via the rnai pathway ,
we next tested whether combining sirnas in dec - lnps would induce gene silencing equivalent to that observed with single sirnas .
a combination of 2ome modified sirnas targeting cd80 , cd86 and cd40 were incorporated into dec - lnps and injected into b6 mice along with adjuvant ( lps ) to activate the dcs .
reduction of each of the targeted costimulatory molecules was observed , with cd80 and cd86 knocked down to basal levels ( figure 5c ) .
gene silencing was similar to that achieved following treatment with dec - lnps containing single sirnas ( e.g. , ~50% for cd86 : compare cd86 dec - lnp , figure 5a with mix dec - lnp , figure 5c right panel ) . to determine functional relevance of this knockdown , we used the robust mlr assay .
b6 mice were injected with dec - lnps encapsulated with either a mix of cd80 , cd86 , and cd40 or control sirnas .
after 24 hours , splenic cd8 dcs that had taken up dec - lnps were isolated , irradiated , and cultured with splenic t cells derived from a balb / c mouse .
maximal proliferation was observed when dcs were isolated from mice injected with lps only , or lps plus dec - lnps containing control sirnas . in contrast , proliferation of t cells cultured with dcs derived from mice injected with lps plus dec - lnps containing cd80 , cd86 , and cd40 sirnas was significantly reduced ( figure 5d ) .
currently , the most advanced lnp platforms use the ionizable cationic lipid dlindma , or its dlin - kc2-dma and dlin - mc3-dma derivatives .
following systemic administration , these lnps accumulate in first pass organs , i.e. , liver and spleen , making them attractive vehicles for hepatic gene knockdown .
furthermore , these lnps have been shown to safely and effectively reduce expression of hepatocyte genes in clinical trials .
however , there is only very limited data demonstrating targeted delivery of potentially clinically relevant lnps to nonhepatic cells .
we have shown that conjugation of a scfv , specific for the dc receptor dec205 , is sufficient to direct delivery of dlindma - formulated dec - lnps to splenic dcs .
similar to conventional nt - lnps , dec - lnps incorporated sirnas with high efficiency ( > 80% ) , and were of uniform diameter ( ~100 nmol / l ) .
we found that coating lnps with only ~50 scfv was sufficient for dc delivery ( figures 1 , 2 , and 3 ) .
we showed that dec - lnp uptake correlated well with dec205 receptor density : dcs > b cells , t cells and macrophages ( dec205 expression is 1050-fold less than on dcs ) .
use of dec205 mice further confirmed that uptake occurred via the dec205 receptor ( figures 2a and 3d ) .
we also showed that the intracellular pathway accessed by dec - lnps was consistent with the dec205 pathway ( figure 2b ) .
importantly , we demonstrated that targeted delivery resulted in effective rnai - mediated gene silencing of one , or several genes , to essentially basal expression levels ( figures 4 and 5a c ) .
this reduction in gene expression was sufficient to inhibit a robust mlr ( figure 5d ) .
coated lnps formulated with the cationic lipid ddab with full - length dec205 antibody for sirna delivery and demonstrated the ability to reduce expression of the costimulatory molecule cd40 in dec205 dcs . while some gene knockdown was achieved , this lipid is known for its adjuvant qualities , and is being developed for applications that require immune response stimulation .
the lnps also proved relatively inefficient at sirna loading ( 10% compared with 85% for dlindma lnps ) .
the sirnas used were also unmodified , which could serve as another possible source of immune stimulation . therefore , the potential for this formulation appears to be limited .
more recently , ramishetti et al . utilized a full - length antibody targeting cd4 to enhance t - cell uptake and induce sirna - mediated gene silencing using a clinically relevant lnp formulation similar to the one we employed in our work .
interestingly , both this study and the work from zheng et al . chose to utilize full - length antibodies for lnp targeting , which may limit their utility when considering the immunogenicity of whole antibodies and their rapid clearance from the circulation by fc - mediated uptake by macrophages .
in fact , in preliminary studies , we coated lnps with full - length dec205 antibody and failed to observe uptake by dec205 dcs . using our dec - lnps
mg / kg ) comparable to that study which used n - acetylgalactosamine ( galnac)-coated lnps to target hepatocytes via the asialoglycoprotein receptor ( asgpr ) in apoe knockout mice .
in contrast , when the ability of nt - lnps to silence antigen presenting cells ( apcs ) , including dcs , was investigated a dose of 5 mg / kg was required to achieve partial silencing : ~10-fold higher when compared with our dec - lnps .
furthermore , the authors used the dlindma derivative , dlin - kc2-dma , which displays improved silencing ability in hepatocytes ( relative ed50 dlin - kc2-dma : 0.1 mg / kg versus dlindma : 1 mg / kg ) . as these lnps were formulated for effective gene silencing in hepatocytes ,
biodistribution studies show that nt - lnps are found in the spleen , although at ~50-fold less than in the liver . while targeting ligands
have been shown to have little effect on the overall biodistribution of nanoparticle formulations , they can enhance cell - specific uptake . therefore , while high doses of nt - lnps can confer partial gene silencing in apcs , at low lnp concentrations , we find that a targeted approach is required to achieve knockdown in a specific dc subset
. injection of low - dose nt - lnps failed to result in detectable uptake / gene silencing .
as the rnai pathway is resident in the cytoplasm , the intracellular pathway used following receptor ligation is an important consideration .
we chose the well - characterized dec205 receptor for targeting our lnps . following binding ,
the internalized receptor is routed to late endosomes and associated cargo gains access to the cytoplasm .
the dlindma lipid fuses with anionic phospholipids present in the endosomal membrane resulting in release of encapsulated cargo into the cytoplasm .
however , it is likely that different routes of uptake will affect the efficiency of delivery and subsequent gene silencing . thus , a logical extension to our approach is to determine whether we observe improved gene silencing using other dc specific targeting agents and/or more effective dlindma derivatives .
we are currently investigating the efficacy of dec - lnps formulated with the dlin - mc3-dma ( ed50 0.03 mg / kg ) .
having demonstrated that we could efficiently target sirnas to dcs , the lack of toxicity of these lnps and their cargo had to be established .
we observed significant dc activation following injection of targeted lnps containing unmodified and 2f modified sirnas , and incorporation of 2ome modifications at distinct residues was required to maintain costimulatory molecules analyzed at basal levels ( figure 4a and supplementary figure s2a ) .
as injecting mice to determine immune stimulation is time and resource intensive , we assessed immune stimulation in murine and human tissue culture systems .
murine bmdcs were refractory to lnp stimulation suggesting they would not serve as a useful surrogate for gauging immunogenicity of lnps .
taken together , these assays demonstrate a requirement for in vivo analysis to uncover the stimulatory capacity of the lnps .
recently , a human whole blood cytokine assay has been described that showed similar activation profiles elicited by lnps when compared with in vivo injection , potentially circumventing a requirement for cumbersome in vivo analyses . for the purposes of inhibiting a multifactorial immune response , such as that observed in graft rejection or autoimmunity , the simultaneous knockdown of several ( co)stimulatory genes
this approach has been used to demonstrate that combining antibodies targeting cd80 and cd86 can reduce disease severity in experimental autoimmune encephalomyelitis and in antigen - induced arthritis .
transfer of dcs treated ex vivo with cd40 , cd80 , and cd86 antisense oligonucleotides ( as - odn ) into nod mice delayed type 1 diabetes ( t1d ) onset .
a follow - up study demonstrated that multiple injections of microspheres containing cd40 , cd80 , and cd86 as - odns were sufficient to prevent t1d , possibly due to reduced expression of the costimulatory molecules by splenic dcs .
we achieve significant reduction in expression of cd40 , cd80 , and cd86 on splenic dcs using our dec - lnps following systemic delivery , and ongoing studies are addressing the durability of gene knockdown and the potential use of these dec - lnps for treating autoimmune diseases . in summary
, we have shown that a clinically relevant lnp can be modified for cell - specific delivery of sirnas . in vivo administration
did not produce overt inflammatory responses , and we achieved significant rnai - mediated gene - specific knockdown at low sirna doses .
these attributes make this an attractive platform to warrant further investigation as a potential therapeutic agent for various autoimmune diseases .
furthermore , judicious selection of the ligand coating the lnp opens up the potential for targeting multiple cell types .
mice . c57bl/6 ( b6 ) and balb / c mice were purchased from taconic ( hudson , ny ) .
dec205 mice were bred to homozygosity on the b6 background ( dec205 ) , and validated by pcr and flow cytometry .
mice were housed in the barrier facility at the institute for animal studies , albert einstein college of medicine ( bronx , ny ) according to institutional animal care and use committee ( iacuc ) guidelines .
the single - chain fragment variable ( scfv ) antibodies constructed for dec205 ( clone nldc145 ) and isotype ( clone gl117 ) were cloned from antibody constructs , as previously described .
the variable regions of the heavy and light chain sequences were fused with a flexible linker ( g4s)6 separating them and cloned into a pet28a ( emd millipore , billerica , ma ) bacterial expression vector .
further modifications to the constructs included a strep - tag - ii ( wshpqfek ) at the n - terminus and a 10-residue histidine tag ( h10 ) with a short flexible linker ( g4s ) near the c - terminus .
a c - terminal unpaired cysteine residue was introduced for scfv conjugation to the lipid dspe - peg - mal .
the scfv constructs were transformed into bl21 ( de3 ) plyss competent cells ( promega , fitchburg , wi ) .
the scfv dec205 or isotype were expressed in escherichia coli strain bl21 ( de3 ) plyss as insoluble inclusion bodies . following bacterial growth in luria broth ( lb ) medium and induction of protein expression with isopropyl 1-thio - d - galactopyranoside
cells were lysed , and insoluble protein pelleted by centrifugation . solubilized inclusion bodies were passed over a ni - nta column and the bound protein was eluted with 8 m guanidine hydrochloride .
the purified proteins were refolded by rapid dilution in refolding buffer , and purified by size - exclusion chromatography using superdex 200 ( ge healthcare life sciences , pittsburgh , pa ) .
proteins were buffer - exchanged in phosphate - buffered saline ( pbs ) and concentrated .
scfvs were tested for endotoxin contamination using a kinetic chromogenic limulous amoebocyte lysate ( lal ) assay ( kinetic - qcl lal assay , lonza , walkersville , md ) .
the free cysteine engineered onto the n - terminus of the scfv was reduced using tris-(2-carboxyethyl ) phosphine hydrochloride ( life technologies , carlsbad , ca ) . following buffer exchange into pbs
, the reduced scfv was reacted with a 10-fold molar excess of alexafluor488 ( af488)-c5-maleimide according to manufacturer instructions ( life technologies , carlsbad , ca ) .
polyacrylamide gel electrophoresis ( sds - page ) and coomassie staining and storm molecular phosphorimager scanning of the stained gel ( ge healthcare , piscataway , nj ) . preparation of lnps .
lnps were formed using the following lipids at a 15:40:5:40 mol % : dspc ( avanti polar lipids , alabaster , al ) , cholesterol ( chol ; sigma - aldrich , mo ) , 1,2-distearoyl - sn - glycero-3-phosphatidylethanolamine - n-(maleimide-(polyethylene glycol)-2000 ) ( dspe - peg - mal , avanti polar lipids , alabaster , al ) , and 1,2-dilinoleyloxy-3-dimethylaminopropane ( dlindma ) synthesized by the chemical biology core facility at albert einstein college of medicine as previously described .
a fluorophore conjugated to a lipid , 1,2-dioleoyl - sn - glycero-3-phosphoethanolamine - n-(lissamine rhodamine b sulfonyl ) ( dope - rhodamine b ; avanti polar lipids , alabaster , al ) was incorporated ( 0.1 % ) to monitor lnp delivery .
lnps were formed using a modified version of the spontaneous vesicle formation by ethanol dilution method .
the lipid mixture was extruded ( mini - extruder and 1 ml gas - tight syringes , avanti polar lipids , alabaster , al ) sequentially with polycarbonate 200 and 80 nm pore size membranes .
the extruded lipid mixture was mixed via rapid pipetting and vortexing with sirna , followed by dialysis into pbs .
for attachment of scfv , lnps were mixed with reduced scfv at room temperature for 1 hour , and free maleimide groups were then quenched with -mercaptoethanol ( me ) .
tangential flow filtration ( microkros filter module ) with a 500 kda mwco ( spectrum laboratories , rancho dominguez , ca ) was used for buffer exchange into pbs and concentration of lnps .
lnps were assayed for size and polydispersity by dynamic light scattering ( dynapro plate reader , wyatt technology , santa barbara , ca ) .
cryo - electron microscopy confirmed lnp size , geometry and lamellarity ( analytical imaging facility ; albert einstein college of medicine , bronx , ny ) .
sirna incorporation was determined by quant - it ribogreen rna assay ( life technologies , carlsbad , ca ) . to determine scfv conjugation efficiency , lnps were resolved on sds - page gels .
a 3 kda difference in migration of the scfv was indicative of binding to dspe - peg - mal ( unconjugated scfv ~30 kda , scfv - dspe - peg - mal ~33 kda ) .
a cholesterol quantification kit ( abcam , cambridge , ma ) was use to determine the total lipid concentration . based on lipid concentration , the surface area per lipid head group ( ~0.6 nm ) , lnp diameter and their unilamellar structure , ~5060 scfvs are conjugated to each lnp under the conditions outlined above .
sirna synthesis . modified and unmodified individual sirna sequences were synthesized on an expedite 8909 dna synthesizer ( biolytic , fremont , ca ) and purified by standard protocols , as described .
dylight 547-phosphoramidite was attached to the 5 end of the sense strand for synthesis of fluorescently labeled sirna ( dy547-sirna ; glen research , sterling , va ) .
sirnas were modified by 2f substitutions at every pyrimidine or by 2ome substitutions at the bolded nucleotides : cd40 sense 5-gaagauuaucccggucauauu-3 ; cd40 anti - sense 5-uaugaccg ; ggauaaucuucuu-3 ; cd80 sense 5-gaauuaccuggcaucaauauu-3 ; cd80 anti - sense 5-uauugaugccagguaauucuu-3 ; cd86 sense 5-caacuggacucuacg acuuuu-3 ; cd86 anti - sense 5-aagucguagaguccaguuguu-3 ; control sense 5-uagcgacuaaacacaucaauu-3 ; control anti - sense 5-uugauguguuuaguc gcuauu-3. luciferase sense 5-gauuauguccgguuauguauu-3 ; luciferase anti - sense 5-uacauaaccggacauaaucuu-3. lnp binding , uptake , and intracellular localization .
cho cells , cho cells that stably express dec205 ( cho - dec ) , a dec205 b lymphoma line ( a20 cells ) and bmdcs , derived from b6 and dec205 mice , were used to assess lnp binding and uptake .
cells were grown in complete medium ( dulbecco 's modified eagle 's medium , 5% fbs , 10 mmol / l 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid , 20 mmol / l l - glutamine ) .
bmdcs were generated from bone marrow derived from the femur and tibia as previously described .
adherent cells ( a20 , cho ) were harvested using ethylenediaminetetraacetic acid to preserve receptor expression and were incubated at 4 c , 30 minutes with 500 nmol / l scfv - lnps ( containing either 0.1% dope - rhodamine b or cy3-labeled sirnas ) . for uptake ,
cells were washed with fluorescence - activated cell sorting buffer ( pbs containing 0.5% bsa , 0.1% na n3 , 2 mmol / l edta ) and analyzed by flow cytometry ( lsrii , becton dickinson , franklin lakes , nj ; flowjo , ashland , or ) . to monitor intracellular localization , a20 cells were incubated with scfv - lnps , containing sirnas labeled with dylight 547 ( ge dharmacon , lafayette , co ) , 1 hour , 37 c .
following one wash with pbs , cells were pipetted onto poly - l - lysine coated coverslips and allowed to adhere .
adhered cells were fixed with 4% paraformaldehyde , washed with pbs and stained with anti - lamp1 ( clone 1d4b ; ebioscience , san diego , ca ) and anti - eea1 ( clone c45b10 ; cell signaling technology , danvers , ma ) .
stained cells were mounted with vectashield mounting media containing 4,6-diamidino-2-phenylindole ( dapi ) ( vector laboratories , burlingame , ca ) and samples were visualized on a leica sp5 aobs confocal microscope ( leica , buffalo grove , il ) .
image processing ( linear adjustments to brightness and contrast ) was performed on image files using nih imagej software ( bethesda , md ) .
lnps ( 0.61 mg / kg ) were injected ( retroorbital ( r.o . ) ) into b6 mice and 25 ng ultrapure lps ( invivogen , san diego , ca ) was injected r.o .
one day later , spleens were harvested , collagenase treated to maximize dc yield , and single cell suspensions subjected to red cells lysis .
cells were stained with cell surface markers and analyzed by flow cytometry to determine lnp uptake and expression of costimulatory molecules .
antibodies were used for detection of the following cell surface markers : cd11c , cd8 , dec205 , cd40 , cd80 , cd86 , cd19 , b220 , cd3 , tcr , f4/80 , cd11b , h2k , dcir2 , cd49b , cd45.1 .
cell viability was monitored using a live / dead exclusion dye , i.e. , live / dead blue ( life technologies , carlsbad , ca ) .
cells were acquired using an lsrii cytometer and data analyzed using flowjo software . to measure gene knockdown , cd11ccd8 splenocytes that had taken up lnps ( i.e. , sirna - dy547 ) were sorted by flow cytometry ( facs aria iii , becton dickinson , franklin lakes , nj ) into rnaprotect ( qiagen , valencia , ca ) to stabilize rna .
total rna was isolated using the rneasy rna isolation kit ( qiagen , valencia , ca ) , reverse transcribed using superscript iii ( life technologies , carlsbad , ca ) and random hexamers ( idt , coralville , ia ) , according to manufacturer 's instructions .
quantitative pcr was performed using platinum taq polymerase ( life technologies , carlsbad , ca ) and sybr green to detect amplified products on an iq5 icycler ( biorad , hercules , ca ) .
reactions were performed in triplicate with the following primers : u6 forward : 5-ctcgcttcggcagcacatatacta-3 ; u6 reverse : 5-acgaatttgcgtgtcatccttgcg-3 ; cd40 forward : 5-cggctgtgcgcgctatg-3 ; cd40 reverse : 5-ggctgagaattcgcctgagtc-3 ; cd80 forward : 5-ctactctcttatcatcctgggcct-3 ; cd80 reverse : 5-cccggaagcaaagcaggtaatcct-3 ; cd86 forward : 5-gccacccacaggatcaattatcct-3 ; cd86 reverse : 5-aaagagagaggctgttggagatac-3. relative amounts of mrna were normalized to u6 mrna and primer specificity verified by melt curve analysis . to detect rnai - mediated cleavage products ,
total rna was isolated from cd11ccd8 splenocytes that had taken up lnps , i.e. , sirna - dy547 ( as for gene knockdown by qpcr ) , ligated at the 5 end to an adapter rna oligonucleotide and first strand cdna was synthesized using a gene - specific primer ( gsp ) .
pcr amplification using an adapter - specific primer and a nested gsp primer will amplify the cleavage product and identify the cut site which corresponds to position 10/11 of the sirna guide strand if mediated via rnai .
the primers and oligonucleotide sequences used : adapter rna : 5-cgacuggagcacgaggacacugacauggacugaaggaguagaaa-3 ; gsp forward : 5-cgactggagcacgaggacactga-3 ; cd80 gsp reverse : 5-tgcgccgaatcctgccccaa-3 ; cd80 reverse nested : 5-atcaggagggtcttctgggg-3 ; cd86 gsp reverse : 5-agtaactgaagctgtaatctccttccaata-3 ; cd86 reverse nested : 5-ctgtgacattatcttgtgatatctgcatgt-3. analysis of immune stimulation .
cd11c murine bmdcs ( day 6 ; macs purified , miltenyi biotec , san diego , ca ) were cultured with dec - lnps and monitored for activation ( cd40 , cd80 , cd86 ) 1 day later by flow cytometry . in vivo stimulation
was assessed by injection of dec - lnps r.o . into b6 mice , followed by analysis of activation markers ( cd40 , cd80 , cd86 ) on splenic dcs after 24 hours by flow cytometry .
the pbmc fraction was used directly or following purification of cd14 cells ( stemcell technologies , vancouver , british columbia , canada ) , monocytes were cultured for 5 days in the presence of granulocyte - macrophage colony - stimulating factor and il4 to generate modcs .
nt - lnps ( 5 , 1 , 0.2 , 0.04 , 0.008 mol / l ) were cultured with pbmcs or modcs overnight and cell culture supernatants collected for cytokine analysis ( ifn , il1 , il2 , il4 , il5 , il6 , il7 , il8 , il10 , il12 , il13 , tnf , granulocyte - macrophage colony - stimulating factor ) using a luminex multiplex format ( life technologies carlsbad , ca ) .
cd11ccd8 splenic dcs were isolated by flow cytometry from b6 mice injected the previous day with dec - lnps containing either a 1:1:1 mix of cd40 , cd80 , and cd86 sirnas or control sirnas .
splenic t cells from balb / c mice were isolated by macs using a pan t cell isolation kit ii ( miltenyi , san diego , ca ) .
macs sorted balb / c t cells were carboxyfluorescein succinimidyl ester labeled according to published protocols , and carboxyfluorescein succinimidyl ester - labeled t cells were cocultured with the irradiated b6 dcs .
three days later , dilution of carboxyfluorescein succinimidyl ester was monitored by flow cytometry to determine t - cell activation .
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due to their ability to knock down the expression of any gene , sirnas have been heralded as ideal candidates for treating a wide variety of diseases , including those involving
undruggable targets . however , the therapeutic potential of sirnas remains severely limited by a lack of effective delivery vehicles .
recently , lipid nanoparticles ( lnps ) containing ionizable cationic lipids have been developed for hepatic sirna delivery .
however , their suitability for delivery to other cell types has not been determined .
we have modified lnps for preferential targeting to dendritic cells ( dcs ) , central regulators of immune responses . to achieve directed delivery , we coated lnps with a single - chain antibody ( scfv ; dec - lnps ) , specific to murine dec205 , which is highly expressed on distinct dc subsets .
here we show that injection of sirnas encapsulated in dec - lnps are preferentially delivered to dec205 + dcs .
gene knockdown following uptake of dec - lnps containing sirnas specific for the costimulatory molecules cd40 , cd80 , and cd86 dramatically decreases gene expression levels .
we demonstrate the functionality of this knockdown with a mixed lymphocyte response ( mlr ) .
overall , we report that injection of lnps modified to restrict their uptake to a distinct cell population can confer profound gene knockdown , sufficient to inhibit powerful immune responses like the mlr .
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Introduction
Results
Discussion
Materials and Methods
Supplementary Material
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according to the mca , only a compromised ability to make a decision in accordance with certain procedural norms , loosely defined with reference to the ability to understand ,
retain and use or weigh relevant information , or an inability to communicate a decision , properly grounds judgments of incapacity ( s. 3(1 ) ) .
in addition , the identified problems of decision - making must be attributed to an impairment of , or disturbance in the functioning of , mind or brain ( s. 2(1 ) ) .
this law aims to protect the individual 's right to pursue their own ends by adopting a capacity test that focuses on the decision - making process rather than the substance of the patient 's decision .
influential liberal thinking holds that it is not the place of the law to pursue certain ends rather , the law should aim to provide a space for individuals to pursue their own ends ( plant , 2011 ) and this position is reflected in english common law relating to mental capacity:[in assessments of capacity ] it is most important that those considering the issue should not confuse the question of mental capacity with the nature of the decision made by the patient , however grave the consequences .
the view of the patient may reflect a difference in values rather than an absence of competence and the assessment of capacity should be approached with this firmly in mind.whether the patient 's decision is consistent with proposed substantive norms in relation to what they should pursue for example that people should pursue good health , or that they should want to live is said to be beyond the proper scope of the incapacity test . in adopting this position
, english law allows that even the most controversial treatment decisions might be explained by the fact that the patient 's evaluation of the situation diverges from a clinical perspective , and perhaps many people 's evaluation of the situation .
[ in assessments of capacity ] it is most important that those considering the issue should not confuse the question of mental capacity with the nature of the decision made by the patient , however grave the consequences .
the view of the patient may reflect a difference in values rather than an absence of competence and the assessment of capacity should be approached with this firmly in mind. in practice then , questions of mental capacity might be thought of as boiling down to a question about whether a decision can be explained in terms of the patient 's particular motivating commitments ( their desires , values , projects , ideas about a good life ) ; or whether the decision is properly explained in terms of a problem in decision - making , which is due to a dysfunction of mind or brain . is the patient 's controversial decision understandable in the light of their commitments ? or is the patient 's ability to understand , retain , and use or weigh what the likely costs and benefits of treatment will be for them , currently compromised ? scrutinizing a patient 's commitments in the context of a capacity assessment raises moral concerns because the possibility that a choice can be explained by idiosyncratic commitments plays a significant role in protecting the patient 's right to make controversial decisions .
perhaps the most straightforward approach to conceiving of commitments , and the one most likely to protect this right , is to say that the person 's commitments are their current commitments those they recognize at the time of the decision .
the problem with conceiving of commitments in this way is that it fails to recognise certain problems of practical decision - making to do with motivational coherence over time .
someone with bipolar disorder , for example , might clearly be choosing or acting in accordance with their current desires during a manic phase , but this perspective fails to capture what seemingly goes wrong with practical decision - making in such cases . as a result , if this understanding of commitments is adopted in assessments of mental capacity the reach of this area of law arguably does not extend as far as it should a person may be judged to have mental capacity where this seems doubtful .
psychopathologies characterised by this kind of problem seem better accounted for in a legal structure that conceives of mental capacity , and in particular , commitments , in diachronic terms .
a diachronic perspective might also help illuminate some other hard cases that are not normally characterised in terms of problems of motivational coherence over time .
for example , someone with anorexia nervosa may be choosing rationally in accordance with their current commitments especially a strong desire not to gain weight , or to exert extreme control over what they eat and so may be judged to have capacity on these grounds . however , an assessment of their evaluative capacities in diachronic terms might yield the conclusion that their decision - making ability is seriously compromised ( craigie , 2011 ) .
their future self may come to the view that they should have been motivated in ways that they could not recognise or could not act on at the time , and this might be a fairly robust feature of people with a diagnosis of anorexia nervosa .
this perspective in relation to questions of mental capacity chimes with the suggestion that particularly in psychiatry , colloquialisms [ such as ] you will thank me later sometimes become the unwritten rules within which the merits of a patient 's consent are assessed ( gostin , 1981 , p. 742 ) .
such a perspective may also offer a solution in cases where the patient does not have a psychiatric diagnosis .
mackenzie and watts discuss a case of a woman who was judged to have the capacity to refuse life - saving treatment , and subsequently died , in a situation where she had recently been left by her husband , following the amputation of both her legs .
they argue that the trauma of these events and her subsequent inability to imagine a life without her partner clouded the woman 's judgment in relation to treatment:the tragedy is that in this case therapy or treatment might have corrected her assumptions regarding future quality of life , and a decision to accept the lifesaving treatment might have resulted. ( 2011 , p. 33
; for a full description of the case , see halpern , 2012)differences between these cases suggest that taking a diachronic perspective in assessments of mental capacity allows for decision - making to be compromised in at least two ways . in the bipolar disorder and anorexia nervosa cases a diachronic perspective is used to argue that a person 's evaluative capacities may be compromised the capacities that determine the desires or values that motivate a decision . in the emotional trauma case , which might equally be applied to cases of depression , a diachronic perspective is used to identify a problem in the person 's ability to assess whether what they want or value is achievable . according to jodi halpern 's original analysis of the emotional trauma case , the woman should have been judged to lack capacity on grounds that her beliefs about the future were unresponsive to evidence , rendering her unable to think through alternatives ( halpern , 2012 , pp .
the tragedy is that in this case therapy or treatment might have corrected her assumptions regarding future quality of life , and a decision to accept the lifesaving treatment might have resulted. ( 2011 , p. 33
; for a full description of the case , see halpern , 2012 ) one problem with adopting a diachronic perspective in assessments of mental capacity is that whether questions of procedural rationality are properly understood in diachronic terms remains a contested issue in the fields of economics , psychology and philosophy .
the functional element of the mca 's incapacity test concerns problems in meeting certain procedural requirements problems in the ability to understand , retain , use or weigh relevant information .
sceptics deny that at least some norms derived from a diachronic perspective fall into this category .
they hold that such requirements are substantive rather than procedural in nature , so i turn now to this theoretical concern .
much of the normative and descriptive work on a diachronic perspective in personal decision - making has taken place in the fields of economics and psychology , but these fields have tended to adopt contrasting positions on the normative question .
economists often treat a person 's weighting of the future as a preference like any other , in a framework that assumes that preferences are not available to rational criticism .
prudence to refer to taking a diachronic perspective in decision - making:prudence can not on this view be explained merely by the perception that something is in one 's future interest ; there must be a desire to further one 's future interests if the perception is to have an effect
there seems little doubt that most people have the desire that makes prudence possible [ however , according to this view we are not ] in any sense required to possess the desires in question : consequently we are not required to act on the specified considerations . if one lacks the relevant desire , there is nothing more to be said .
( nagel , 1970 , p. 28)psychologists , on the other hand , often assume that people are rationally required to give significant weight to the future personal consequences of choices , the strongest version being that people ought to be temporally neutral in their decision - making .
it is widely accepted within this camp that there are at least some good reasons to give less weight to future consequences , particularly in order to take into account an expected depreciation in their future value .
. however , foreseeable changes in one 's circumstances can also be relevant : 50 might be much more valuable to a final year law student than 100 will be for them in the following year , when they reasonably expect to be working for a corporate lawyer . on these grounds
it might be rational for the student to choose 50 now , foregoing 100 in a year 's time .
likewise , it is argued that uncertainly with respect to future consequences can provide a good reason to choose a certain outcome now , over a more valuable but uncertain outcome in the future ( baron , 1994 , pp .
i am using the term temporally neutral to refer to decisions that give equal weight to future and present utility , excepting the above kinds of considerations .
the pressing question is why people ought to be impartial with respect to all parts of their lives once these kinds of consideration are taken into account .
prudence can not on this view be explained merely by the perception that something is in one 's future interest ; there must be a desire to further one 's future interests if the perception is to have an effect
there seems little doubt that most people have the desire that makes prudence possible [ however , according to this view we are not ] in any sense required to possess the desires in question : consequently we are not required to act on the specified considerations . if one lacks the relevant desire , there is nothing more to be said .
( nagel , 1970 , p. 28 ) the arguments used to defend this position in the psychological literature tend to take the form that a temporally neutral orientation advances the goals of practical decision - making .
choosing an outcome merely because it will happen sooner is said to be irrational because if one option achieves your goals better , then you should choose that one , regardless of when you decide ( baron , 1994 , p. 514 ) . using the classic marshmallow experiments as an example ,
if a child would rather eat two marshmallows than one , then they should choose to wait and be given two marshmallows sometime later rather than one marshmallow now because overall this achieves the child 's goals better .
this kind of argument considers the question of goal satisfaction from a position that is not situated at a particular point in time , and prescribes that people ought to take this perspective in their decision - making : they should be just as concerned about themselves a year from now as they are about themselves this minute ( p. 516 )
. individuals should give weight to the implications for their future self , so the argument goes , for their own overall good .
nagel argues that prudence is a requirement of practical reason , which is secured because the personal consequences of a man 's action concern his future ( 1970 , p. 42 ; his italics ) .
according to nagel , this constraint on practical decision - making follows from the person 's awareness that they persist over time .
it reflects the individual 's conception of himself as a temporally persistent being : his ability to identify with past and future stages of himself and to regard them as forming a single life .
failure to be susceptible to prudence entails radical dissociation from one 's future , one 's past , and from oneself as a whole , conceived as a temporally extended individual ( p. 58 ) .
it is a mistake in practical reasoning because it comes at the cost of dissociation from one 's temporally extended self ( p. 69 ) .
the conception of self as extended throughout a human lifetime is powerful in the context of questions of procedural rationality because it offers grounds for claiming that a person ought to give weight to considerations that they may not currently care about .
it offers an answer to what i have characterised as the economists ' view , that if a person has no interest in the future personal consequences of their choice there is nothing more to be said , rationally speaking .
applied to the context of treatment decision - making , this kind of perspective is powerful because it offers grounds for calling a patient 's mental capacity into question , with reference to their ability to consider their future and value it appropriately .
rejections of this kind of rational requirement are therefore often based on a rejection of the idea of the self as something that necessarily extends throughout a lifetime , and derek parfit 's account of personal identity is often used to make this argument ( baron , 1994 , p. 516 ; frederick , 2006 ; maclean , 2006 ) .
parfit holds that the persistence of a person over time must be understood in terms of their
psychological continuity the connectedness of psychological features such as memories , character , interests and preferences across a lifetime ( parfit , 1983 ; 1984 ) . on this view , whether the future inhabitant of one 's body will be the same person can be a matter of degree . psychological features sometimes change so radically over a human lifetime that a person now will not necessarily be the same person who occupies that body in the future . and
not giving weight to the interests of a future self is irrational only to the degree that one shares the relevant psychological characteristics with that self .
more recently , galen strawson has defended a similar position in his rejection of narrative accounts of the self .
strawson argues that although experiencing oneself as one person throughout a lifetime what he calls a
diachronic self - experience may be more common , some people experience themselves in what he describes as a more episodic way . unlike the more diachronic person ,
the more episodic person has little or no sense that the self that one is was there in the ( further ) past and will be there in the future , although one is perfectly aware that one has long - term continuity considered as a whole human being ( strawson , 2005 , p. 65 ) . [ predominantly ] episodic individuals may sometimes connect to charged events in their pasts in such a way that they feel those events happened to them embarrassing memories are a good example and anticipate events in their futures in such a way that they think those events are going to happen to them thoughts of death can be a good example
however , the episodic person has no great or special interest in [ their ] past , and nor do they have a great deal of concern for [ their ] future ( p. 67 ) .
strawson holds that even a strongly episodic self is within the normal range of human experience , and that it is normatively on a par with a more diachronic self - experience . on his view
, theorists such as nagel assert that selves extend over a lifetime , and diachronic norms of procedural rationality are derived from this claim .
parfit and strawson dispute this assumption , and conclude that disregard for the future personal consequences of a choice may simply reflect an alternative way of living a human life , at least when we consider the question of reasons for action in non - moral terms .
however , all of the theorists canvassed thus far reason from premises about the extension of self over a lifetime , to conclusions about what reasons individuals have , and it will be argued here that this conception of the relationship between the requirements of practical rationality and the metaphysics of self is the wrong way around . as described above , the kind of requirement in question is often defended by nagel and others on the grounds that not giving weight to the future undermines one 's own interests . however , at times a different kind of argument is suggested in nagel 's work .
the suggestion seems to be that a unified self over a lifetime is not something that is automatically present , but rather something that is generated by giving weight to future personal consequences in decision - making .
the proposed requirement is justified , not because it deploys the correct conception of self , or because one 's future self will otherwise regret the decision , but because adherence plays a role in bringing the self into being , by enabling the decision - maker to reach towards something outside himself ( nagel , 1970 , p. 43 ) .
michael bratman is particularly clear about the role that he proposes giving weight to the future plays in the generation of the self , and how this in turn grounds a diachronic perspective on questions of procedural rationality , so it is his view that i will focus on here .
self - governing policies which are integral to temporally extended agency lie at the core of human agency because it is through these that a person 's identity emerges .
these processes are essential for understanding what it is for an agent to take a stand what it is for an agent to recognise certain desires as one 's own and it is this feature in particular that distinguishes human agency from the agency of other purposive creatures ( bratman , 2000 , pp .
adopting broadly the same approach to personal identity as parfit begins with , bratman suggests that the forward - looking psychological ties that play a role in constituting a unified self over time can be generated through reflective and planning processes :
i can help ensure appropriate psychological continuities and connections by sticking with and executing my prior plans and policies , and by monitoring and regulating my motivational structures in favor , say , of my continued commitment to philosophy
such processes play an important role in the constitution and support of continuities and connections characteristic of the identity of the agent over time.
indeed, bratman suggests , this is what plans and policies are for ( 2000 , p. 47
by emphasising the role that planning and policy - making play in generating the psychological ties that are proposed to underpin personal identity over time , bratman describes a picture of human agency in which the agent plays an active role . the temporally extended self develops through the agent 's exercise of certain psychological capacities . on this point bratman
makes a significant departure from parfit and strawson , who seem to hold that the degree to which the relevant psychological ties extend over time is a fixed fact about the individual , from which rational requirements on their action may be derived .
the claim that engaging in planning and policy - making processes is rationally required is defended primarily on the non - instrumental grounds that these processes play a central role in developing and sustaining the self .
bratman 's argument takes the form of an answer to the problem that agents who reflect on their first - order desires and form second - order desires do not yet exhibit what is distinctive about human agency , because there is nothing to distinguish these new desires as those that the agent identifies with ; second - order desires seem to be
just one more wiggle in the psychic stew ( bratman , 2000 , p. 38 ) .
the solution that bratman offers is that where an agent stands with respect to a particular first - order desire is established through the exercise of planning and policy - making practices : self - governing policies might , so to speak , crystallize pressures from various elements of one 's psychic stew into a more decisive attitude that can , in the relevant context , establish where one stands ( 2000 , p. 51 ) .
these mental processes that underpin temporally extended agency give authority to particular desires , so it is through these that the self the distinctive feature of human agency emerges . on this view , a compromised ability to engage in planning and policy - making processes threatens not the interests of the future self , but the very constitution of the self .
this is what grounds the procedural requirement that one must give weight to the future personal consequences of actions . and this , we might suggest , is what makes the ability to consider and value the future an appropriate consideration to include in a mental capacity test .
bratman 's account offers a story about the self that fits well with a developmental understanding of personhood that is often appealed to in contemporary medical law and ethics .
his account of the relationship between temporally extended agency and the self also fits well with the difficulties associated with certain psychopathologies .
it is no accident , i suggest , that disorders such as dementia , bipolar disorder and depression are associated with seriously compromised capacities for diachronic agency . among many other factors , in a complex picture , problems of diachronic agency surely make a contribution to our sense that these constellations of behavioural and experiential features are appropriate targets for the label of disorder .
a sense of the self as compromised in the context of problems of diachronic agency is also found in first - person reports of dementia ( addis and tippett , 2004 ; however , see caddell and clare , 2012 ) and depression ( fuchs , 2008 ; ratcliffe , 2012 ) .
it would seem that the functioning of capacities for diachronic agency is connected to our sense of others as having well - functioning human agency , as well as our subjective sense of selfhood , and for these reasons
i find it implausible that a strongly episodic self - experience is just a less common way of living a human life .
strawson defends a view along these lines , claiming that he himself is strongly episodic ( in his sense ) .
yet he has a successful academic career which could not have been achieved without meeting a wide range of commitments that each extended over considerable periods of time , for example in completing academic qualifications and writing books .
it seems to me that these achievements evidence considerable skills in diachronic agency . while for strawson a diachronic self - experience is connected to a narrative conception of the self , diachronicity in the sense concerned here
a life lived in the moment could still be a life lived with diachronic agency , as i will argue at the end of the following section .
i now turn back to questions of mental capacity and what implications a diachronic perspective such as bratman 's would have .
the motivation for investigating these theoretical questions about procedural rationality was the idea that a diachronic perspective might enable a mental capacity test such as that adopted in the mca to deal with certain hard cases , particularly in the context of mental disorder , without undermining its value - neutral approach .
the arguments above suggest that a diachronic perspective on questions of mental capacity is consistent with a procedural understanding of the functional element of the mca 's incapacity test .
given this , could a diachronic understanding of what it is to have mental capacity help by justifying the inclusion of a requirement such as the ability to consider the future as a part of what it means to be able to understand , retain , use or weigh information in assessments of mental capacity ?
something like this requirement already seems to be assumed in the condition that the patient must understand the consequences of the treatment options .
the relevant information necessarily concerns the future as well as the present , due to the fact that understanding the likely effects of deciding one way or the other is a part of this requirement ( mental capacity act 2005 code of practice , s. 4.16 ) .
for example , a person with a severe learning disability might be judged to lack the capacity to consent to a blood test on grounds that they do not understand that the test is necessary for their future physical well - being .
the same might be said in a case of a delirious patient who is resisting treatment for a serious injury . in such cases the person 's connection to the world , in the sense of their ability to understand how things are likely to be in the future ,
what to do is limited in a way that can be compared to john stuart mill 's paradigm case of justified paternalistic intervention , where a person is about to cross a bridge that unbeknownst to them is unsafe
. the person in the example does not understand that they risk falling into water if they attempt to cross .
the widely accepted conclusion is that the person is not in a position to assess whether to cross the bridge , and this justifies the use of proportional physical force to stop them from crossing , when this is necessary to inform them about the state of the bridge .
the same reasoning might be applied in the case of the woman who has recently suffered a serious emotional trauma , on grounds that her ability to imagine a range of future possibilities is severely limited ( halpern , 2012 ) .
the patient can not currently conceive of alternative futures to the desperately unhappy one she imagines , and perhaps this justifies taking the decision out of her hands until she is seeing things more clearly .
the problem that returns here is that understanding concerns not simply how things are likely to be , as in the fact that an attempt to cross the bridge is likely to result in a fall into water , or that a certain proportion of people who have both legs amputated learn to walk again using prosthetic limbs .
it concerns understanding how the relevant state of affairs will be for this particular patient .
this question about the future necessarily involves an evaluative judgment that , according to a value - neutral approach , only the patient is in a position to make .
so while it might sometimes be clear that a patient 's ability to consider the future is compromised , for example when someone denies that their condition is life - threatening when it clearly is , in many cases assessing understanding in relation to the future will have no equivalent point of reference .
in such a context there seems to be no standard against which the functional element of the mental capacity test the part concerning the patient 's ability to understand , retain , use or weigh information can be directly assessed .
therefore , any conclusions drawn about this capacity would seem to be based on the fact that the patient has suffered an emotional trauma . removing a patient 's right to make their own treatment decision under these circumstances may seem justified given what we know about how people who lose limbs adapt , as reflected in self - reported measures of well - being , and given the known psychological effects of emotional trauma
. however , an appeal to this kind of knowledge in an assessment of mental capacity would involve using generalised observations to draw a conclusion about a particular person 's capacity to assess how things will be for them in the future .
the legislation is clear that an assessment of decision - making ability should not be based solely on features such as a patient 's diagnosis .
it must be shown that this particular person 's mental capacities are relevantly impaired , not merely that they belong to a diagnostic category . however
, this requirement can not be met in the case of this capacity , because there is no way to directly assess it while remaining value neutral in accordance with the aspirations of this area of law . what about the possibility raised at the beginning of this article , that a person 's evaluative capacities might be called into question when viewed from a diachronic perspective ?
drawing on bratman 's account , planfulenss and self - governing policies are rationally required because of the role they play in developing and sustaining the self .
caring about the interests of the future inhabitant of one 's body , and having at least some grip on what those interests will be , seem like prerequisites for engaging successfully in these processes . and perhaps this offers part of an explanation about how decision - making can be compromised in the context of disorders such as depression and bipolar disorder .
very crudely , the idea would be that as part of a much bigger picture the capacities necessary for diachronic agency are not functioning properly and this undermines the constitution of the self .
i have argued that , understood in this way , these considerations are procedural rather than substantive in nature .
but would the implementation of these considerations in practice nonetheless come into conflict with the goal of preserving patient autonomy ? assessing a person 's grip on what interests the future inhabitant of their body will have runs into the same difficulties that occurred in the context of assessing understanding in the emotional trauma case . without making substantive assumptions
judging that a person is likely to lack these capacities in the context of a manic episode may seem justified , because of what we know about the trajectory of bipolar disorder that fairly reliably people 's commitments during a manic episode radically undermine their overall interests .
but whether this kind of consideration provides grounds for a judgment of incapacity in english law depends on what evidence is considered appropriate for assessing the functional element of the incapacity test .
however , as genevra richardson points out , existing case law indicates that courts will take a flexible approach to the legal definition [ of capacity ] to enable them to reach their preferred outcome ( richardson , this volume , p. 00 ) .
this may mean that in practice significant weight is given to a person 's diagnosis .
however , this solution undermines the protections of patient autonomy conferred by making an individual assessment of functional capacities , rather than diagnosis , the proper grounds for infringements on self - determination .
a related possibility considered at the beginning of this article was that a diachronic perspective might be used to justify giving weight to a reasonable prediction of what the patient 's future self would want in retrospect , in assessments of mental capacity .
however , it does not follow from the view offered here , even given the sure knowledge that a patient 's future self would retrospectively endorse a medical intervention , that their present self is irrational in refusing it . in the imagined case there is an unambiguous conflict between what the patient currently wants ( no treatment ) and what the future self will want in retrospect ( treatment ) . using this conflict to doubt the patient 's current capacity to make the treatment decision assumes that the future self 's perspective should be given greater weight in the current self 's decision - making .
however , there is nothing in the present view that would justify resolving the conflict in this way .
the proposed account requires that the agent exercise planfulness and self - governing policies in decision - making , which would seem to involve giving equal weight at most , not greater weight , to one 's expected future desires .
moreover , humans have a well - established time preference for negative experiences to be in the past rather than the present or future , and this makes it easier for the patient to endorse involuntary treatment when it is in the past ( doherty , 2011 ) .
this would seem to weigh in favour of privileging the perspective of the self who is facing the infringement on liberty . in the absence of any reasons to believe that the future self 's perspective should be privileged
, this form of justification for doubting mental capacity looks to be question - begging .
its favouring of the future self 's perspective already assumes that the current self 's decision - making capacity is relevantly impaired .
favouring the future self 's perspective might seem justified on grounds that , for example , evaluative capacities are often compromised in the context of certain diagnoses or during experiences such as emotional trauma .
however , at least in theory , this evidence alone does not justify a judgment of incapacity under current english law , for reasons of preserving patient autonomy as argued above .
finally , i turn to the question of caring about future personal interests , especially in the context of life - threatening choices .
a central difficulty associated with including a consideration of this kind in questions of mental capacity is the problem of how to assess caring simpliciter , independently of caring about a particular outcome . according to the account offered here , procedural rationality requires that we care about the future personal consequences of choices , but what in the future we should give weight to is left open .
specifying this in assessments of mental capacity would mean that the test was not value neutral .
the case of the committed smoker ( exemplified by david hockney ) can be used to illustrate the point .
one criticism often levelled at smokers is that they are irrational because they do not care sufficiently about the future personal consequences of their smoking .
however , this criticism assumes that future health , or a long life , is what the person should care about . considering a person 's decision to keep smoking from a value - neutral perspective , which makes no assumptions of this kind ,
are they being short - sighted ? or do they care about things other than health and long life in a way that makes their decision rational ?
bratman 's account shows how asking this second question can be consistent with a diachronic perspective on rationality .
it is clear that the committed smoker is very likely undermining the health of the future inhabitant of his body .
however , it does not follow that he is not engaging in the psychological processes that underpin temporally extended agency .
it may be a central feature of the person 's life plan that he should not be overly concerned about living a long life
. his deep and enduring commitment might be to a hedonistic lifestyle that is incompatible with being concerned about health in old age .
for such a person , not living fully enough is a more likely source of future regrets than health - endangering behaviours . caring about one 's future might even require , in such a case , life - threatening or health - threatening choices .
parallel arguments could be made in the case of a decision by a jehovah 's witness to refuse a blood transfusion ; for a political activist 's hunger strike ; for a person who risks their life in an attempt to save a loved one ; and a freediver 's commitment to their dangerous extreme - sports lifestyle .
these kinds of decision do not seem characterized by a lack of planfulness about the future . rather than undermining the sense of oneself as a temporally persisting agent
, decision - making in line with these kinds of commitment looks likely to contribute to the overlapping webs of cross - temporal connections and continuities that are proposed to play a role in constituting human agency ( bratman 2000 , pp .
the future self taken into consideration in these cases may be a nearer self than in the case of a person who is invested in living a long life because the artist who is a committed smoker , for example , might not expect to or want to live a long life
nothing in the above account enjoins us to aim for a life of a particular length .
what it requires is that , at least to some degree , choices be made with a reflectivity about the future , such that insofar as we are agents that agency is temporally extended .
this perspective allows us to reconcile the ideas that mental capacity requires that we care about the future personal consequences of our choices , and that life - ending decisions can be made with mental capacity .
however , against this theoretical background , resolving questions about whether a patient 's capacity to care about the future is compromised would seem to require looking beyond their procedural capacities .
answers to this question will rest on views about whether or not the life - threatening decision is consistent with the person 's commitments , understood from a diachronic perspective .
these factors are taken into account in an assessment of best interests once it is decided that a patient lacks mental capacity ( mca s. 4(6 ) ) .
however , if the ability to care about the future is included as part of what it means to have mental capacity , then consideration of a patient 's commitments from a diachronic perspective will also be central to this prior decision .
it has been argued here that a diachronic perspective on mental capacity is relevant to questions about when an infringement on treatment decision - making liberty is justified . however , including considerations derived from a diachronic perspective in assessments of capacity comes into conflict with central patient autonomy - preserving features of the mca .
it 's worth reflecting , then , on why a diachronic perspective might be morally relevant in this context .
i will suggest that the answer lies in a common view about what makes patient autonomy valuable what makes it the case that for the most part , people 's treatment decisions , especially their refusals , ought to be respected .
the importance of allowing patients to be self - determining in relation to their own medical care is often justified along lines that it
protects people 's general capacity to lead their lives out of a distinctive sense of their own character , a sense of what is important to and for them ( dworkin , 1994 , p. 224 ) .
different theorists argue for the value of patient autonomy on importantly different grounds , but a common theme is that treatment decisions should be respected because of their assumed connection to the self .
however , in a case where human agency is seriously compromised in the way described by bratman , selfhood is undermined .
capacities for diachronic agency that play a central role in establishing what 's important for the person where the person stands are compromised . and
in the context of such problems , the importance placed on patient autonomy in liberal societies would seem to give medical professionals a reason to do what they can to restore selfhood , perhaps even if this requires an infringement on personal liberty .
this might involve overriding a refusal of treatment , for example , though the justification for doing so would only warrant interventions of a particular kind
those that are likely to restore the general capacity that the principle of patient autonomy aims to protect .
this suggests that in some cases , especially in the context of mental illness , the importance placed on patient autonomy in contemporary medical ethics presents the law with a dilemma . on the one hand
, it seems right that laws which license infringements on decision - making liberty should equally apply to everyone , not just to particular groups ; and that if a patient has mental capacity in relation to a particular treatment decision then they should be free to direct the course of their medical care .
consequently , it seems we ought to do away with dedicated mental health laws that legalise detention and involuntary treatment without reference to mental capacity only in the context of a mental disorder . on the other hand , the way in which diachronic agency is compromised in some instances especially in the context of mental disorder
seems to justify or perhaps even require an infringement on liberty to restore the self .
these grounds are difficult to take into consideration in assessments of mental capacity without compromising features of the mca that are designed to preserve patient autonomy .
so while it might be possible to implement the mca in a way that sufficiently accounts for the problems of decision - making and selfhood that are sometimes present in mental disorders , doing so comes at the price of what is seen to be a central virtue of the legislation .
in this article my focus was requirements of rationality derived from a diachronic perspective , and the question of what kinds of consideration this perspective would enable mental capacity assessments to include . it was an attempt to explore the limits of the mca 's incapacity test , given its value - neutral approach .
it was argued that considerations derived from a diachronic perspective are relevant to questions of treatment decision - making liberty , and are consistent with a procedural understanding of the functional element of the test .
however , in practice , appeal to these considerations in assessments of mental capacity undermines the role played by the functional element of the test in preserving patient autonomy . while involuntary treatment without reference to mental capacity is often assumed to undermine the principle of respecting patient autonomy , it was argued here that this is not necessarily so .
when human agency is seriously compromised , preserving patient autonomy may sometimes require overriding a treatment decision to restore selfhood , on grounds that are not easily accounted for under the mca without compromising the value - neutral aspirations of this area of law .
in considering a move to abandon dedicated mental health law , legislators must therefore resolve a bind .
the central motivation for such a move is concern about unjustified infringements on liberty in the context of psychiatric diagnoses .
the worry is that the protections of patient autonomy in the mca do not currently extend to the treatment of mental disorders , because mental health legislation allows people to be treated for a mental disorder even if they refuse with mental capacity .
however , considerations derived from a diachronic perspective that seem relevant to when an infringement on decision - making liberty is justified are especially present in mental disorder .
the inclusion of these considerations seems necessary for adequately caring for those with a mental disorder under universal capacity legislation . yet including these factors in assessments of mental capacity undermines the protections of patient autonomy that supporters of a shift to universal capacity legislation hope to gain .
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calls for the adoption of a universal capacity approach to replace dedicated mental health law are motivated by the idea that the measures designed to protect patient autonomy in legislation such as the mental capacity act 2005 should apply to everyone , including people with a psychiatric diagnosis . in this article
it is argued that a diachronic perspective on questions of mental capacity is necessary if capacity law is to play this broader role , but that employing this perspective in assessments of capacity undermines central patient autonomy preserving features of the legislation , which presents a moral dilemma .
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Mental capacity and the ability to consider the future
Are we rationally required to value the future?
Including the ability to consider the future in assessments of mental capacity
A dilemma about what respect for patient autonomy requires
Conclusion
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exacerbation is an important life - threatening event for patients with copd , and can lead to hospitalization and death.14 patients who suffer frequent and repeated exacerbations within 1 year have a poor prognosis,5 characterized by worsening of health - related quality of life ( hrqol),6,7 a rapid decline in lung function,810 and high mortality.11 frequent exacerbators also carry a high risk of further exacerbation and hospitalization.11,12 however , it has been suggested that japanese patients with copd may have fewer exacerbations , and they also may have a higher proportion of elderly patients , those with emphysema , and those with a lower body mass index in comparison to westerners.1215 the prognosis of japanese patients with copd who suffer frequent and repeated exacerbations is unclear .
we conducted a 1-year prospective observational trial in a daily - life setting involving 90 japanese patients with copd to investigate whether previous moderate - to - severe exacerbations are associated with future exacerbations in this patient population .
we conducted a 1-year prospective observational trial in accordance with good clinical practice ( gcp ) guidelines and approved by the ethics committee of kurume university and chikugo city hospital ( gcp 11 - 127 , september 2012august 2014 ) .
consecutive patients for whom medical records were available covering a period of at least 1 year since provision of informed consent were selected for the study ; information on previous annual copd - related exacerbations and hospitalizations was collected on the basis of those medical records .
copd patients were divided into three groups , based on the total number of moderate and severe exacerbations within the last year before enrollment in the study , ie , non- ( previous moderate and severe exacerbations , 0/year ) , infrequent ( one exacerbation / year ) , and frequent ( two or more exacerbations / year ) exacerbator groups , in accordance with a previous report.16 in addition , patients with previous hospitalizations were classified as having a subphenotype with severe exacerbation ( severe exacerbators ) .
the data collected for each patient included baseline data for previous moderate and severe exacerbations and hospitalizations ; clinical parameters included age , sex , body mass index , smoking habits , smoking index , comorbidities , duration of copd , 5-grade modified medical research council ( mmrc ) dyspnea scale score,17 total copd assessment test ( cat ) score,18,19 frequency scale for symptoms of gastroesophageal reflux disease ( gerd ) ( fssg),20 center for epidemiologic studies depression ( cesd ) scale score,21 medications , blood pressure and heart rate , lung function and blood parameters , and chest computed tomography .
duration of copd was defined as the period ( years ) since the patient had been diagnosed by a physician as having copd , emphysema , and/or chronic bronchitis.22,23 after stable status for at least 4 weeks had been confirmed , each patient was required to regularly visit the hospital every 2 months , and to request emergency admission when the symptoms worsened .
regular respiratory medications were not changed during the period of the study , which was conducted in a daily - life setting .
the diagnosis of copd was based on age 40 years , smoking index > 10 pack - years , forced expiratory volume in 1 second ( fev1)/forced vital capacity ( fvc ) ratio of < 0.7 after bronchodilator administration , and the spirometric gold ( global initiative for chronic obstructive lung disease)-stage classification , ie , stage i ( fev1 80% predicted ) , ii ( 50% fev1 < 80% predicted ) , iii ( 30% fev1 < 50% predicted ) , and iv ( fev1 < 30% predicted).1 chest computed tomography confirmed that all patients had low - attenuation areas .
to exclude any patients with asthma , patients who had past symptoms of repeated spasmodic wheezes and medications for asthma and a classification of fev1 > 200 ml or > 12% after bronchodilation were excluded.24 diagnosis of asthma
copd overlap syndrome ( acos ) was made on the basis of a history of dyspnea and wheezing attacks at rest , large variations in daily symptoms , a fixed fev1/fvc ratio of < 0.7 , marked reversibility of fev1 after administration of bronchodilators ( > 15% and > 400 ml ) , and a peripheral eosinophil count of > 600/mm , in accordance with a previous report.25 baseline data for the mmrc scale , total cat , fssg scale , and cesd scale scores were obtained only once , based on a self - report completed by each patient after written informed consent had been obtained.1721 information about comorbidities was obtained from the patients by interview , and the diagnoses were confirmed by physicians .
however , patients who had moderate - to - severe comorbidities associated with poor prognosis , such as active malignancies , depression ( cesd scale > 16 points),21 liver cirrhosis , digestive ulcers , persistent arrhythmia , congestive heart disease , coronary artery disease , lung fibrosis and bronchiectasis , chronic renal failure requiring dialysis , and central nervous system disorders , including palsy and dementia , were excluded , in accordance with previous studies.26,27 hypertension ( systolic > 140 mmhg or diastolic > 90 mmhg blood pressure or use of medications ) , dyslipidemia ( serum low-[14 ] or high- [ < 4 ]
15 mg / l , or use of medications ) , diabetes ( blood hemoglobin a1c 6.5% national glycohemoglobin standardization program value or use of medications)28 without triopathy ( neuropathy , retinopathy , and nephropathy ) , and gerd ( fssg scale > 7 points)20 were accepted as comorbidities .
previous and prospective exacerbations documented in the medical records made by physicians were accepted as moderate and severe events .
moderate exacerbations that required a prescription for antibiotics and/or systemic corticosteroids were defined on the basis of symptom - based diagnosis , such as increased cough and sputum production , a change in sputum color , and worsening of dyspnea from a stable state and beyond normal day - to - day variations , ie , showing acute onset and necessitating a change in regular medication , in accordance with previous reports.1,29,30 copd - related and other causes of death and hospitalization were prospectively followed for 1 year .
hospitalization was decided by each examining physician when hypoxemia required additional or intensive oxygen and/or assisted ventilation therapy , performance status was 3 , and unconsciousness occurred with copd exacerbation.1,23,31 however , pneumonia was also recognized as exacerbation .
mild and unreported exacerbations were not considered to have been equal in severity to previous and prospective exacerbations . however , mild exacerbations were defined as those that improved naturally without any medication or administration of inhaled short - acting bronchodilators .
unreported exacerbations were considered to be those to which patients had been insensitive , or those that had been self - controlled in spite of worsening of respiratory symptoms .
data are expressed as mean standard deviation ( sd ) and number ( percentage ) of non- , infrequent , and frequent exacerbators at baseline .
characteristics were compared using analysis of variance , fisher s exact test , -test for trend , and tukey
subsequent moderate and severe exacerbation events during 1 year of prospective observation were recognized as future risk indicators , and baseline parameters were chosen and modified in accordance with a previous report.16 kaplan meier analyses and log - rank tests for subsequent moderate and severe exacerbations were performed in all three groups of patients . for patients who were observed throughout the study period , the odds ratio ( 95% confidence interval [ ci ] ) of baseline parameters was determined to predict factors for future risk ( subsequent exacerbations once or more and twice or more ) using univariate and logistic multivariate regression analyses .
medians of age , body mass index , smoking index , and duration of copd were 68 years , 21.4 kg / m , 53.5 pack - years , and 4 years , respectively , and each median was used as the cutoff value for the analysis .
statistical analysis was performed by the jmp version 9.0 statistical software package ( sas institute inc , cary , nc , usa ) .
we conducted a 1-year prospective observational trial in accordance with good clinical practice ( gcp ) guidelines and approved by the ethics committee of kurume university and chikugo city hospital ( gcp 11 - 127 , september 2012august 2014 ) .
consecutive patients for whom medical records were available covering a period of at least 1 year since provision of informed consent were selected for the study ; information on previous annual copd - related exacerbations and hospitalizations was collected on the basis of those medical records .
copd patients were divided into three groups , based on the total number of moderate and severe exacerbations within the last year before enrollment in the study , ie , non- ( previous moderate and severe exacerbations , 0/year ) , infrequent ( one exacerbation / year ) , and frequent ( two or more exacerbations / year ) exacerbator groups , in accordance with a previous report.16 in addition , patients with previous hospitalizations were classified as having a subphenotype with severe exacerbation ( severe exacerbators ) .
the data collected for each patient included baseline data for previous moderate and severe exacerbations and hospitalizations ; clinical parameters included age , sex , body mass index , smoking habits , smoking index , comorbidities , duration of copd , 5-grade modified medical research council ( mmrc ) dyspnea scale score,17 total copd assessment test ( cat ) score,18,19 frequency scale for symptoms of gastroesophageal reflux disease ( gerd ) ( fssg),20 center for epidemiologic studies depression ( cesd ) scale score,21 medications , blood pressure and heart rate , lung function and blood parameters , and chest computed tomography .
duration of copd was defined as the period ( years ) since the patient had been diagnosed by a physician as having copd , emphysema , and/or chronic bronchitis.22,23 after stable status for at least 4 weeks had been confirmed , each patient was required to regularly visit the hospital every 2 months , and to request emergency admission when the symptoms worsened .
regular respiratory medications were not changed during the period of the study , which was conducted in a daily - life setting .
the diagnosis of copd was based on age 40 years , smoking index > 10 pack - years , forced expiratory volume in 1 second ( fev1)/forced vital capacity ( fvc ) ratio of < 0.7 after bronchodilator administration , and the spirometric gold ( global initiative for chronic obstructive lung disease)-stage classification , ie , stage i ( fev1 80% predicted ) , ii ( 50% fev1 < 80% predicted ) , iii ( 30% fev1 < 50% predicted ) , and iv ( fev1 < 30% predicted).1 chest computed tomography confirmed that all patients had low - attenuation areas .
to exclude any patients with asthma , patients who had past symptoms of repeated spasmodic wheezes and medications for asthma and a classification of fev1 > 200 ml or > 12% after bronchodilation were excluded.24 diagnosis of asthma
copd overlap syndrome ( acos ) was made on the basis of a history of dyspnea and wheezing attacks at rest , large variations in daily symptoms , a fixed fev1/fvc ratio of < 0.7 , marked reversibility of fev1 after administration of bronchodilators ( > 15% and > 400 ml ) , and a peripheral eosinophil count of > 600/mm , in accordance with a previous report.25
baseline data for the mmrc scale , total cat , fssg scale , and cesd scale scores were obtained only once , based on a self - report completed by each patient after written informed consent had been obtained.1721
information about comorbidities was obtained from the patients by interview , and the diagnoses were confirmed by physicians .
however , patients who had moderate - to - severe comorbidities associated with poor prognosis , such as active malignancies , depression ( cesd scale > 16 points),21 liver cirrhosis , digestive ulcers , persistent arrhythmia , congestive heart disease , coronary artery disease , lung fibrosis and bronchiectasis , chronic renal failure requiring dialysis , and central nervous system disorders , including palsy and dementia , were excluded , in accordance with previous studies.26,27 hypertension ( systolic > 140 mmhg or diastolic > 90 mmhg blood pressure or use of medications ) , dyslipidemia ( serum low-[14 ] or high- [ < 4 ]
15 mg / l , or use of medications ) , diabetes ( blood hemoglobin a1c 6.5% national glycohemoglobin standardization program value or use of medications)28 without triopathy ( neuropathy , retinopathy , and nephropathy ) , and gerd ( fssg scale > 7 points)20 were accepted as comorbidities .
previous and prospective exacerbations documented in the medical records made by physicians were accepted as moderate and severe events .
moderate exacerbations that required a prescription for antibiotics and/or systemic corticosteroids were defined on the basis of symptom - based diagnosis , such as increased cough and sputum production , a change in sputum color , and worsening of dyspnea from a stable state and beyond normal day - to - day variations , ie , showing acute onset and necessitating a change in regular medication , in accordance with previous reports.1,29,30 copd - related and other causes of death and hospitalization were prospectively followed for 1 year .
hospitalization was decided by each examining physician when hypoxemia required additional or intensive oxygen and/or assisted ventilation therapy , performance status was 3 , and unconsciousness occurred with copd exacerbation.1,23,31 however , pneumonia was also recognized as exacerbation .
mild and unreported exacerbations were not considered to have been equal in severity to previous and prospective exacerbations . however , mild exacerbations were defined as those that improved naturally without any medication or administration of inhaled short - acting bronchodilators .
unreported exacerbations were considered to be those to which patients had been insensitive , or those that had been self - controlled in spite of worsening of respiratory symptoms .
data are expressed as mean standard deviation ( sd ) and number ( percentage ) of non- , infrequent , and frequent exacerbators at baseline .
characteristics were compared using analysis of variance , fisher s exact test , -test for trend , and tukey
subsequent moderate and severe exacerbation events during 1 year of prospective observation were recognized as future risk indicators , and baseline parameters were chosen and modified in accordance with a previous report.16 kaplan meier analyses and log - rank tests for subsequent moderate and severe exacerbations were performed in all three groups of patients . for patients who were observed throughout the study period ,
the odds ratio ( 95% confidence interval [ ci ] ) of baseline parameters was determined to predict factors for future risk ( subsequent exacerbations once or more and twice or more ) using univariate and logistic multivariate regression analyses .
medians of age , body mass index , smoking index , and duration of copd were 68 years , 21.4 kg / m , 53.5 pack - years , and 4 years , respectively , and each median was used as the cutoff value for the analysis .
statistical analysis was performed by the jmp version 9.0 statistical software package ( sas institute inc , cary , nc , usa ) .
ninety of 110 patients who provided informed consent were finally analyzed ; 32 ( 35.6% ) patients had suffered previous moderate and/or severe exacerbations during the last year .
the numbers of patients with non- , infrequent , and frequent exacerbations were 58 ( 64.4% ) , 12 ( 13.3% ) , and 20 ( 22.2% ) , respectively ( figure 1 ) . at baseline , frequent exacerbators had a significantly lower body mass index , were less likely to be male or current smokers , had higher mmrc - scale grades and higher total cat score , had a higher proportion of spirometric stages iii and iv , gerd , and use of inhaled corticosteroid ( ics)/long - acting muscarinic antagonist or ics / long - acting 2-agonist combination therapy , and lower lung - function parameters , including fev1 , fvc , and fev1/fvc ratio before and after bronchodilator use compared with nonexacerbators , but not in comparison with infrequent exacerbators ( table 1 ) .
frequent and infrequent exacerbators had significantly higher mmrc - scale grades and total cat scores compared with nonexacerbators ( table 1 ) .
there was no change in the frequency of previous annual hospitalizations per patient between those with frequent and infrequent exacerbations ( table 1 ) . during the 1-year prospective observation period , one frequent exacerbator , one infrequent exacerbator , and two nonexacerbators died , with causes of death respiratory failure with copd exacerbation , cerebrovascular attack , and malignancies ( small - cell lung cancer and colon cancer ) , respectively , whereas one frequent exacerbator and six nonexacerbators dropped out for private reasons . as a result , 78 patients ( 17 frequent , eleven infrequent , and 50 nonexacerbators ) completed the study ( figure 1 ) .
figure 2 also shows that among the frequent exacerbators , the number of patients who subsequently suffered severe exacerbations ( requiring one or more hospitalizations ) was two ( 11.8% ) , and frequent ( two or more exacerbations / year ) , infrequent ( one exacerbation / year ) , and no moderate or severe exacerbations were seven ( 42.1% ) , six ( 35.3% ) , and four ( 23.5% ) , respectively , whereas seven ( 63.6% ) of eleven infrequent and 37 ( 74.0% ) of 50 nonexacerbators experienced no further exacerbation .
the proportions of frequent exacerbators who subsequently experienced frequent and infrequent exacerbations were significantly higher than those of nonexacerbators ( odds ratio [ 95% ci ] 2.94 [ 1.217.17 ] , p=0.0340 ; 2.94 [ 1.725.03 ] , p=0.0004 , respectively ) , but not in comparison with infrequent exacerbators ( 1.51 [ 0.494.63 ] , p>0.05 ; 2.01 [ 0.924.80 ] , p=0.053 , respectively ) . among five ( 5.6% ) of the severe exacerbators , two ( 40.0% ) had subsequently suffered severe exacerbations .
the number of patients who had subsequent frequent , infrequent , and no moderate or severe exacerbations was two ( 40% ) , 0 , and three ( 60% ) , respectively ( figure 2 ) .
in subanalysis , there was no difference in the proportion of patients who had subsequently suffered severe exacerbations between the frequent and severe exacerbator groups ( p>0.05 ) .
in addition , one severe exacerbator died due to copd - related respiratory failure during the 1-year prospective observation period .
the numbers of patients who reported pneumonia ( n=78 ) and those who regularly used ics ( n=15 ) were seven ( 9% ) and three ( 20% ) , respectively .
patients who regularly used ics had higher but not significant contraction of pneumonia than those who did not use ics ( p>0.05 ) .
figure 3a shows that the mean annual frequencies ( sd , exacerbations / year ) of future total ( 1.41.2 , p=0.0020 ) and moderate ( 1.31.1 , p=0.0057 ) exacerbations in frequent exacerbators were significantly higher than those in nonexacerbators ( 0.40.8 and 0.40.7 , respectively ) , but not in infrequent exacerbators ( 0.91.4 and 0.81.4 , respectively ) .
figure 3b shows the kaplan meier analysis of the period until the first moderate and severe exacerbations .
the median ( mean sd ) periods ( days ) until the first moderate and severe exacerbations for frequent , infrequent , and nonexacerbators were 266 ( 23528 ) , 365 ( 29330 ) , and 365 ( 32313 ) days , respectively ( log - rank test , p=0.0012 ) .
there was no significant difference in the annual frequencies of severe exacerbation ( hospitalization ) among non- ( 0.00.3 ) , infrequent ( 0.10.39 ) , and frequent ( 0.10.3 ) exacerbators ( p>0.05 ) .
the median ( mean sd ) periods until the first severe exacerbation were 365 ( 3632 ) , 365 ( 3650 ) , and 365 ( 3605 ) days , respectively ( p>0.05 ; data not shown ) .
univariate analysis revealed that a low fev1 ( < 50% ) had the highest odds ratio for risk of future exacerbation ( one or more exacerbations / year ) , followed in order by regular use of ics , two or more previous exacerbations in the last year , presence of gerd , pneumococcal vaccination , one or more previous exacerbations in the last year , low body mass index ( 21.4 kg / m ) , a high total cat score ( 10 points ) , a high mmrc scale grade ( 2 ) and older age ( 68 years ) , whereas in patients who suffered two or more exacerbations , the highest odds ratio was observed due to the presence of gerd , followed in order by regular use of ics , low fev1 predicted , pneumococcal vaccination , older age , a high mmrc - scale grade , long duration of copd ( 4 years ) , and two or more and one or more previous exacerbations in the last year ( table 2 ) .
however , 13 ( 86.7% ) of 15 patients who had received ics had also received pneumococcal vaccination .
the independent risk factors for both one or more and two or more future exacerbations were low fev1 predicted , presence of gerd , and regular use of ics , whereas a low body mass index was an independent risk factor for one or more but not for two or more future exacerbations ( table 3 ) .
ninety of 110 patients who provided informed consent were finally analyzed ; 32 ( 35.6% ) patients had suffered previous moderate and/or severe exacerbations during the last year .
the numbers of patients with non- , infrequent , and frequent exacerbations were 58 ( 64.4% ) , 12 ( 13.3% ) , and 20 ( 22.2% ) , respectively ( figure 1 ) .
at baseline , frequent exacerbators had a significantly lower body mass index , were less likely to be male or current smokers , had higher mmrc - scale grades and higher total cat score , had a higher proportion of spirometric stages iii and iv , gerd , and use of inhaled corticosteroid ( ics)/long - acting muscarinic antagonist or ics / long - acting 2-agonist combination therapy , and lower lung - function parameters , including fev1 , fvc , and fev1/fvc ratio before and after bronchodilator use compared with nonexacerbators , but not in comparison with infrequent exacerbators ( table 1 ) .
frequent and infrequent exacerbators had significantly higher mmrc - scale grades and total cat scores compared with nonexacerbators ( table 1 ) .
there was no change in the frequency of previous annual hospitalizations per patient between those with frequent and infrequent exacerbations ( table 1 ) .
during the 1-year prospective observation period , one frequent exacerbator , one infrequent exacerbator , and two nonexacerbators died , with causes of death respiratory failure with copd exacerbation , cerebrovascular attack , and malignancies ( small - cell lung cancer and colon cancer ) , respectively , whereas one frequent exacerbator and six nonexacerbators dropped out for private reasons . as a result , 78 patients ( 17 frequent , eleven infrequent , and 50 nonexacerbators ) completed the study ( figure 1 ) .
figure 2 also shows that among the frequent exacerbators , the number of patients who subsequently suffered severe exacerbations ( requiring one or more hospitalizations ) was two ( 11.8% ) , and frequent ( two or more exacerbations / year ) , infrequent ( one exacerbation / year ) , and no moderate or severe exacerbations were seven ( 42.1% ) , six ( 35.3% ) , and four ( 23.5% ) , respectively , whereas seven ( 63.6% ) of eleven infrequent and 37 ( 74.0% ) of 50 nonexacerbators experienced no further exacerbation .
the proportions of frequent exacerbators who subsequently experienced frequent and infrequent exacerbations were significantly higher than those of nonexacerbators ( odds ratio [ 95% ci ] 2.94 [ 1.217.17 ] , p=0.0340 ; 2.94 [ 1.725.03 ] , p=0.0004 , respectively ) , but not in comparison with infrequent exacerbators ( 1.51 [ 0.494.63 ] , p>0.05 ; 2.01 [ 0.924.80 ] , p=0.053 , respectively ) . among five ( 5.6% ) of the severe exacerbators , two ( 40.0% ) had subsequently suffered severe exacerbations .
the number of patients who had subsequent frequent , infrequent , and no moderate or severe exacerbations was two ( 40% ) , 0 , and three ( 60% ) , respectively ( figure 2 ) . in subanalysis , there was no difference in the proportion of patients who had subsequently suffered severe exacerbations between the frequent and severe exacerbator groups ( p>0.05 ) .
in addition , one severe exacerbator died due to copd - related respiratory failure during the 1-year prospective observation period .
the numbers of patients who reported pneumonia ( n=78 ) and those who regularly used ics ( n=15 ) were seven ( 9% ) and three ( 20% ) , respectively .
patients who regularly used ics had higher but not significant contraction of pneumonia than those who did not use ics ( p>0.05 ) .
figure 3a shows that the mean annual frequencies ( sd , exacerbations / year ) of future total ( 1.41.2 , p=0.0020 ) and moderate ( 1.31.1 , p=0.0057 ) exacerbations in frequent exacerbators were significantly higher than those in nonexacerbators ( 0.40.8 and 0.40.7 , respectively ) , but not in infrequent exacerbators ( 0.91.4 and 0.81.4 , respectively ) .
figure 3b shows the kaplan meier analysis of the period until the first moderate and severe exacerbations .
the median ( mean sd ) periods ( days ) until the first moderate and severe exacerbations for frequent , infrequent , and nonexacerbators were 266 ( 23528 ) , 365 ( 29330 ) , and 365 ( 32313 ) days , respectively ( log - rank test , p=0.0012 ) .
there was no significant difference in the annual frequencies of severe exacerbation ( hospitalization ) among non- ( 0.00.3 ) , infrequent ( 0.10.39 ) , and frequent ( 0.10.3 ) exacerbators ( p>0.05 ) .
the median ( mean sd ) periods until the first severe exacerbation were 365 ( 3632 ) , 365 ( 3650 ) , and 365 ( 3605 ) days , respectively ( p>0.05 ; data not shown ) .
univariate analysis revealed that a low fev1 ( < 50% ) had the highest odds ratio for risk of future exacerbation ( one or more exacerbations / year ) , followed in order by regular use of ics , two or more previous exacerbations in the last year , presence of gerd , pneumococcal vaccination , one or more previous exacerbations in the last year , low body mass index ( 21.4 kg / m ) , a high total cat score ( 10 points ) , a high mmrc scale grade ( 2 ) and older age ( 68 years ) , whereas in patients who suffered two or more exacerbations , the highest odds ratio was observed due to the presence of gerd , followed in order by regular use of ics , low fev1 predicted , pneumococcal vaccination , older age , a high mmrc - scale grade , long duration of copd ( 4 years ) , and two or more and one or more previous exacerbations in the last year ( table 2 ) .
however , 13 ( 86.7% ) of 15 patients who had received ics had also received pneumococcal vaccination . the independent risk factors for both one or more and two or more future exacerbations were low fev1 predicted ,
presence of gerd , and regular use of ics , whereas a low body mass index was an independent risk factor for one or more but not for two or more future exacerbations ( table 3 ) .
the frequent - exacerbator phenotype is important to consider in the management of copd patients , who require future exacerbation - related hospitalization associated with high mortality.16 a previous western study suggested that frequent exacerbators tend to have had exacerbations during the previous year , a lower fev1 , more severe hrqol , a history of gerd , and a higher peripheral white blood - cell count in comparison with infrequent exacerbators and nonexacerbators.12 another western study demonstrated that the characteristics of frequent exacerbators include a more severe mmrc - scale grade , lower fev1 predicted , comorbid cardiovascular disease , depression or osteoporosis , and female sex as independent risk factors.32,33 we conducted the present study to observe moderate and severe exacerbations 1 year before and after baseline to investigate the characteristics of japanese copd patients who were frequent exacerbators . based on exacerbations during the previous year , we found that frequent exacerbators were more likely to be female , have a lower body mass index , have a significantly lower fev1 predicted , have a higher mmrc - scale grade ( lower exercise tolerance ) , and have a lower total cat score ( lower hrqol ) .
we also found that the characteristics of frequent exacerbators were similar between japanese and westerners , except for body mass index , as reported previously.12,16,32 interestingly , univariate analysis did not show that previous frequent exacerbators would become future frequent exacerbators ( table 2 ) .
indeed , 60% of previous frequent exacerbators did not suffer subsequent exacerbations , whereas conversely 14% of patients who had not previously suffered exacerbations subsequently did so ( figure 2 ) .
investigation of factors predicting the change in frequency of exacerbations is critically important , as they are still unclear.34 a total of 78 ( 86.7% ) of our 90 patients completed the 1-year prospective study period . among five severe exacerbators , one ( 20% ) died , two ( 40% ) again developed severe exacerbation , and two ( 40% )
therefore , severe exacerbators appear to have a poor prognosis . however , our study - sample size was small and the observational period short .
frequent exacerbators had a significantly higher frequency of future exacerbations and a shorter period until the next exacerbation than nonexacerbators , and also had a significantly poorer prognosis than the latter , thus confirming the findings of a previous study.12 previous studies have demonstrated that over half of copd patients have unreported exacerbations .
such unreported exacerbations are thought to be an important component of hrqol decline.7,35,36 in our present study , to make the conditions for previous and future exacerbations uniform , daily journals for symptoms were not accepted .
previous studies have demonstrated that the frequency of annual moderate or severe exacerbations per patient in japanese individuals may be lower than that in the usa and europe.13,14,3537 in our study , the mean ( sd [ range ] ) of previous annual total and severe exacerbations were 0.841.48 ( 06 ) and 0.060.23 ( 01 ) exacerbations / year , respectively for all patients ( data not shown ) , whereas those of future total and severe exacerbations were 0.711.08 ( 04 ) and 0.060.28 ( 02 ) exacerbations / year , respectively .
our data for the frequency of exacerbations seem to indicate a slightly lower incidence in japanese than in westerners.1215,31,32,38 previous japanese reports on the frequency of exacerbations have been scarce.13,14 japanese copd patients may be slightly older and thinner on average than westerners.1215,31,32,38 although the discrepancy between japanese and westerners is still unclear , the difference in the frequency of exacerbations may be associated with different populations of phenotypes with emphysema , locality , and understanding of both the physician and the patient about diseases and exacerbations , such as convenient use of ics , and in definition of exacerbation .
patients with copd have a wide variety of numerous comorbidities , which are strongly associated with mortality and exacerbation.1,12,26 we assessed baseline comorbidities based on interviews with patients and the physicians diagnosis , and partly through examinations or questionnaires .
several comorbidities , such as depression21,39 assessed by the cesd questionnaire , acos27,40 based on previous criteria , and congestive status with heart failure and cor pulmonale based on history or medical signs,41 were carefully excluded .
gerd , although not moderate to severe , was a common major comorbidity , and the proportion of frequent exacerbators with gerd was significantly higher than that of nonexacerbators .
gerd was an independent , and the highest , risk factor for future frequent exacerbations , although all patients with gerd were receiving proton - pump inhibitors .
our results confirmed previous reports.12,42 a low fev1 predicted was an independent risk factor for future exacerbations .
the guidelines1,43 recommend long - term treatment with ics and pneumococcal vaccination for patients with severe and very severe copd , and frequent exacerbations may not be adequately controlled by long - acting bronchodilators . in our study
regular use of ics , but not pneumococcal vaccination , was an independent risk factor for future frequent exacerbations .
sixteen ( 17.8% ) of our patients used ics , although none had received ics without long - acting bronchodilators at baseline .
users of ics included four nonexacerbators with gold stage ii , seven patients with frequent exacerbations , and the remaining five had severe or very severe disease .
the risk of ics for respiratory infection including pneumonia is a concern.4446 among four nonexacerbators with gold stage ii and users of ics during the prospective observation period , two patients had frequent exacerbations and one had infrequent exacerbations .
this risk must be weighed against the benefits when prescribing ics to patients with copd .
first , the study population was small and the observation period rather short for analyzing severe exacerbations and mortality .
balcells et al demonstrated that female sex was the highest risk factor for exacerbation,33 although in the present study there was no difference in the frequency of exacerbations between males and females .
both physicians and patients were aware of the severity and frequency of previous exacerbations at baseline .
we carefully removed severe comorbidities , such as cardiovascular disease and depression , in accordance with previous reports.26,27 however , patients with asthma and acos may have been included , because we did not investigate airway responsiveness and inflammation or serum total immunoglobulin ( ig ) e levels , although we carefully excluded patients with asthma based on symptoms and spirometry
. however , no patients had received medications for osteoporosis and low peripheral lymphocyte counts , although latent osteoporosis and hiv infections were not tested for .
fourth , the contents of medicines could not be ethically unified in a clinical setting in japan , and in our study adherence to pharmacological medicines and compliance with inhalation techniques were not assessed or included .
the effects of respiratory medications , including inhaled medicines , on previous and future exacerbations were investigated .
the presence of gerd , regular use of ics , and low fev1 may be associated with frequent exacerbations .
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backgroundthe prognosis of japanese patients with copd who suffer repeated exacerbations is unclear , although westerners with such episodes have a poor prognosis.materials and methodswe conducted a 1-year prospective observational trial involving 90 japanese patients with copd : 58 nonexacerbators , 12 infrequent exacerbators , and 20 frequent exacerbators classified on the basis of exacerbation frequency ( zero , one , and two or more exacerbations / year ) , respectively , during the previous year were observed prospectively for 1 year .
the characteristics of frequent exacerbators , the frequency of exacerbation , and the period until the first event were then compared among the groups.resultsa total of 78 patients completed the study .
frequent exacerbators had a significantly higher risk of frequent exacerbation in the following year than the case for nonexacerbators ( odds ratio [ 95% confidence interval ] 2.94 [ 1.217.17 ] , p=0.0340 ) , but not in comparison with infrequent exacerbators ( 1.51 [ 0.494.63 ] , p>0.05 )
. the mean annual frequency of exacerbations in the following year was significantly ( p=0.0020 ) higher in the frequent exacerbators ( 1.4 exacerbations / year ) than in the nonexacerbators ( 0.4 ) , but not in the infrequent exacerbators ( 0.9 , p>0.05 ) .
the mean period until the first exacerbation was significantly shorter in the frequent exacerbators than in the infrequent or nonexacerbators ( p=0.0012 ) .
independent risk factors for future frequent exacerbation included the presence of gastroesophageal reflux disease , more severe airflow obstruction , and use of inhaled corticosteroids.conclusionour present results indicate that japanese copd patients suffering frequent exacerbation have a poor prognosis .
the characteristics of japanese and western copd patients suffering frequent exacerbation are similar .
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Introduction
Materials and methods
Study design
Diagnosis and spirometric classification of COPD
Assessment of mMRC scale, total CAT, FSSG scale, and CESD
Comorbidities
Frequency and date of mortality, hospitalizations, and exacerbations
Statistical analysis
Results
Study subjects
Baseline characteristics
Stability of the frequent and severe exacerbation phenotype in patients who completed the study
Comparison of annual exacerbation and hospitalization among non-, infrequent, and frequent exacerbators
Baseline characteristics of patients suffering further exacerbations (one or more/year) and frequent exacerbations (two or more/year)
Discussion
Conclusion
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it is not uncommon to see bleeding symptoms in patients in outpatient or hospital - based practice .
postpartum hemorrhage complicates pregnancy and accounts for significant morbidity and mortality , particularly in underdeveloped countries.1,2 menorrhagia is a common clinical challenge and is often associated with secondary anemia , excessive fatigue , and a negative effect on health - related quality of life.3 postoperative bleeding is one of the more common complications of surgery .
trauma is a leading cause of morbidity and mortality in the younger population.4 while bleeding symptoms may be commonly seen by physicians of multiple specialties , it is unclear how frequently these symptoms belie an underlying undiagnosed congenital or acquired bleeding disorder . in the us ,
the most common congenital bleeding disorders include von willebrand disease , which affects approximately 1% of the population ( males and females equally),5 and hemophilia a and b combined , which affect approximately 20,000 persons ( essentially all males , with rare exception).6 medications can also affect coagulation or platelet function,7 as can certain herbal supplements.8 trauma and surgery can lead to blood loss , and critical reduction in coagulation factors can lead to additional non - surgical bleeding complications ( coagulopathic bleeding).9 the most ubiquitous method for evaluating coagulation is prothrombin time ( pt)/international normalized ratio ( inr ) and activated partial thromboplastin time ( aptt ) .
typically , they are ordered to monitor anticoagulant therapy ( pt / inr for warfarin , aptt for heparin ) , to evaluate coagulation preoperatively , or in response to hemorrhagic symptoms .
the pt / inr provides an assessment of the extrinsic ( tissue factor - dependent ) and final common pathways of the coagulation cascade , while the aptt provides an assessment of the intrinsic ( tissue factor - independent ) and final common pathways.10 an example of a potentially life - threatening cause of unexplained recent onset or acute bleeding associated with a prolonged aptt is acquired hemophilia , with an incidence of approximately 1 to 4 per million / year.11 acquired hemophilia primarily affects older adults,12 an ever - growing segment of the population that presents for medical evaluation and care , but may also occur during pregnancy as postpartum hemorrhage or in association with other underlying diseases , including cancer and autoimmune disorders.13 prompt diagnosis is a primary determinant of prognosis in acquired hemophilia14 because initiation of definitive therapy ( ie , hemostatic and immunosuppressive ) is delayed until the diagnosis is made .
acquired hemophilia - related bleeding does not respond to the typical management algorithms used for hemorrhaging in a patient and , therefore , is associated with high morbidity and mortality .
severe bleeds occur in up to 90% of patients with acquired hemophilia,11 and the reported overall mortality rate in these patients ranges from 8% to 22%.1517 given the rarity of acquired hemophilia , combined with the general lack of familiarity of nonhematologists with this condition , the diagnosis of acquired hemophilia poses a clinical challenge , even in patients presenting with straightforward bleeding and an isolated , prolonged aptt .
a survey was conducted of physicians across a number of specialties to identify potential barriers to the effective recognition and management of this rare but important cause of serious bleeding .
the survey , based on an actual case found to be the result of acquired hemophilia , focused on participants stepwise evaluation and management of a case patient who presented to the hospital with recent - onset bleeding .
the survey also assessed participants history with regard to consulting hematologists , discovering or diagnosing underlying bleeding disorders , and encountering acquired hemophilia in clinical practice .
findings pertaining to the interpretation and follow - up of abnormal coagulation studies , enlistment of hematology consultation , and the diagnostic decision process in an actively bleeding case patient were the primary areas assessed in this analysis .
results were evaluated across specialties to determine any specialty - specific practice trends that might hinder effective recognition and management of an actively bleeding patient with coagulopathy , including acquired hemophilia .
physicians within the specialties of hematology , hematology / oncology , emergency medicine , geriatrics , internal medicine , rheumatology , obstetrics and gynecology , critical care medicine , and general surgery were randomly sampled from the american medical association physician masterfile .
physicians who were part of the harris interactive online physician panel were invited via email to participate in the survey , while physicians who were not part of the panel were invited via first class mail .
the invitation provided a uniform resource locator address and password for one - time use to log on to the survey site , where invitees first encountered questions to determine eligibility to participate in the survey .
eligible physicians were required to be actively practicing , with additional specialty - specific requirements .
in particular , hematologists , hematologist / oncologists , general surgeons , and emergency medicine and critical care physicians were required to be practicing at an acute care hospital . for internists and geriatricians ,
at least 60% of their practice had to consist of patients older than 60 years of age .
obstetricians / gynecologists were required to have an active obstetrics practice . upon confirmation of eligibility , participants completed the online survey , the average duration of which was approximately 10 minutes .
each specialty was recruited to a final sample of 50 complete responses to ensure a sufficient number of responses to generate reasonable hypotheses about specialists behavior to test in face - to - face interviews .
findings from the surveys completed by the physicians specializing in hematology , hematology / oncology , emergency medicine , geriatrics , internal medicine , rheumatology , and critical care medicine are presented herein .
general surgeons and obstetrics and gynecology physicians each completed different surveys from the aforementioned specialties ; the findings of those surveys are not included here .
survey questions focused on the diagnostic and general therapeutic approach to the management of a case patient ( figure 1 ) whose presentation and clinical course were based on those of a real patient who experienced significant delays in diagnosis despite the involvement of multiple specialists . at each juncture in the case , participants were given lists of actions that included diagnostic tests ( laboratory , radiology , other ) , consultations , and potential treatments addressing bleeding or anemia ( local hemostasis , transfusion ) . since familiarity with the upper limits of normal of the pt / aptt tests was being tested in this study , upper limits of normal were not specified , and aptts were 25% to 30% above the upper limits of normal and set at a value that respective specialties would recognize as abnormal based on their experience with ordering and interpreting this laboratory test .
the survey did not specify whether laboratory testing , imaging studies , or consultations in this hypothetical case had to be available immediately or locally , and the assumption was that respondents would answer based on their best medical judgment in an ideal practice situation .
rheumatologists were only asked about an initial emergency department / urgent care presentation because it was expected that if a bleeding disorder developed in one of their patients it would present in an outpatient or emergency department / urgent care setting .
conversely , critical care specialists started at presentation 2 , since their involvement in a recurrent epistaxis workup would be unlikely .
vital signs were adjusted to make it more likely that a potential intensive care unit admission would require their evaluation . since emergency medicine physicians would most likely have ordered an imaging study initially to define the patient s retroperitoneal hematoma , we allowed for the option of a general surgery consultation even though the computed tomography findings were never presented .
hematology / oncology respondents were given the option of ordering additional specialty coagulation laboratory tests ( eg , mixing studies ) . participants were additionally questioned about their experiences diagnosing underlying bleeding disorders and , in the case of nonhematologist specialists , consulting hematologist colleagues .
conversely , hematology and hematology / oncology specialists were asked about being on the receiving end of consultations , specifically from emergency medicine personnel , and the reasons for those consultations .
a total of 302 physicians ( 5051 per specialty ) participated in the case - based survey reported herein .
demographics of the specialty groups were consistent : mean age ranged from 45.6 to 50.6 years , and mean years of practice experience ranged from 14.5 to 19.3 .
faced with an older adult female patient with recurrent epistaxis , nearly 90% of physicians in each of the surveyed specialties indicated they would have ordered a complete blood count and coagulation studies ( pt / inr and aptt ) as part of the initial evaluation ( figure 2a ) . despite abnormal results of the aptt at 42 seconds ( with an upper limit of normal typically ranging from 35 to 39 seconds , although not specified in the case ) , less than half the physicians in most specialties would have chosen to repeat the coagulation studies as one of the next steps ( figure 2b ) . in contrast , 67% of hematologists would have repeated these studies . less than 45% of surveyed physicians in all nonhematology specialties would have consulted a hematologist after reviewing the initial coagulation study results ( figure 2b ) .
rheumatologists were most likely to obtain a consult ( 43% ) , although they were presented with an initial aptt that was more abnormal ( 50 seconds ) than the aptt initially presented to the other specialists .
after the patient s second presentation several weeks later with bruising and abdominal / back pain , again nearly 90% or more of respondents ( including critical care specialists , who began their case review at this juncture in the patient s clinical course ) would have ordered both a complete blood and coagulation studies as part of their initial evaluation ( figure 2c ) .
when these results revealed a clearly abnormal and markedly changed aptt of 63 seconds , the majority of respondents indicated they still would not have repeated coagulation studies ( figure 2d ) .
approximately 75% of internal medicine and geriatrics physicians would have consulted a hematologist at this point , compared with 47% and 50% of emergency medicine and critical care specialists , respectively ( figure 2d ) .
sixty percent of hematologists and hematologists / oncologists surveyed would have evaluated the patient s peripheral blood smear at this point .
in addition to the aforementioned hematologic laboratory tests , other diagnostic tests and consultations that participants would have recommended from a series of options as part of their evaluation after the patient s second presentation are shown in figure 3 .
participants across specialties clearly preferred computed tomography of the abdomen ( 80%84% ) over abdominal ultrasound ( 9%28% ) , upper gastrointestinal endoscopy ( 2%16% ) , or colonoscopy ( 0%16% ) .
emergency medicine physicians demonstrated the greatest breadth of testing , with 82% additionally recommending urinalysis and 92% recommending a stool guaiac test .
requests for gastroenterology consultations ranged from 10% to 43% and were highest for the internal medicine group , which also had the highest proportion of physicians recommending endoscopy ( 16% each for upper gastrointestinal endoscopy and colonoscopy ) . for the purposes of assessing multiple specialties , the next 12 hours of observation in the case patient s clinical course were described as having occurred in the emergency department . by the end of this observation period ,
the aptt had further increased to at least twice the upper limit of normal , while the hemoglobin level had decreased .
internists and geriatricians were most inclined to repeat the laboratory studies at this juncture ( figure 4 ) .
in contrast with previous time points , by this point in the patient s clinical presentation , 73% to 94% of respondents would have consulted a hematologist .
the majority of hematologists would have ordered 1:1 mixing studies of patient and normal plasma to rule out coagulation inhibitors ( 97% ) and fibrinogen / fibrin split products testing ( 75% ) in response to the laboratory results obtained after 12 hours of observation , as part of a diagnostic evaluation of the prolonged aptt .
emergency medicine physicians were given the additional option of consulting general surgery practitioners ; 2% would have done so after the first set of laboratory results at the second presentation , and 20% would have done so in response to the second set of laboratory results . the percentages of physicians in each specialty who would have recommended admission of this case patient to a general hospital floor or back to a skilled nursing facility or nursing home after initial presentation with subsequent abnormal aptt of 42 seconds are shown in figure 5a .
across the majority of specialties , less than 20% of physicians would have recommended overnight admission to a general hospital floor , while more than 50% of physicians in these specialties would have recommended discharge to a skilled nursing facility or a nursing home .
in contrast with respondents from other specialties , a slightly higher proportion of hematologists and hematologist / oncologists would have endorsed a higher level of care at this point ; 36% of physicians in this group indicated they would have favored overnight hospital admission over discharge to a skilled nursing facility .
the majority of physicians faced with the second presentation would have recommended that the patient be admitted to the hospital .
the differences among specialties were most noticeable in allocation to a general hospital floor versus an intensive care unit after review of initial laboratory results or the second set of laboratory results obtained after 12 hours of observation ( figure 5b ) . while approximately 80% or more of physicians in each specialty would have recommended hospital admission in response to both sets of laboratory results , the proportion recommending admission specifically to the intensive care unit increased as the laboratory values deteriorated ( worsening anemia and coagulopathy ) . based on the laboratory results obtained after 12 hours of observation ,
nearly 50% or more of physicians in each specialty would have recommended admission to the intensive care unit in lieu of a general hospital floor , compared with approximately 40% or less across specialties after the initial laboratory results obtained during the patient s second presentation .
the difference was most noticeable for physicians in emergency medicine ( 35%73% ) , the specialty most likely to refer the patient to an inpatient medical / hospitalist or critical care service .
more than 85% of emergency medicine and critical care physicians reported having ever discovered an underlying bleeding disorder , while 100% of hematologists and hematologist / oncologists reported having ever diagnosed one ( figure 6a ) .
the percentage of physicians in other specialties who had ever discovered an underlying bleeding diathesis ranged from 47% to 65% . among those physicians who had previously diagnosed or discovered a bleeding disorder , 14% to 77% had specifically encountered acquired hemophilia ( figure 6b ) , although this quantitative survey did not assess whether they understood this disorder .
hematologists and hematologists / oncologists accounted for the highest percentage in this group , while internal medicine specialists accounted for the lowest percentage .
table 2 shows the frequencies with which various specialties had ever consulted a hematologist when they encountered a patient with an abnormal pt / inr and aptt and no history of a bleeding diathesis or medications that affect coagulation .
the distribution of reasons for which respondents would have consulted a hematologist is also shown in table 2 . in the majority of specialties ( emergency medicine , geriatrics , and internal medicine ) ,
the highest percentage of physicians had consulted a hematologist 1 to 2 times for a patient with an unexplained prolonged pt / inr or aptt .
in contrast , more than 80% of rheumatologists had consulted a hematologist 3 , 4 , or 5 or more times for the same reason . the highest percentage ( 39% ) of critical care physicians
the most common reason for consulting a hematologist was abnormal coagulation study results in the setting of clinical bleeding ( table 2 ) , which was parallel to the findings for the 100 additional surgeons and obstetricians surveyed ( data not shown ) .
the most common response among critical care specialists to the open - ended question , what are the circumstances around which you would call for a hematology consult ? was an unexplained abnormal lab or bleeding ( 33 like or similar mentions ) , followed by any unexplained bleeding disorder
conversely , the frequency with which hematologists had ever been consulted by an emergency medicine provider for an abnormal pt / inr or aptt in a patient with no history of bleeding diathesis or medications that affect coagulation is outlined in table 2 .
the majority ( 57% ) of hematologists had been consulted a minimum of 5 times by their colleagues in emergency medicine .
the majority of these consults pertained to significantly abnormal coagulation studies with or without clinical bleeding ( table 2 ) .
faced with an older adult female patient with recurrent epistaxis , nearly 90% of physicians in each of the surveyed specialties indicated they would have ordered a complete blood count and coagulation studies ( pt / inr and aptt ) as part of the initial evaluation ( figure 2a ) . despite abnormal results of the aptt at 42 seconds ( with an upper limit of normal typically ranging from 35 to 39 seconds , although not specified in the case ) , less than half the physicians in most specialties would have chosen to repeat the coagulation studies as one of the next steps ( figure 2b ) . in contrast , 67% of hematologists would have repeated these studies .
less than 45% of surveyed physicians in all nonhematology specialties would have consulted a hematologist after reviewing the initial coagulation study results ( figure 2b ) .
rheumatologists were most likely to obtain a consult ( 43% ) , although they were presented with an initial aptt that was more abnormal ( 50 seconds ) than the aptt initially presented to the other specialists .
after the patient s second presentation several weeks later with bruising and abdominal / back pain , again nearly 90% or more of respondents ( including critical care specialists , who began their case review at this juncture in the patient s clinical course ) would have ordered both a complete blood and coagulation studies as part of their initial evaluation ( figure 2c ) .
when these results revealed a clearly abnormal and markedly changed aptt of 63 seconds , the majority of respondents indicated they still would not have repeated coagulation studies ( figure 2d ) .
approximately 75% of internal medicine and geriatrics physicians would have consulted a hematologist at this point , compared with 47% and 50% of emergency medicine and critical care specialists , respectively ( figure 2d ) .
sixty percent of hematologists and hematologists / oncologists surveyed would have evaluated the patient s peripheral blood smear at this point .
in addition to the aforementioned hematologic laboratory tests , other diagnostic tests and consultations that participants would have recommended from a series of options as part of their evaluation after the patient s second presentation are shown in figure 3 .
participants across specialties clearly preferred computed tomography of the abdomen ( 80%84% ) over abdominal ultrasound ( 9%28% ) , upper gastrointestinal endoscopy ( 2%16% ) , or colonoscopy ( 0%16% ) .
emergency medicine physicians demonstrated the greatest breadth of testing , with 82% additionally recommending urinalysis and 92% recommending a stool guaiac test .
requests for gastroenterology consultations ranged from 10% to 43% and were highest for the internal medicine group , which also had the highest proportion of physicians recommending endoscopy ( 16% each for upper gastrointestinal endoscopy and colonoscopy ) . for the purposes of assessing multiple specialties , the next 12 hours of observation in the case patient s clinical course were described as having occurred in the emergency department . by the end of this observation period ,
the aptt had further increased to at least twice the upper limit of normal , while the hemoglobin level had decreased .
internists and geriatricians were most inclined to repeat the laboratory studies at this juncture ( figure 4 ) .
in contrast with previous time points , by this point in the patient s clinical presentation , 73% to 94% of respondents would have consulted a hematologist .
the majority of hematologists would have ordered 1:1 mixing studies of patient and normal plasma to rule out coagulation inhibitors ( 97% ) and fibrinogen / fibrin split products testing ( 75% ) in response to the laboratory results obtained after 12 hours of observation , as part of a diagnostic evaluation of the prolonged aptt .
emergency medicine physicians were given the additional option of consulting general surgery practitioners ; 2% would have done so after the first set of laboratory results at the second presentation , and 20% would have done so in response to the second set of laboratory results .
the percentages of physicians in each specialty who would have recommended admission of this case patient to a general hospital floor or back to a skilled nursing facility or nursing home after initial presentation with subsequent abnormal aptt of 42 seconds are shown in figure 5a . across the majority of specialties ,
less than 20% of physicians would have recommended overnight admission to a general hospital floor , while more than 50% of physicians in these specialties would have recommended discharge to a skilled nursing facility or a nursing home .
in contrast with respondents from other specialties , a slightly higher proportion of hematologists and hematologist / oncologists would have endorsed a higher level of care at this point ; 36% of physicians in this group indicated they would have favored overnight hospital admission over discharge to a skilled nursing facility . the majority of physicians faced with the second presentation would have recommended that the patient be admitted to the hospital .
the differences among specialties were most noticeable in allocation to a general hospital floor versus an intensive care unit after review of initial laboratory results or the second set of laboratory results obtained after 12 hours of observation ( figure 5b ) . while approximately 80% or more of physicians in each specialty would have recommended hospital admission in response to both sets of laboratory results , the proportion recommending admission specifically to the intensive care unit increased as the laboratory values deteriorated ( worsening anemia and coagulopathy ) . based on the laboratory results obtained after 12 hours of observation ,
nearly 50% or more of physicians in each specialty would have recommended admission to the intensive care unit in lieu of a general hospital floor , compared with approximately 40% or less across specialties after the initial laboratory results obtained during the patient s second presentation .
the difference was most noticeable for physicians in emergency medicine ( 35%73% ) , the specialty most likely to refer the patient to an inpatient medical / hospitalist or critical care service .
more than 85% of emergency medicine and critical care physicians reported having ever discovered an underlying bleeding disorder , while 100% of hematologists and hematologist / oncologists reported having ever diagnosed one ( figure 6a ) .
the percentage of physicians in other specialties who had ever discovered an underlying bleeding diathesis ranged from 47% to 65% . among those physicians who had previously diagnosed or discovered a bleeding disorder , 14% to 77% had specifically encountered acquired hemophilia ( figure 6b ) , although this quantitative survey did not assess whether they understood this disorder .
hematologists and hematologists / oncologists accounted for the highest percentage in this group , while internal medicine specialists accounted for the lowest percentage .
table 2 shows the frequencies with which various specialties had ever consulted a hematologist when they encountered a patient with an abnormal pt / inr and aptt and no history of a bleeding diathesis or medications that affect coagulation .
the distribution of reasons for which respondents would have consulted a hematologist is also shown in table 2 . in the majority of specialties ( emergency medicine , geriatrics , and internal medicine ) , the highest percentage of physicians had consulted a hematologist 1 to 2 times for a patient with an unexplained prolonged pt / inr or aptt .
in contrast , more than 80% of rheumatologists had consulted a hematologist 3 , 4 , or 5 or more times for the same reason .
the highest percentage ( 39% ) of critical care physicians had consulted a hematologist at least 5 times for such a patient .
the most common reason for consulting a hematologist was abnormal coagulation study results in the setting of clinical bleeding ( table 2 ) , which was parallel to the findings for the 100 additional surgeons and obstetricians surveyed ( data not shown ) .
the most common response among critical care specialists to the open - ended question , what are the circumstances around which you would call for a hematology consult ? was an unexplained abnormal lab or bleeding ( 33 like or similar mentions ) , followed by any unexplained bleeding disorder
conversely , the frequency with which hematologists had ever been consulted by an emergency medicine provider for an abnormal pt / inr or aptt in a patient with no history of bleeding diathesis or medications that affect coagulation is outlined in table 2 .
the majority ( 57% ) of hematologists had been consulted a minimum of 5 times by their colleagues in emergency medicine .
the majority of these consults pertained to significantly abnormal coagulation studies with or without clinical bleeding ( table 2 ) .
bleeding is commonly encountered in outpatient and hospital - based medical practice and can have a wide variety of underlying causes .
while bleeding may be relatively common , most acquired and congenital bleeding disorders are uncommon , and some , such as acquired hemophilia , are rare . nevertheless , the consequences of failure to recognize promptly and treat properly a bleeding disorder may be significant.18 in the case of acquired hemophilia , morbidity and mortality rates are particularly high : severe bleeding is experienced by up to 90% of affected patients , and mortality rates
are as high as 22%.11 this survey provided a step - wise methodology to tease out specialty - specific patterns of interpretation of clinical data to identify barriers to the diagnosis and treatment of underlying bleeding disorders .
the sample size obtained across specialties was sufficient to generalize these findings , at least to the point of identifying specific issues for education and development of clinical decision - making pathways .
when presented with a clinical picture that includes a recent history or symptoms of active bleeding , clinicians typically obtain coagulation times , such as the pt / inr and aptt , as part of the initial diagnostic evaluation .
proper interpretation of laboratory test results includes recognition of abnormal values and , more important , the potential clinical significance of such results . a common pitfall in the interpretation of coagulation times is failure to appreciate that even mildly abnormal values may represent a serious underlying coagulation deficit .
another important observation is to identify how an abnormal laboratory value may have changed over time , which can be facilitated by the ability of electronic medical record systems to display data trends . in the absence of iatrogenic causes , even a mildly elevated pt / inr or aptt may be indicative of true coagulopathy and should not be ignored or dismissed , particularly when there is evidence of bleeding , as was the case with this patient , even at initial presentation . after excluding laboratory error ,
the differential diagnosis of an isolated prolongation of aptt includes heparin effect , lupus anticoagulant , and deficiency of or antibody against an intrinsic pathway factor ( viii , ix , xi , or xii).19,20 a detailed history , focusing on factors such as heparin exposure,19,20 history of thromboembolism ( lupus anticoagulant),21 and prior personal or family history of bleeding,22 may provide diagnostic clues .
laboratory testing should include a 1:1 mixing test of patient plasma with control plasma to determine whether a prolonged aptt is the result of an intrinsic pathway factor deficiency or an inhibitor that continues to block the activity of the intrinsic system even in the presence of control plasma .
the majority of inhibitor antibodies identified in this manner will turn out to be lupus anticoagulants .
although far less common , this is the same diagnostic pathway that leads to the identification of the antifactor viii antibodies associated with acquired hemophilia.23 unlike acquired hemophilia , lupus anticoagulants typically do not present with bleeding , and the abnormal aptt is due to interference with phospholipid - dependent coagulation reactions .
once an acquired antifactor viii antibody is suspected , confirmatory testing includes measuring factor viii activity , which should be significantly reduced , and the bethesda assay,24 which is used to quantitate antifactor viii antibodies inhibitor activity .
we found a general lack of appropriate consideration and response to the presenting symptom of bleeding and the prolonged aptt throughout this case study .
this is consistent with data from the european acquired haemophilia registry ( each-2 ) , which reported a median delay of 3 days between onset of bleeding symptoms and the diagnosis of acquired hemophilia and a median delay of one day between the first abnormal aptt test in those same patients and the established diagnosis.23 in addition , we found that emergency medicine and critical care physicians were reluctant to consider a bleeding disorder as the primary explanation for this patient s clinical presentation .
the disposition of a patient with active hemorrhage and evidence of coagulopathy should be based on several factors , including the patient s current condition and anticipated clinical course , taking into account the presenting vital signs and evolving laboratory findings . at the time of the patient
s second presentation , vital signs were notable for mild tachycardia and a pulse pressure at the upper limit of normal , and subsequent laboratory findings indicated a decreasing hemoglobin level and an increasing aptt .
these findings alone prompted hospital admission , although the exact location ( general floor versus intensive care unit ) of admission may vary , based on the level of monitoring and nurse - to - patient ratios in a particular hospital .
another important variable is the anticipated potential for clinical deterioration , which is based in large part on clinician appreciation of the seriousness of the diagnosis .
we found a consistent tendency to consider admission to a general floor bed with the second presentation , even though this was ultimately an unstable , critically ill patient with an undiagnosed bleeding disorder .
we also found that the physicians who participated in this survey were reluctant to consult a hematologist as they worked through this case scenario , particularly given that options for additional testing ( liver function , disseminated intravascular coagulation ) were not available to evaluate for common causes of coagulopathy .
relative to their reported historical experience with hematology consultations during an average practice experience of approximately 20 years , this survey finding was somewhat surprising .
rheumatologists and critical care specialists reported a greater frequency of hematology consultation relative to the other specialties ( table 2 ) .
we would expect the emergency medicine physicians to be most likely to consult a hematologist , yet 16% of them reported never having consulted a hematologist .
the highest percentage ( 46% ) had only consulted a hematologist 1 or 2 times , and almost one quarter of geriatricians and internists had never consulted a hematologist , even though these specialists would be expected to first encounter patients with undiagnosed bleeding disorders , including acquired hemophilia .
one potential reason for not seeking hematology consultation might be the lack of availability of hematology / oncology specialists with expertise in coagulation disorders , including in rural and community hospitals .
a limitation of this survey was that one can not interpret the thinking behind the responses of the individual participants .
therefore , 31 qualitative 45-minute interviews were conducted subsequent to the quantitative study and focused particularly on critical care ( n = 7 ) , emergency medicine ( n = 6 ) , hematology / oncology ( n = 4 ) , or hematology ( n = 2 ) physicians to understand the reasoning behind their decisions and depth of knowledge ( unpublished data ) . we found that the physicians focus was generally on finding the source ( location ) of bleeding and not on finding the underlying reason for bleeding .
this could potentially lead to surgical intervention in the face of an underlying bleeding disorder , with subsequent adverse outcomes . in a series of 67 patients with acquired hemophilia at a single center in bonn , germany ,
4 of 5 deaths were the result of surgical intervention for bleeding at outside hospitals in the setting of a delayed diagnosis of acquired hemophilia.13 when queried about their experience encountering and/or diagnosing underlying bleeding disorders , particularly acquired hemophilia , more than 85% of physicians in hematology , hematology / oncology , emergency medicine , and critical care medicine reported having ever discovered or diagnosed an underlying bleeding disorder , compared with 65% , 54% , and 47% of rheumatologists , internists , and geriatricians , respectively . while reports of ever specifically having encountered acquired hemophilia were high , it is unclear from this study whether the participants truly understood the diagnosis .
this seems unlikely , given the rarity of acquired hemophilia , relative to the reported frequency of having encountered it .
except for hematology and/or oncology ( 77% ) and critical care ( 36% ) specialists , approximately one quarter of surveyed physicians had ever encountered acquired hemophilia .
although they accounted for the highest percentage of physicians who had ever encountered this condition , nearly one quarter of hematologists had never encountered acquired hemophilia .
subsequent unpublished data from the aforementioned qualitative research further suggest that , compared with hematology practitioners , specialists in hematology / oncology , who likely practice mostly oncology , might be able to identify mixing studies and inhibitors but might not fully understand the underlying pathophysiology that constitutes acquired hemophilia , making it hard for them to recognize the condition .
given the survey findings reflecting the infrequency with which most physicians have encountered these conditions , consultation with a hematologist may facilitate the diagnostic evaluation and proper management of a hemorrhaging patient suspected of having an underlying bleeding diathesis , particularly acquired hemophilia .
the consulting hematologist can provide specific guidance , leading to the prompt diagnosis and optimal management of an actively bleeding patient with acquired hemophilia , including initiation of immunosuppression , which is usually necessary to eradicate the inhibitor and to prevent additional bleeding episodes .
however , this requires a level of familiarity and expertise in treating acquired hemophilia and other rare bleeding disorders that is not often seen outside of an academic hematology practice .
this represents yet another barrier to the effective diagnosis and management of this rare yet serious bleeding diathesis .
for the hospitalist or intensivist charged with the care of a bleeding patient , immediate stabilization is the initial priority and should take precedence over determination of a specific etiology of bleeding .
however , in the presence of underlying coagulopathy , particularly acquired hemophilia or other rare disorders , traditional measures of stabilization may not be as effective as expected .
a prolonged pt / inr or aptt in the absence of iatrogenic causes should never be ignored , even with only minimally prolonged values , and determination of the cause of an abnormal coagulation study should carry at least equal weight to looking for the anatomic site of bleeding . any delay in establishing the diagnosis of a bleeding diathesis such as acquired hemophilia can result in significant morbidity or even death .
while hospitalists and intensivists should be able to conduct a thorough differential of bleeding and eliminate most common etiologies , consultation with a hematologist ( particularly one with specific expertise in coagulation disorders ) may facilitate the evaluation of coagulopathic patients and subsequent interpretation of diagnostic findings , as well as initiation of appropriate treatment . given the rarity of acquired hemophilia , as exemplified by the findings of this survey
, physicians must harbor a high index of suspicion to diagnose this condition promptly in patients who present with recent - onset or acute bleeding . given the high morbidity and mortality
the insights from this survey highlights knowledge and practice gaps that could be the focus of targeted educational initiatives , including diagnostic algorithms , to ensure proper and efficient workup of the abnormal laboratory studies that characterize acquired hemophilia .
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background : bleeding symptoms commonly seen by multiple physician specialties may belie undiagnosed congenital or acquired bleeding disorders . acquired hemophilia is a potentially life - threatening cause of unexplained acute bleeding manifested by an abnormal activated partial thromboplastin time ( aptt ) that does not correct with 1:1 mixing with normal plasma.methods:practicing physicians ( hematology / oncology , emergency medicine , geriatrics , internal medicine , rheumatology , obstetrics and gynecology , critical care medicine , and general surgery ) completed an online survey based on a hypothetical case scenario.results:excluding surgeons and obstetrician / gynecologist respondents , 302 physicians ( about 50 per specialty ) were presented with an older adult woman complaining of recurrent epistaxis .
nearly 90% ordered a complete blood count and coagulation studies ( aptt , prothrombin time [ pt]/international normalized ratio [ inr ] ) . despite a prolonged aptt of 42 seconds ,
< 50% of nonhematologists would repeat the aptt , and < 45% would consult a hematologist ; emergency medicine physicians were least likely ( 10% ) and rheumatologists were most likely ( 43% ) to consult .
after presentation weeks later with bruising and abdominal / back pain , 90% of physicians within each specialty ordered a complete blood count or pt / inr / aptt . despite an aptt of 63 seconds
, the majority did not repeat the aptt . at this point ,
approximately 75% of internal medicine and geriatric physicians indicated they would consult a hematologist , versus 47% in emergency medicine and 50% in critical care .
all participants preferred abdominal computed tomography ( 80%84% ) .
after 12 hours of additional observation , 73% to 94% of respondents consulted a hematologist .
complete blood count revealed anemia and an aptt twice the upper limit of normal ; emergency medicine physicians remained least likely to request a consult.conclusion:determining the cause of an abnormal coagulation study result should carry equal weight as looking for the site of bleeding and could be facilitated by consultation with a hematologist .
insight from this survey highlights knowledge and practice gaps that could be the target of focused educational initiatives .
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Introduction
Materials and methods
Results
Initial presentation: recommended diagnostic evaluations and hematology consultation
Second presentation: recommended diagnostic evaluations and hematology consultation
Recommendations regarding case patient disposition
Familiarity with bleeding disorders
History of hematology consultations
Discussion
Conclusion
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polyarginine peptides
are known as one of the widely used classes
of cell - penetrating peptides ( cpps ) and cellular delivery tools .
it has been reported that the presence of the
guanidine group in the side chain of arginine plays a key role for
improved ability of arginine - rich peptides to cross the cell membrane .
various systematic structural investigations have been performed
to determine the required number of arginine residues and the length
of the peptide for the optimization of cellular uptake .
short polyarginine peptides containing less than six arginine residues
did not exhibit significant cellular uptake in several previously
reported investigations .
thus , the presence of more than six arginine residues in the structure
of polyarginine peptides is critical for their efficient cell - penetrating
functions .
however , several investigations were conducted to
increase the
cellular uptake of polyarginines by attaching the fatty acid to the n - terminal of the peptide .
it has been previously reported
that the acylation of the n - terminal by fatty acids
can facilitate the intracellular uptake of polyarginines .
for instance , katayama et al . synthesized acylated octa - arginines
and discovered that the introduction of hydrophobic fatty acid enhanced
the intracellular uptake of octa - arginine peptide and its conjugated
ubiquitin .
furthermore , lee et al . designed
a class of lipopeptides carrying 715 arginine residues . among
them ,
myristoylated - hendeca - arginine ( c14r11 ) was found to be the most efficient cell - penetrating peptide . however , the fatty acylated polyarginine peptides
that contain 715 arginine residues can potentially cause toxicity ,
and they can be easily degraded by proteases . moreover , linear
peptides carrying l - form are not stable
in serum and therefore have a limited application for in vivo studies ( figure s2 , supporting information ) . replacing l - form amino acids
with d - form to improve the peptide stability leads to high
cost production .
thus , the synthesis and development of cyclic
cpps containing short amino acid sequence with less toxicity is desired . herein , we designed acylated cyclic polyarginine peptides ( acpps )
containing five arginine residues and investigated their ability as
cell - penetrating peptides .
we compared acpps with a corresponding
acylated linear polyarginine peptide ( alpp ) and a nonacylated cyclic
polyarginine as controls .
we hypothesize that the combination of acylation
and cyclization of short polyarginine peptides having less than six
arginine residues will increase the intracellular uptake and can generate
peptides with molecular transporter properties . for convenience , square
brackets [ ] and parentheses ( )
phosphopeptide ptyr - glu - glu - ile ( pyeei )
is an optimal peptide ligand for binding to the src tyrosine kinase
sh2 domain . in this study , we used negatively charged fluorescein - labeled
phosphopeptide f-gpyeei as a model cell - impermeable compound .
all peptides were synthesized by
fmoc / tbu solid - phase peptide synthesis strategy either
manually or using rainin ps3 synthesizer ( protein technologies inc . ) .
manual reactions were carried out in a glass reaction vessel with
a sintered glass frit by mixing under nitrogen bubbling at room temperature .
fmoc - l - amino acid building blocks , fatty acids , and preloaded
h - arg(pbf)-2-chlorotrityl resin were used as starting materials .
2-(1h - benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate
( hbtu ) , hydroxybenzotriazole ( hobt ) , and n , n - diisopropylethylamine ( dipea ) in n , n - dimethylformamide
( dmf ) were used as coupling and activating reagents , respectively ,
for manual synthesis . in the case of using peptide synthesizer , 0.4
m n - methylmorpholine in dmf was used instead of dipea .
piperidine in dmf ( 20% ) was employed to deprotect fmoc group at each
step . to cleave the linear peptide from the resin , a mixture
of trifluoroacetic acid ( tfa)/triisoproylsilane ( tis)/water ( 92.5/5/2.5 ,
however , in the synthesis of cyclic peptides , the
side chain protected linear peptides were first cleaved from the resin
by using a cleavage cocktail , containing 2,2,2-trifluoroethanol ( tfe)/acetic
acid / dichloromethane ( dcm ) ( 2:1:7 , v / v / v ) .
cyclization reaction was
performed by employing a mixture of 1-hydroxy-7-azabenzotriazole ( hoat )
and n , n-diisopropylcarbodiimide
( dic ) in anhydrous dmf / dcm for 12 h. after solvent evaporation , the
peptide was deprotected and cleaved from the resin by using a cleavage
cocktail reagent
r , containing tfa / thioanisole/1,2-ethanedithiol
( edt)/anisole ( 90:5:3:2 , v / v / v / v ) for 23 h. the crude peptides
were precipitated and washed with cold diethyl ether . to purify the
crude peptides , we used a reversed - phase high pressure liquid chromatography
( rp - hplc ) system using shimadzu lc-8a preparative liquid chromatography
on a phenomenex gemini c18 column ( 10 m , 250 21.2 mm )
with a gradient 0100% of acetonitrile ( ch3cn ) containing
0.1% tfa ( v / v ) and water containing 0.1% tfa ( v / v ) for 1 h with a
flow rate at 15.0 ml / min at the wavelength of 214 nm . as a representative
example , the synthesis of dodecanoyl-[r5 ] is described
here .
h - arg(pbf)-2-chlorotrityl resin ( 660
mg , 0.35 mmol , 0.53 mmol / g ) was swelled in dmf for 40 min by n2 .
fmoc - arg(pbf)-oh ( 681 mg , 1.05 mmol , 3 equiv ) was coupled
to the n - terminal of the resin , using hbtu ( 398 mg ,
1.05 mmol , 3 equiv ) , hobt ( 142 mg , 1.05 mmol , 3 equiv ) , and dipea
( 366 l , 2.1 mmol , 6 equiv ) in dmf ( 15 ml ) by agitating the
resin for 1 h using n2 .
after the coupling , the resin was
washed with dmf , followed by fmoc - deprotection with piperidine in
dmf ( 20% ) .
the subsequent three fmoc - arg(pbf)-oh couplings and one
dde - lys(fmoc)-oh ( 559 mg , 1.05 mmol , 3 equiv ; dde = 1-(4,4-dimethyl-2,6-dioxocyclohex-1-ylidene)ethyl )
coupling was carried out in the same manner , respectively . after removing
the fmoc group in side chain of lysine , dodecanoic acid (
210 mg , 1.05
mmol , 3 equiv ) was coupled using hbtu , hobt , and dipea .
then dde protection
group at n - terminal of peptide was removed by 2%
hydrazine in dmf , followed by washing with dmf and dcm .
the side chain
protected linear peptides were cleaved from the resin by using a cleavage
cocktail , tfe / acetic acid / dcm ( 2:1:7 , v / v / v ) , for 1 h. the filtrate
was evaporated , and the residue was dried overnight in a vacuum .
the
cyclization was conducted under a dilute condition with anhydrous
dmf / dcm ( 5:3 , v / v , 250 ml ) , using hoat ( 190 mg , 1.4 mmol , 4 equiv )
and dic ( 240 l , 1.54 mmol , 4.4 equiv ) , and stirred for 12 h
under nitrogen atmosphere .
after cyclization , the solvent was evaporated ,
and the side chain deprotection was carried out by the addition of
reagent r for 2 h. the crude dodecanoyl-[r5 ] was precipitated and washed with cold diethyl ether and purified
by preparative rp - hplc system as described above .
fluorescein - labeled
peptides were synthesized with the same protocol
before the attachment of fatty acid .
we used fmoc-12-aminododecanoic
acid instead of dodecanoic acid , and after removing fmoc group , 5(6)-carboxyfluorescein
diisobutyrate ( cfdi ) was attached using 7-azabenzotriazol-1-yl - oxytris(pyrrolidino)phosphonium
hexafluorophosphate ( pyaop ) , hoat , and dipea .
as an example , we started
fluorescein - labeled peptide synthesis in smaller scale with h - arg(pbf)-2-chlorotrityl
resin ( 208 mg , 0.11 mmol , 0.53 mmol / g ) .
fmoc - arg(pbf)-oh ( 214 mg ,
0.33 mmol , 3 equiv ) , dde - lys(fmoc)-oh ( 176 mg , 0.33 mmol , 3 equiv ) ,
and fmoc-12-aminododecanoic acid ( 144 mg , 0.33 mmol , 3 equiv ) were
used to couple each building block to the resin using hbtu ( 125 mg ,
0.33 mmol , 3 equiv ) , hobt ( 45 mg , 0.33 mmol , 3 equiv ) , and dipea ( 115
l , 0.66 mmol , 6 equiv ) in dmf .
cfdi ( 170 mg , 0.33 mmol , 3 equiv )
was coupled with amino group of 12-aminododecaonic chain using pyaop
( 172 mg , 0.33 mmol , 3 equiv ) , hoat ( 45 mg , 0.33 mmol , 3 equiv ) , and
dipea .
after removal of dde protecting group with 2% hydrazine in dmf
and washing with dmf and dcm , side chain protected fluorescein linear
peptides were cleaved from resin using tfe / acetic acid / dcm ( 2:1:7 ,
v / v / v ) .
the molecular weights of final products were confirmed by
an axima performance matrix - assisted laser desorption / ionization - time - of - flight
( maldi - tof ) mass spectrometer ( shimadzu corporation ) .
dodecanoyl-(r6 ) : maldi - tof ( m / z ) c48h96n22o8 calcd . 1108.7781 ; found 1109.7308 [ m + h ] .
f-dodecanoyl-(r5 ) : maldi - tof ( m / z ) c69h107n23o14 calcd . 1481.8368 ;
found 1482.7586 [ m + h ] .
w - dodecanoyl-[r5 ] : maldi - tof ( m / z ) c61h107n25o9 calcd .
human ovarian adenocarcinoma
( sk - ov-3 ) ,
leukemia ( ccrf - cem ) , and embryonic kidney ( hek 293 t ) cells were purchased
from american type culture collection .
the sk - ov-3 and hek 293 t cells
were grown in eagle s minimum essential medium ( emem ) , and
rpmi-1640 medium ( atcc , manassas , va ) was used for ccrf - cem cells
in a humidified atmosphere of 5% co2 at 37 c .
both
media were supplemented with fetal bovine serum ( fbs , 10% ) and penicillin
streptomycin
solution ( 1% , 10,000 units of penicillin and 10 mg of streptomycin
in 0.9% nacl ) .
cytotoxicity
of peptides was examined
by mts proliferation assay in two human cancer cell lines ( sk - ov-3
and ccrf - cem ) and one human normal cell line ( hek 293 t ) .
cells were
seeded into 96-well plates ( sk - ov-3 ( 5 10 cells / well ) ,
ccrf - cem ( 1 10 cells /
well ) , and hek 293 t ( 1
10 cells / well ) ) .
then , the cells were incubated with complete
media ( 100 l ) overnight .
different concentrations ( 0600
m ) of peptide solution ( 10 l ) were added to cells and
incubated at 37 c with 5% co2 for 24 h. then , celltiter
96 aqueous solution ( 20 l ) was added to each well and incubated
for 14 h. the absorbance was measured at 490 nm using microplate
reader .
sk - ov-3
cells were grown in 6-well plates ( 2 10 cells / well )
with complete emem media 24 h prior to the experiment .
the fluorescein - labeled
peptide stock solution ( 1 mm ) was prepared in water and diluted in
gibco opti - mem i reduced serum medium to obtain the final concentration
of 5 m .
the media were removed , and the mixture containing
fluorescein - labeled peptide solution ( 5 m ) was added .
after
1 h incubation , trypsin edta solution was added to detach cells
from the plate s surface and remove cell surface binding peptides .
after 5 min , the complete media ( 2 ml ) were added to neutralize the
trypsin .
finally ,
the cells were analyzed by bd facscalibur or facsverse flow cytometer
using fitc channel .
5(6)-carboxyfluorescein ( fam ) was used as a negative control . for examination
of the cellular uptake mechanism of f-dodecanoyl-[r5 ] at low temperature
, the assay was carried out at 4 c
to inhibit the energy - dependent cellular uptake pathways .
the sk - ov-3
cells were preincubated at 4 c for 15 min and incubated with
the fluorescein - labeled peptide for 1 h at 4 c .
cells were collected
and analyzed using flow cytometry with the previously described protocol
above .
the data collected at 37 c were used as the control .
to perform the atp - depletion assay ,
cells were incubated with sodium
azide ( 10 mm ) and 2-deoxy - d - glucose ( 50 mm ) for 1 h before
adding the fluorescein - labeled peptide . during the incubation time
( 1 h ) , the fluorescein - labeled bicyclic peptide ( 5 m )
was prepared
in the opti - mem i reduced serum medium in the presence of sodium azide
( 10 mm ) and 2-deoxy - d - glucose ( 50 mm ) .
then , the cells were
incubated with this solution for 1 h. the following sample preparation
and flow cytometry analysis protocol was the same as described above .
sk - ov-3 cells
were seeded with complete emem on coverslips in 6-well plate ( 1
10 cells / well ) and kept until 50% confluency .
the media
were removed , and cells were incubated with f-dodecanoyl-[r5 ] ( 10 m ) and f-dodecanoyl-(r5 ) ( 10
m ) in gibco opti - mem i reduced serum medium ( life technologies ,
grand island , ny ) for 1 h at 37 c . then cells were washed with
1 phosphate buffered saline with calcium and magnesium ( pbs ) for three times .
the coverslips were mounted on microscope
slides , and images were obtained using carl zeiss lsm 700 system with
a 488 nm argon ion laser excitation and a bp 505530 nm band - pass
filter .
sk - ov-3 and ccrf - cem cells were seeded in 6-well plates ( 2
10 cells / well for sk - ov-3 and 1 10cells / well
for ccrf - cem ) and grown with complete emem media ( rpmi-1640 for ccrf - cem )
overnight .
a mixture of fluorescein - labeled phosphopeptides f-gpyeei
( 5 m ) and peptides ( 10 m ) were prepared in opti - mem
i reduced serum medium at room temperature and incubated for 15 min .
then the cells were incubated with the premixed solution at 37 c
with 5% co2 for 1 h. the sample preparation for facs analysis
was carried out by previously mentioned protocol described before .
in this assay ,
the
acylated cyclic polyarginine peptides
were synthesized by fmoc / tbu solid - phase peptide
synthesis method .
fmoc - l - arg(pbf)-oh was coupled on h - arg(pbf)-loaded
2-chlorotrityl resin in the presence of hbtu , hobt , and dipea in dmf .
then dde - lys(fmoc)-oh was attached , and a fatty acid was coupled to
the side chain of lysine .
dde protecting group was removed by 2% hydrazine
in dmf , and a cleavage cocktail containing tfe / acetic acid / dcm ( 2:1:7
v / v / v ) was used for 1 h to cleave the side - chain protected linear
peptides from the resin .
cyclization of linear peptides was carried
out in the presence of a mixture of hoat and dic in anhydrous dmf / dcm
for 624 h. the side - chain deprotection of cyclic peptide was
carried out by a cleavage cocktail reagent
r for 2
h. the crude peptides were precipitated and purified with rp - hplc
as described above .
as a representative example , the synthesis of
dodecanoyl-[r5 ] is shown in scheme 1 .
a corresponding acylated
linear polyarginine peptide ( alpp ) was
synthesized for comparative studies with the cyclic peptide ( acpp ) .
moreover , a cyclic polyarginine without fatty acid [ r5 ]
was also synthesized to investigate the effect of the fatty acid on
cyclic peptide and its effect on molecular transporting efficiency .
to investigate whether the peptide alone can enter into cells , fluorescein - labeled
f-dodecanoyl-[r5 ] and f-dodecanoyl-(r5 ) , where f = fluorescein , were synthesized for facs
and microscopy investigations .
chemical structures of synthetic peptides
used in this study ( f ,
fluorescein - labeled ; [ ] , cyclic peptide ; ( ) , linear peptide ) .
the cytotoxicity
of all peptides were tested in two different cancer cell lines , adherent
( sk - ov-3 ) and nonadherent ( ccrf - cem ) cells , and a normal cell line
( hek 293 t ) using mts assay ( figure 2 ) .
cyclic
polyarginine [ r5 ] without fatty acid was used to explore
the effect of n - terminal acylation on cytotoxicity
and cellular uptake .
alpp ( dodecanoyl-(r5 ) ) and [ r5 ] peptides showed consistently less cytotoxicity in all three
cells compared to acpps ( dodecanoyl-[r5 ] and dodecanoyl-[r6 ] ) .
after 24 h incubation , acpps showed approximately 20%
toxicity in cells at a concentration of 25 m in ccrf - cem cells .
however , dodecanoyl - linear ( r5 ) and [ r5 ] did
not exhibit significant cytotoxicity at 25 m and showed less
than 20% toxicity at the concentration of 100 m . in sk - ov-3
cells , dodecanoyl-(r5 ) and [ r5 ] showed more
than 80% cell viability at the concentration of 25 m .
in normal
cells ( hek 293 t ) , all peptides exhibited less than 5% toxicity at
25 m .
this differential behavior of the peptides in normal ,
and cancer cells can be possibly rationalized through the interaction
between polyarginine peptides and cell membranes .
the membrane of
cancer cells holds more negative charges compared to that in normal
cells because of the presence of anionic lipids such as phosphatidylserine .
therefore , polyarginine peptides can be interacted
with cancer cells more effectively compared to normal cells .
these data indicated
that acpps are more toxic than alpp and a nonacylated cyclic peptide
[ r5 ] , especially at concentration of 25 m .
thus , a noncytotoxic concentration of 510 m was used
in cell - based assays .
comparison of cytotoxicity between cyclic and linear acylated
polyarginine
peptides and nonacylated cyclic peptide [ r5 ] at various
concentrations against ccrf - cem , sk - ov-3 , and hek 293 t after 24 h. the intracellular uptake studies of fluorescein - labeled
acylated cyclic and linear pp , f-dodecanoyl-[r5 ] and f-dodecanoyl-(r5 ) , was carried out in sk - ov-3
cells by using flow cytometry and confocal laser scanning microscopy
( clsm ) methods .
fluorescein ( fam , f ) alone was selected as
a negative control . as it is shown in figure 3 ,
the f-dodecanoyl-[r5 ] and f-dodecanoyl-(r5 ) showed approximately 13.7- and 10.3-fold higher cellular
uptake than that of control 5,6-carboxyfluorescein ( fam ) , respectively ,
in sk - ov-3 cells .
the cellular uptake of f-dodecanoyl-[r5 ] was confirmed by clsm images ( figure 4 ) .
f-dodecanoyl-[r5 ] showed higher fluorescence intensity
compared to that of f-dodecanoyl-(r5 ) in sk - ov-3
cells .
therefore , acpp f-dodecanoyl-[r5 ] was found
to be a more efficient cell - penetrating peptide compared to its linear
counterpart .
as shown in figure 4 , the fluorescence
signal is extended through the whole cells , suggesting that f-dodecanoyl-[r5 ] can get localized in the nucleus as well as cytoplasm .
comparative
cellular uptake of f-dodecanoyl-[r5 ] and f-dodecanoyl-(r5 ) ( 5 m ) in sk - ov-3
cells ( 1 h ) .
confocal laser scanning
microscope image of ( a ) f-dodecanoyl-[r5 ] and ( b )
f-dodecanoyl-(r5 ) .
the peptides
were incubated for 1 h in sk - ov-3 cells at 10 m concentration .
the mechanism
of the cellular internalization of
f-dodecanoyl-[r5 ] was investigated by a temperature
control assay at 4 c along with atp depletion assay .
these two
assays have been widely used to examine the energy - dependent endocytosis .
facs results showed that the intracellular uptake
of f-dodecanoyl-[r5 ] was significantly reduced
at 4 c , indicating that the mechanism of internalization was
mainly dependent on the endocytosis pathways ( figure 5 ) .
furthermore , atp depletion
assay was performed to investigate receptor - mediated endocytosis . to induce atp depletion ,
sk - ov-3 cells were
preincubated with sodium azide ( 10 mm ) and 2-deoxy - d - glucose
( 50 mm ) for 1 h prior to the experiment , and a similar concentration
was maintained during the incubation ( 1 h ) with f-dodecanoyl-[r5 ] .
the results showed that the cellular uptake of f-acpp
was inhibited in the presence of sodium azide and 2-deoxy - d - glucose , suggesting that receptor - mediated endocytosis is involved
for the cellular uptake of f-acpp . in atp depletion assay ,
the basic cellular uptake of fam was higher compared to that in temperature
control assay .
however , this is evident that the intracellular uptake
of f-dodecanoyl-[r5 ] was inhibited , and there was
no significant difference between the cells treated with fam compared
to those treated with f-dodecanoyl-[r5 ] in temperature
control and atp depletion assays , suggesting that endocytosis is the
major pathway for the cellular uptake of f-dodecanoyl-[r5 ] .
cellular uptake of f-dodecanoyl-[r5 ] ( 5 m )
in sk - ov-3 cells in temperature control assay at 37 and 4 c ,
and atp depletion assay with nan3 ( 10 mm ) and 2-deoxy - d - glucose ( 50 mm ) analyzed by flow cytometry .
cells with no
treatment were used as control . the ability
of acpps as a molecular transporter was evaluated and compared by
selecting a fluorescein - labeled phosphopeptide , f-gpyeei ,
as a molecular cargo .
phosphopeptide , pyeei ( ptyr - glu - glu - ile ) is
an optimal peptide template for the sh2 domain of src tyrosine kinase .
several analogues of this peptide have been synthesized as potent
ligands for this target . because of the presence of the
negatively charged amino acid residues in the structure of the peptide
including phosphorylated tyrosine
moreover , the internalization of the negatively charged phosphopeptide
in cancer cells by diffusion is more difficult because cancer cell
membranes are composed of more negatively charged lipids .
thus , cellular
delivery of cell - impermeable negatively charged phosphopeptides is
significantly challenging .
we have previously reported different peptide - based
carriers for the intracellular delivery of negatively charged phosphopeptides
as model cell - impermeable drugs in several cell lines . in this study ,
the intracellular uptake
of f-gpyeei was monitored in the presence and absence of synthetic
peptides after 1 h incubation by flow cytometry .
as it is exhibited
in figure 6 , the acpps ( dodecanoyl-[r5 ] and dodecanoyl-[r6 ] ) delivered the phosphopeptide more
efficiently compared to alpps , dodecanoyl-(r5 ) and -[r5 ] .
the intracellular uptake of f-gpyeei in the presence
of dodecanoyl-[r5 ] and dodecanoyl-[r6 ] was enhanced
by 3.4- and 5.5-fold higher than the uptake in the absence of acpps .
however , dodecanoyl-(r5 ) and -[r5 ] only improved
1.3- and 1.4-fold intracellular uptake , respectively .
the results
showed that acylated and cyclized polyarginine peptides can deliver
the phosphopeptide effectively .
however , the intracellular uptake
of the phosphopeptide did not improve significantly in the presence
of either acylated linear polyarginine peptide or cyclic [ r5 ] .
these data suggest that a combination of acylation and cyclization
would improve the molecular transporting efficiency of the polyarginine - based
peptide ( containing less than six arginines ) for the intracellular
delivery of a cell - impermeable phosphopeptide .
this has been previously
reported that the acylated linear octa - arginine increased the cellular
uptake of molecular cargoes by just adding fatty acid to the n - terminal of octa - arginine .
however , we discovered that both cyclization and acylation in a
short penta - arginine can significantly improve the delivery of a cell - impermeable
phosphopeptide in sk - ov-3 cells .
cellular uptake of f-gpyeei ( 5
m ) in the presence
of dodecanoyl-[r5 ] and -[r5 ] , dosecanoyl-(r5 ) , and dodecanoyl-[r6 ] ( 10 m ) in sk - ov-3
cell line .
phosphopeptide delivery efficiency of dodecanoyl-[r5 ] were compared with known cpps ( r7 , crrrrrrr ;
tat , ygrkkrrqrrr ) .
the major driving forces for the intracellular delivery are
presumed
to be structural rigidity through cyclization of the peptide and the
interaction of the fatty acid with the cell membrane .
it has been
previously reported that the cellular uptake of the peptide can be
increased due to the structural rigidity by cyclization of arginine - rich
peptides .
they proposed that the maximal
distance between guanidine groups of arginine residue can lead to
an efficient transduction of arginine - rich peptides .
our investigations
showed that dodecanoyl-[r6 ] is able to deliver more efficiently
by 1.6-fold higher f-gpyeei uptake compared to that of dodecanoyl-[r5 ] . increasing
the number of positively charged arginine residues
can enhance the cellular uptake through ionic interactions with the
negatively charged phosphopeptide and/or phospholipid in the cell
membrane through ionic interactions .
however , the higher number of
arginine residue is not the only responsible element for the efficient
cellular internalization .
for example , it has been reported that polyarginine
containing 11 amino acids ( r11 ) showed higher cellular
uptake compared to the polyarginine containing 13 amino acids ( r13 ) . at the same time
, r11 was found to be a more
potent transporter compared to r9 in prostate cancer cells .
these investigations showed that an optimal
number of arginine residues are required for the highest degree of
functionality .
however , the greater number of amino acid residues
in cyclic peptides can decrease the structural rigidity , which lowers
the ability of the peptide to get into cells .
dodecanoyl-[r5 ] was also compared with several commonly
cpps , such as cr7 and tat ( ygrkkrrqrrr ) peptides .
the acpp
improved the cellular uptake of the phosphopeptide by 1.4- and 1.8-fold
higher than those of cr7 and tat , respectively ( figure 6 ) .
these results revealed that although other peptides
containing arginine can deliver the molecular cargo , acpp dodecanoyl-[r5 ] with a shorter peptide sequence than cr7 and
tat can work as a molecular transporter with higher efficiency .
chemical structures
of acpps with different length of fatty acid
chains ( c8 , c12 , and c16 ) . to investigate the effect
of the chain length on the cell penetration
potency , we synthesized octanoyl-[r5 ] , dodecanoyl-[r5 ] , and hexadecanoyl-[r5 ] ( figure 7 ) .
acpps showed less than 20% toxicity in cells at the concentration
of 25 m ( figure 8a ) .
the in
vitro toxicity results showed that increasing the fatty acid
chain length caused enhanced toxicity in cells as hexadecanoyl-[r5 ] was more cytotoxic than dodecanoyl-[r5 ] and octanoyl-[r5 ] .
these data indicate that the fatty acid chain length could
alter the interaction with the cell membrane and disturb the membrane
integrity . on the basis of the cytotoxicity data ,
( a ) cytotoxicity
assay of cyclic polyarginine peptide - fatty acid
conjugates against sk - ov-3 cells ( 24 h incubation ) .
( b ) cellular uptake
of a phosphopeptide , f-gpyeei ( 5 m ) , in the presence
of peptide - fatty acid conjugates ( 10 m ) in sk - ov-3 cells .
it has been previously reported that there was a relationship
between
the length of fatty acid in polyarginines and their cellular uptake , meaning that the optimal length of fatty acid
is required for optimal functionality based on the peptide sequence
and cell type .
the results exhibited that the cellular uptake of f-gpyeei
was improved in the order of octanoyl-[r5 ] < dodecanoyl-[r5 ] < hexadecanoyl-[r5 ] ( figure 8b ) .
the cellular uptake of f-gpyeei in the presence
of hexadecanoyl-[r5 ] was 9.3- and 6.0-fold higher than
that in the presence of octanoyl-[r5 ] and dodecanoyl-[r5 ] , respectively .
these data suggest that the length of the
attached fatty acid chain in the structure significantly influences
the efficiency of the peptide as a molecular transporter . sixteen - carbon
chain length ( c16 )
was found to be an optimized length
for the intracellular delivery of f-gpyeei in sk - ov-3 cells .
after acpps were found to act as cpp and molecular
transporter , a systematic investigation was performed to modify the
fatty acid to another hydrophobic moiety .
w4-[r5 ] ,
the whole fatty acid chain was replaced with four tryptophan residues .
the second conjugate w - dodecanoyl-[r5 ] had one more tryptophan
at the end of the dodecanoyl fatty acid chain .
w4-[r5 ] enhanced intracellular delivery of f-gpyeei by 4.1-fold
higher compared to that of dodecanoyl-[r5 ] in sk - ov-3 .
however , w - dodecanoyl-[r5 ] improved the uptake of f-gpyeei
by 1.3-fold higher compared to that of w4-[r5 ] in ccrf - cem cells ( figure 9 ) .
thus , it is not straightforward
to compare the cellular uptakes in sk - ov-3 and ccrf - cem .
however ,
the results could be assessed indirectly by relative comparison with
the cellular uptake by w4-[r5 ] .
these data suggest
that the presence of hydrophobic tryptophan moieties could enhance
the molecular transporting efficiency . in other words ,
appropriate
modification of hydrophobic moiety in cpps can increase the drug delivery
ability of acpp .
thus , cyclic nature , short length of polyarginine ,
and hydrophobic segments were found to be critical elements to generate
a peptide with an optimized molecular transporter efficiency .
cellular uptake
assay of a phosphopeptide , f-gpyeei ( 5
m ) , in the presence of w4-[r5 ] and w - dodecanoyl-[r5 ] ( 10 m ) against sk - ov-3 and ccrf - cem cell lines ( 1
h incubation ) .
cells with no treatment
were used as control . to evaluate
whether the presence of peptides can enhance the pharmacological effect
of a molecular cargo , the antiproliferative activity of doxorubicin
( dox )
was examined in the presence of dodecanoy-[r6 ] ( [ r6]-c12 ) in mcf-7 cells in a time - dependent assay .
as it is shown in figure 10 ,
the antiproliferative
potency of dox ( 5 m ) was enhanced by 7% , 11% , and 14% in the
presence of [ r6]-c12 ( 5 m ) when compared
with that of the drug alone after 24 , 48 , and 72 h incubation , respectively .
these results showed that the cell proliferation of mcf-7 cells was
inhibited in a time - dependent manner suggesting a sustained drug release
by peptide leading to a higher inhibitory effect .
the presence of
the peptide possibly improved the cellular uptake of dox and enhanced
the antiproliferative activity .
time - dependent antiproliferative activity
of dox ( 5 m ) in
the presence and absence of [ r6]-c12 ( 5 m ) .
in
conclusion , acylated cyclic polyarginine peptides were synthesized
and examined as cpps and potential molecular transporters .
acpps showed
higher potency as molecular transporter compared to the corresponding
linear counterpart and cyclic polyarginine without fatty acid .
the
mechanism of the peptide internalization was found to be energy - dependent
endocytosis .
cyclization and acylation reactions on the structure
of the peptide enhanced the intracellular uptake of polyarginine peptides
although they carry a short length of sequence .
this intracellular
delivery property of acpps can be optimized by modifying the length
of fatty acid chain . to the best of our knowledge ,
this is the first
report of cyclic fatty acylated polyarginine peptide as molecular
transporter of a cell - impermeable phosphopeptide .
this study provided
insights about how a combination of the cyclic nature and acylation
can improve the cell internalization of polyarginines .
further investigations
are undergoing to determine whether conjugation of cell - impermeable
hydrophobic drugs to acylated cyclic polyarginines can be an efficient
method for designing novel drug delivery systems .
|
many
of the reported arginine - rich cell - penetrating peptides ( cpps )
for the enhanced delivery of drugs are linear peptides composed of
more than seven arginine residues to retain the cell penetration properties .
herein , we synthesized a class of nine polyarginine peptides containing
5 and 6 arginines , namely , r5 and r6 .
we further
explored the effect of acylation with long chain fatty acids ( i.e. ,
octanoic acid , dodecanoic acid , and hexadecanoic acid ) and cyclization
on the cell penetrating properties of the peptides .
the fluorescence - labeled
acylated cyclic peptide dodecanoyl-[r5 ] and linear peptide
dodecanoyl-(r5 ) showed approximately 13.7- and 10.2-fold
higher cellular uptake than that of control 5,6-carboxyfluorescein ,
respectively .
the mechanism of the peptide internalization into cells
was found to be energy - dependent endocytosis .
dodecanoyl-[r5 ] and dodecanoyl-[r6 ] enhanced the intracellular uptake
of a fluorescence - labeled cell - impermeable negatively charged phosphopeptide
( f-gpyeei ) in human ovarian cancer cells ( sk - ov-3 ) by 3.4-fold
and 5.5-fold , respectively , as shown by flow cytometry
. the cellular
uptake of f-gpyeei in the presence of hexadecanoyl-[r5 ] was 9.3- and 6.0-fold higher than that in the presence of
octanoyl-[r5 ] and dodecanoyl-[r5 ] , respectively .
dodecanoyl-[r5 ] enhanced the cellular uptake of the phosphopeptide
by 1.42.5-fold higher than the corresponding linear peptide
dodecanoyl-(r5 ) and those of representative cpps , such
as hepta - arginine ( cr7 ) and tat peptide .
these results
showed that a combination of acylation by long chain fatty acids and
cyclization on short arginine - containing peptides can improve their
cell - penetrating property , possibly through efficient interaction
of rigid positively charged r and hydrophobic dodecanoyl moiety with
the corresponding residues in the cell membrane phospholipids .
|
Introduction
Experimental
Section
Results and Discussion
Conclusions
|
as of 2014 , the worldwide prevalence of type 2 diabetes mellitus ( t2 dm ) was estimated to be 9% among adults aged 18 years with great impact on mortality , particularly in low- and middle - income countries ( lmic ) [ 1 , 2 ] . moreover , globally , approximately 25% to 75% of diabetes cases remain undiagnosed [ 3 , 4 ] , until further complications , especially at the macro- and micro - vascular level , manifest clinically . in latin america ,
an important strategy to prevent or delay t2 dm complications is the early identification of those with undiagnosed diabetes ; yet , universal screening for diabetes at the population level is not practical in resource - limited settings .
the american diabetes association recommends the use of glucose test as t2 dm screening in people with overweight and obesity as well as in those with other risk factors . as a result
, risk assessment scores have been developed to address this problem in a simple and inexpensive way .
most of the available algorithms for diabetes screening have been developed in caucasian [ 79 ] and asian populations [ 1013 ] and very few in other ethnic groups [ 14 , 15 ] . to date , one diabetes risk score has been developed and validated in latin america so far which was derived from one urban area in brazil , thus bearing limited generalizability to the wider region .
furthermore , it is well established that before adopting existing risk scores as screening tools in different populations and ethnic groups , their performance needs to be evaluated , calibrated , or validated in local settings . as the american diabetes association , the peruvian ministry of health recommends diabetes screening in general population with fasting glucose in adults aged 40 to 70 years with risk factors .
however , fasting glucose is not always available in primary care settings , especially in semiurban and rural areas . as a result , a major challenge to be overcome in many countries is the implementation of a simple , fast , and laboratory - free based screening method .
consequently , we aimed to develop a simple laboratory - free risk score to identify people with undiagnosed diabetes and incident diabetes in peru , a latin american country that spans coastal , andean , and rainforest settings . in order to do so
, this work benefited from two large - scale population - based surveys : the first one , representative at the national level , was used to develop the score , and the second one , a cohort study , was utilized for external validation .
the national survey of nutritional and biochemical indicators for noncommunicable diseases ( eninbsc in spanish ) , conducted by the peruvian national institute of health , was used to develop our predictive model .
this was complemented with the cronicas cohort study , whose baseline and longitudinal information was used to validate the risk score .
the eninbsc is a national population - based survey carried out in peru between august 2004 and april 2005 , designed to estimate the prevalence of hypertension , type 2 diabetes mellitus , and other risk factors for noncommunicable diseases at the national and regional level .
potential participants were those aged 20 years , habitual residents in the study area , and able to provide consent for their participation in the study .
pregnant women and those currently breastfeeding were excluded from the study . as per design , the eninbsc sample was stratified according to peru 's five major regions of the country : lima , rest of the coast , urban highlands , rural highlands , and jungle . in each stratum , cluster of blocks
the cronicas cohort study is an ongoing cardiopulmonary project aimed to estimate the prevalence and incidence of hypertension , diabetes mellitus , and obesity in four different settings in peru that differ in terms of their urbanicity and altitude : pampas de san juan de miraflores , in the highly urbanized lima , puno in the altitude ( 3,825 meters above the sea level ) contributing with rural and urban areas , and tumbes , a semiurban area in the northern coast of peru .
the study started in september 2010 and a follow - up visit was completed in march 2014 .
a sex- and age - stratified sample was selected at random for each of the settings and all participants aged 35 years , full time residents in the study area , and able to consent , were enrolled .
follow - up data used for this analysis was collected , on average , at 30 months after baseline .
the first one lasted on average 40 minutes and was carried out to apply a face - to - face questionnaire regarding data about household characteristics , demographics , lifestyles behaviors , risk factors , and blood pressure measurements .
the second visit lasted 30 minutes on average and was planned to have an appropriate period of fasting for blood sampling for glucose , total cholesterol , hdl - cholesterol , and the remaining anthropometric measures ( height , weight , and waist circumference ) using standard procedures .
similarly , the procedures of the cronicas study has been published elsewhere . in brief , participants responded to a face - to - face questionnaire applied by trained community health workers .
data collected comprised risk factors for cardiovascular disease based on a modified version of the who step approach questionnaire for surveillance of noncommunicable disease .
a period of 8 to 12 hours of fasting was required for blood sampling to collect fasting glucose , total cholesterol , and hdl - cholesterol .
height , weight , and waist circumference were also assessed , and blood pressure was measured in triplicate after five minutes of resting using an automatic monitor ( omron hem-780 ) previously validated in adult 's population . in both studies ,
diabetes was defined as any of the following conditions : fasting glucose 7.0 mmol / l ( 126 mg / dl ) and/or self - report of physician diagnosis .
fasting glucose was assessed by an enzymatic colorimetric method ( glucose oxidase god - pap ) in both studies . after excluding individuals without known diabetes , undiagnosed diabetes
variables included in the analyses were built to guarantee similarities between both studies : sex ; age ( < 55 and 55 years ) ; education ( in years ) ; self - reported smoking ( current versus never / former smoker ) ; alcohol use ( user versus never user ) ; self - reported diabetes in first - degree relatives ( participant 's parents and/or siblings ) ; and levels of physical activity ( low versus moderate / high levels , based on the transport - related domain of the ipaq ) .
anthropometric measurements included in the analysis were body mass index ( ( bmi ) , < 25 , 2529.9 , and 30 kg / m ) , waist circumference ( < 90 , 9099.9 , and 100 cm ) , waist - to - height ratio ( < 0.50 , 0.500.59 , 0.600.69 , and 0.70 ) , and hypertension ( measured or previously diagnosed ) . a total of 4,206 participants were enrolled in the eninbsc , but only 2,472 were included in this analysis .
reasons for exclusion were 1,524 because of age < 35 years to make both databases comparable , 129 because of no data about fasting plasma glucose levels being available , and 81 because of known diagnosis of diabetes . in the cronicas study , 3,601 participants were enrolled at baseline but only 2,948 records were analyzed as 465 had no data about glucose levels , and 188 were excluded because of previous diagnosis of diabetes .
in addition , only data from 2,577 participants was used in the longitudinal assessment of the risk score ( comparison of baseline characteristics among those included and excluded from longitudinal analysis is shown in online supplement : e - table 1 ; see supplementary material available online at http://dx.doi.org/10.1155/2016/8790235 ) .
initially , population characteristics of both studies were tabulated using proportions in the case of categorical variables and means and standard deviation ( sd ) with numerical variables .
then , the prevalence and 95% confidence intervals ( 95% ci ) of total diabetes and undiagnosed diabetes were estimated in each study .
the risk score was derived from data of the eninbsc survey taking into account the multistage sampling strategy of the study .
each potential risk factor ( i.e. , sex , age , family history of diabetes , etc . ) was assessed in bivariate models using logistic regression and undiagnosed diabetes as the dependent variable .
then , risk factors with a p value < 0.10 in the bivariate analysis were included in a multiple logistic regression model using stepwise backward elimination with a significance level of 5% . the hosmer - lemeshow goodness - of - fit test was used to assess
how well the predicted prevalence matched the observed prevalence of undiagnosed diabetes ( i.e. , p values over 0.20 indicate that model fits well ) . as we sought for an easily applicable and implementable algorithm ,
the risk factors in the final model were each assigned a weighted score by rounding up all regression coefficients in the final model to the nearest integer as in a previous report .
for the evaluation of the risk score , the area under the receiver operating characteristic ( roc ) curve , sensitivity , specificity , and positive and negative predictive values ( ppv and npv ) were calculated .
the optimal cut - point was determined using the youden index , a single statistic that captures the performance of a diagnostic test ( i.e. , sensitivity + specificity 1 ) .
as one of the main aims of a nonlaboratory risk score is to identify people who warrant having a blood test ( i.e. , fasting glucose , glycated haemoglobin , etc . ) , the cut - point with the highest sensitivity was also estimated and described .
we assessed the performance of our score using bootstrap techniques as well as carrying out an external validation using the cronicas cohort study .
the resulting 1,000 prediction models were then assessed to estimate the bootstrap auc using the bias - corrected version of the confidence intervals .
in addition , using baseline data from the cronicas cohort study , validation measures ( auc , sensitivity , specificity , predictive values , and likelihood ratios ) were estimated . to evaluate the performance of our algorithm ,
the peruvian risk score was compared to previously published models for undiagnosed diabetes including the brazilian risk score , the qingdao score , the indian risk score , the kuwaiti risk score , the patient self - assessment score , and the rotterdam risk score using the c - statistic .
finally , using the follow - up data of the cronicas cohort study , the risk score was also evaluated to detect incident cases of t2 dm by excluding those with diabetes diagnosis at baseline .
analyses were performed using stata 13.0 ( statacorp , college station , tx , usa ) . the protocol and informed consent forms of the eninbsc study were reviewed and approved by the instituto nacional de salud and the centro nacional de alimentacin y nutricin , both part of the ministry of health in lima , peru . in the case of the cronicas cohort study , protocol and consent forms were reviewed and approved by the institutional review boards of the universidad peruana cayetano heredia and the ngo asociacin benfica prisma in lima , peru , and the johns hopkins university in baltimore , usa .
overall , participants from the cronicas study were 5 years older , reported consuming lower levels of alcohol , and were less physically active than those from the eninbsc survey .
the overall prevalence of diabetes was 5.1% ( 129/2538 ; 95% ci : 4.2%5.9% ) in the eninbsc survey and 8.7% ( 272/3135 ; 95% ci : 7.7%9.7% ) in the cronicas cohort study 's baseline .
after excluding those with known diabetes , undiagnosed diabetes was present in 2.0% ( 48/2457 ; 95% ci : 1.4%2.5% ) in the eninbsc survey and in 2.9% ( 85/2948 ; 95% ci : 2.3%3.5% ) in the cronicas cohort study .
after stepwise backward logistic regression , age , diabetes in first - degree relatives , and waist circumference were independently associated with undiagnosed diabetes ( table 2 ) .
the hosmer - lemeshow test showed that the final model fitted relatively well ( p = 0.21 ) .
the peruvian diabetes risk score was constructed based on the coefficients of that final regression model .
the score gave an auc of 0.73 ( 95% ci : 0.650.78 ) , and the optimal cut - point for undiagnosed diabetes using the youden index was 2 ( figure 1 ) .
with this cut - point , about 34.8% of participants were categorized as at high risk of diabetes : sensitivity 69.6% , specificity 65.8% , and ppv and npv of 3.9% and 99.1% , respectively . with a cut - point 1
, 69.8% of participants would be at high risk of diabetes with improved sensitivity ( 93.5% ) but lower specificity ( 30.6% ) .
table 3 shows the performance of the risk score for detecting undiagnosed diabetes at different cut - points .
when bootstrap was used , the performance of our risk score was similar to the obtained in the development model ( auc = 0.72 ; 95% ci : 0.650.78 ) .
in addition , when the risk score was evaluated by applying the score to the cronicas cohort study 's population , the auc for undiagnosed diabetes was 0.68 ( 95% ci : 0.620.73 ) . at the suggested cut - point of 2 ,
42% would be categorized as undiagnosed diabetes with sensitivity , specificity , ppv , and npv of 70.2% , 58.9% , 4.8% , and 98.5% , respectively ( table 4 ) . on the other hand , with a cut - point 1 ,
80% would be categorized as undiagnosed diabetes with sensitivity , specificity , ppv , and npv of 94.0% , 20.0% , 3.3% , and 99.1% , respectively . when previous published algorithms for undiagnosed diabetes
were applied to the cronicas cohort study , the performance of the rotterdam score ( p < 0.001 ) , indian score ( p < 0.001 ) , and qingdao score ( p
< 0.01 ) was poorer than our score ; however , our algorithm performed similar to the other assessed models , such as the brazilian risk score ( p = 0.93 ) , the kuwaiti score ( p = 0.26 ) , and the patient self - assessment score ( p = 0.74 ) , but having only three variables .
the performance of this risk score was also assessed to predict incident cases of diabetes using the longitudinal data from the cronicas cohort study .
one hundred twenty - one new cases of diabetes were found accounting for 6,207 person - years at risk , with an overall incidence of 1.95 ( 95% ci : 1.632.33 ) cases per 100 person - years of risk .
the auc of the score was 0.66 ( 95% ci : 0.610.71 ) . with a cut - point 2 ,
42.5% of participants were categorized as at high risk of developing diabetes : sensitivity , specificity , ppv , and npv were 69.4% , 58.9% , 7.8% , and 97.4% , whereas , for a cut - point 1 , the respective values were 79.9% , 91.9% , 20.7% , 5.5% , and 98.1% .
using a national population - based survey , a simple nonblood based risk score based on age , history of diabetes in first - degree relatives , and waist circumference was built and shown to perform moderately in detecting undiagnosed diabetes when externally validated .
moreover , the performance of the score was almost similar for detecting incident cases of diabetes in the peruvian population .
a relatively recent systematic literature search found 23 different blood - free prevalent diabetes risk scores : ten from europe , nine for asian populations , two from the united states , and two from middle east .
in addition , and not included in the aforementioned review , only one risk score was developed in latin america using brazilian urban population .
the same systematic review reported that auc for these predictive models was greater in the development studies ( range : 0.65 to 0.88 ) than in the validation studies ( range : 0.63 to 0.80 ) , similar to our findings .
another systematic review found that several noninvasive algorithms were created using variables such as age , gender , waist circumference and/or bmi , and family history of diabetes in the final model .
as impracticality due to use of the algorithms was a common barrier to the uptake of risk scores by healthcare staff and individuals , our model , created with three of these more common variables , reached a moderate - to - high sensitivity depending on the used cut - point .
moreover , two of these variables are easily evaluable during medical appointment or through individual 's self - assessment , and only a measuring tape and no calculations are required to be implemented in clinical practice or at the population level . from a cross - sectional point of view , with a cut - point 2 , from 1000 participants assessed by the peruvian diabetes risk score , a total of 420 would be classified as undiagnosed diabetes with the detection of 20 cases and only 6 will be missing . on the other hand , with a cut - point 1 , from 1000 screened individuals , a total of 804 would be categorized as having undiagnosed diabetes with the detection of 27 cases and only 7 will be missing .
thus , the reduction of the cut - point of the risk score would increase sensitivity but reducing the specificity and imposing the need of performing a confirmatory test ( i.e. , fasting glucose ) to almost the double of individuals , with the benefit of having only 7 more people diagnosed .
longitudinally , the same risk score would detect an important number of participants at risk of developing diabetes : 43% of screened individuals would be classified at high risk of diabetes , and of them , 8% would develop diabetes in the next 2.5 years . according to a previous study
, 17 reports described a noninvasive model to predict the development of diabetes and included a median of six risk predictors , ranging from 2 to 11 .
although our score did not perform as good as other well - known longitudinal models in the literature such as the findrisc or the aric scores [ 35 , 36 ] , it only included three variables and was built using cross - sectional information .
in addition , some variables used in the aforementioned studies are difficult to standardize within a country as peru , that is , food portions , physical activity , or sedentarism , limiting therefore its use on a wider scale and in a simple pragmatic fashion .
our algorithm performed better than the rotterdam , the indian , and the qingdao risk scores in our population , which highlights the need of calibration and/or development of a specific score for different ethnic groups before its adoption .
as there are ethnic differences in risk factors for diabetes and peru is considered a multiethnic country , it is necessary to create specific scores or recalibrate existing algorithms before applying in specific contexts .
in addition , with only three variables included , the performance of our predictive model was similar to the other assessed scores included in the analyses .
taken together , the score developed has the potential to augment , in a pragmatic manner , initial rapid screening for diabetes , especially at various nonspecialized primary healthcare services .
our findings also demonstrate that approximately 35% of cases of t2 dm ( 39% in the eninbsc survey and 33% in the baseline of the cronicas cohort study ) are not aware of their disease .
results are similar to those reported in previous studies in our context and in similar settings in latin america .
as the developed risk score is simple , it does not require a blood test or laboratory services , and it might be easily implemented in clinical practice .
moreover , because our score asks for general information in the form of age and diabetes in first - degree relatives and is complemented by a simple anthropometric measure of waist , there is potential for the score to be self - administered . according to our results , any patient aged 55 years and above and having at least one first - degree relative with t2 dm has greater probability of having undiagnosed diabetes but also is at risk of developing diabetes in the future .
in addition , a greater central obesity , that is , 100 cm or more , independent of the other terms of the score is alone a good predictor of diabetes as reported in previous studies .
our algorithm included waist circumference instead of body mass index as other risk scores , providing a better indicator of accumulation of visceral fat and metabolic dysfunction in our context .
recently , the peruvian ministry of health has published the guide of clinical practice for diagnosis , treatment and control of diabetes mellitus in primary care and only recommends screening in general population with plasma glucose among adults between 40 and 70 years with obesity or overweight as suggested by the american diabetes association . as in other lmic , plasma glucose is not always available in primary care , especially in semiurban and rural areas ; therefore , a major challenge to be overcome in many countries is the implementation of a simple , fast , and laboratory - free based screening method .
moreover , within the peruvian context , no risk score has been proposed as part of the aforementioned guide .
thus , our algorithm might fill a gap to facilitate further specialized assessment of high risk individuals for diabetes , an approach that may be of utility to various other countries facing similar challenges .
the strengths of this study include the use of a national population - based survey , including urban and rural areas across major geographical regions , to develop the peruvian diabetes risk score , as well as its validation using bootstrap but also an independent longitudinal cohort study .
additionally , it is only based on three variables ensuring its simplicity to be used and implemented . however , the study has also some limitations .
first , we have utilized fasting plasma glucose as the gold standard for diagnosing diabetes instead of an oral glucose tolerance test ( ogtt ) .
although the ogtt is more sensitive and specific than the fasting plasma glucose , more cases would have been detected with the overload of glucose ; it is rarely performed as part of the routine clinical practice .
second , the cronicas cohort study did not include information from the amazon rainforest as did the eninbsc survey . when a sensitivity analysis was performed excluding individuals from the jungle from eninbsc data ,
in addition , the score was created using a national survey to be applicable to the entire peruvian population .
third , some variables were not assessed in our logistic regression model such as dietary intake or history of gestational diabetes as such data was not available . as a result
, some caution should be made when our algorithm is compared to other risk scores .
fourth , our model is based on the idea of risk stratification instead of individualisation ; for instance , variables were categorized instead of being preserved as numerical .
nevertheless , the performance of our score did not change when age and waist circumference were treated as numerical variables ( data not shown ) .
moreover , our idea was to develop a simple and easily applicable score instead of a complex algorithm for predicting undiagnosed and incident diabetes .
finally , as other diabetes risk scores , the model warrants further scrutiny before it can be used in other populations .
the peruvian diabetes risk score , built using age , self - reported diabetes in first - degree relatives , and waist circumference , proves to be a simple pragmatic screening tool for undiagnosed and incident cases of diabetes in peru .
this experience in generating such simple , easy - to - use approaches for the identification of t2 dm can serve to inform other similar lmic efforts who are on early stages of diabetes prevention .
this tool , due to its simplicity , can facilitate various initiatives oriented to introduce and scale up early preventative and management strategies on a wider scale .
|
objective . to develop and validate a risk score for detecting cases of undiagnosed diabetes in a resource - constrained country .
methods . two population - based studies in peruvian population aged 35 years were used in the analysis : the eninbsc survey ( n = 2,472 ) and the cronicas cohort study ( n = 2,945 ) . fasting plasma glucose 7.0 mmol / l was used to diagnose diabetes in both studies .
coefficients for risk score were derived from the eninbsc data and then the performance was validated using both baseline and follow - up data of the cronicas cohort study . results .
the prevalence of undiagnosed diabetes was 2.0% in the eninbsc survey and 2.9% in the cronicas cohort study .
predictors of undiagnosed diabetes were age , diabetes in first - degree relatives , and waist circumference .
score values ranged from 0 to 4 , with an optimal cutoff 2 and had a moderate performance when applied in the cronicas baseline data ( auc = 0.68 ; 95% ci : 0.620.73 ; sensitivity 70% ; specificity 59% ) .
when predicting incident cases , the auc was 0.66 ( 95% ci : 0.610.71 ) , with a sensitivity of 69% and specificity of 59% . conclusions . a simple nonblood based risk score based on age , diabetes in first - degree relatives , and waist circumference can be used as a simple screening tool for undiagnosed and incident cases of diabetes in peru .
|
1. Introduction
2. Methods
3. Results
4. Discussion
5. Conclusions
|
when deposited on various
substrate surfaces , rod - like , -conjugated ,
small organic molecules are well - known for their tendency to form
highly anisotropic crystal shapes , which are frequently called fibers
or needles . whereas the epitaxial growth has been studied on various substrates ,
in particular anisotropic surfaces seem favorable to conserve the
highly anistropic morphology and optical properties , for example ,
polarized emission or adsorption provided by a parallel molecular
orientation obtained by self - assembly . consequently ,
cu(110 ) , tio2(110 ) and muscovite mica(001 ) are frequently chosen as a proper fundament to study the epitaxial
growth of rod - like small molecules . in this paper , the epitaxial
growth of 2,2:6,2-ternaphthalene
( nnn ) on muscovite mica(001 ) is reported . as indicated in figure 1a ,
the molecule is built from three naphthalene
units , which are linked together by c
the coexistence of needle - like structures
and island - like crystallites is verified .
structural analysis reveals
two different crystal orientations . whereas island - like structures
are built up by upright standing molecules orientated with a ( 001 )
contact plane relative to the muscovite mica substrate ( see figure 1b ) ,
needles consist of nnn molecules with a ( 111 )
lying orientation ( see figure 1c ) .
both crystal
configurations provide a well - defined azimuthal alignment , which is
discussed based on force field simulations and a recently reported
growth model .
the azimuthal alignment
of island like structures is explained by ledge directed
( b ) a side view of nnn molecules packed in the observed crystal
structure . each unit cell houses two nnn molecules .
molecules are
approximately standing on the s ( 001 ) contact plane , which is indicated
in blue .
( c ) a side view along the long molecular axis visualizing
the edge - on / flat - on herringbone stacking of nnn .
the blue area represents
the orientation of the b ( 111 ) contact plane where molecules are aligned
in almost in lying configuration .
this all - aromatic compound could be obtained in high yield by coupling
2 equiv of 2-naphthaleneboronic acid ( 1 ) with 1 equiv of 2,6-dibromonaphthalene
( 2 ) , as described in the supporting information .
the final product , 2,2:6,2-ternaphthalene
( nnn ) , was obtained as a colorless product , which appears to be highly
insoluble in common solvents and could only be recrystallized from
1,2,4-trichlorobenzene ( colorless platelets ) .
the material was checked
with gas chromatography and mass spectroscopy and found to be > 99%
pure before thermal sublimation .
all samples have
been fabricated
on muscovite mica(001 ) substrates ( spi , structure probe , inc . ) .
muscovite
mica is a representative of sheet silicate minerals and provides a
layered structure of aluminum silicate sheets weakly bound by layers
of potassium ions .
each layer is characterized by a high symmetry
direction identified by parallel aligned surface grooves . between
the individual sheets , the high symmetry direction alternates by 120
leading to a periodic stacking sequence
along direction . immediately after
cleaving , the mica substrates were transferred to the hot wall epitaxy
( hwe ) chamber .
the hwe technique was applied for the deposition
of the organic material , which allows us to perform the growth process
close to thermodynamic equilibrium , and in further consequence relatively
high vapor pressure of the organic deposit in the substrate region
can be achieved . therefore , the requirements concerning vacuum conditions
are reduced compared with , for example , molecular beam epitaxy .
the source material nnn was purified twice by
thermal sublimation before filling it into the quartz tube of the
hwe reactor .
muscovite mica substrates were transferred into the deposition
chamber via a load lock and subsequently preheated at the deposition
temperature ( 80 c ) for 30 min to ensure a stable temperature
during the whole deposition process .
the deposition was performed
thereafter under a base pressure of 9 10 mbar .
optical microscope images
have been acquired by a nikon labophot 2a microscope in combination
with a nikon type 115 digital camera .
scanning force microscopy ( sfm )
studies of the deposited organic films were performed using a digital
instruments dimension 3100 in the tapping mode .
the 10 10 m images have been acquired at scan speeds of 46 m / s
using sic tips ( masch , hq : nsc15/al bs ) exhibiting a cone angle
of 40. nominal values for resonance frequency and tip radius
are 325 khz and 10 nm , respectively .
x - ray diffraction ( xrd )
measurements were carried out on a philips xpert x - ray diffractometer
using cr k radiation ( = 2.29 ) and a secondary
graphite monochromator
. please note that the monochromator is transparent
for , /2 , /3 , etc .
, so despite the weak intensity
of the bremsspektrum , it can give clear bragg peaks due to the scatting
on the single crystalline mica substrate .
specular scans were performed
in bragg brentano configuration by varying the z - component of the scattering vector q. consequently
it is possible to detect lattice planes that are parallel to the sample
surface .
pole
figures were acquired by measuring at a constant length of q and only varying its direction .
the unit cell parameters of nnn ,
which were used for analysis , are defined by a =
8.148 0.005 , b = 5.978 0.005
, c = 19.45 0.2 , and
= 94.6 0.2 describing a monoclinic lattice ( p21/a ) . the
unit houses two nnn molecules in planar configuration .
the van der waals ( vdw ) interaction
between the organic molecule and the dielectric substrate is modeled
by lennard - jones - type potentials .
corresponding parameters are taken
from the universal force field implemented
in a matlab program .
the molecules and substrates are assumed to be
rigid where the internal structure of an isolated nnn molecules is
determined from the crystal structure .
simulations were performed for the adsorption of a single nnn molecule
as well as a crystal stack . by assuming that a single nnn molecule
prefers to lie flat on the surface , the energy minimization procedure
is simplified in the following way : we consider only four molecular
degrees of freedom , the x- , y-
,
and z - positions of the molecular center of mass and
the angle . the angle defines the azimuthal molecular
alignment and is probed by rotating the nnn molecule around the z - axis ( surface normal ) . we perform a grid - based optimization
to search for the best molecular adsorption geometry using a grid
of 81 81 points for the lateral position .
the adsorption angle was tested
with a step size of = 1. the surface structure
of the substrate has been assumed to be the same as in the bulk where
the corrugation is about 0.2 . the substrate surface is assumed
to be terminated by the tetrahedral layer of muscovite mica .
for the
simulation of the 7 2 nnn stack , the molecular packing has
been deduced from the crystal structure of a ( 111 ) orientated nnn crystallite . due to the presence of flat
and edge - on molecules ,
the adsorption distance has been optimized ,
yielding a distance of 1.6 of the lowest h atom of edge - on
nnn molecules to the substrate surface . because energetic minima are
significantly narrow for the molecular stack , an angular resolution
of = 0.5 has been chosen for the calculations .
in a first
step , nnn was deposited by hot wall epitaxy ( hwe ) on muscovite mica(001 ) .
whereas the substrate temperature was kept constant at 80 c ,
the deposition time was continuously increased .
scanning force microscopy
was chosen to study the sample morphology versus deposition time ,
and obtained images ( 10 10 m ) are depicted
in figure 2 . as the maximum height scale z0 significantly changes with increasing deposition
time , the corresponding values are indicated for each sample .
the
morphology of all samples is dominated by the presence of several
micrometer long needle like structures that are aligned along multiple
orientations . as exemplified by the cross - section ( 1 ) ,
these fibers
reach height levels up to 200 nm and are characterized by
similar dimensions in width .
a more detailed analysis is provided
in figure 2a , plotting the mean needle height
versus growth time . as indicated by the solid line
, the growth rate
can be approximated by a linear fit , yielding a slope of 17.8
1.2 nmmin .
interestingly , the fibers
surface coverage is approximately constant for the whole sample series
yielding a value of 12.5% .
the sfm analysis reveals
that flat islands start to nucleate at the side walls of the fibers
and continuously fill the substrate surface between the fibers with
increasing deposition time .
the latter observation is underlined by
analyzing the islands surface coverage , which is depicted
in figure 2b as a function of growth time .
the solid line , which represents a guide for the eye , indicates an
asymptotic approach to 85% of the surface area .
again , a representative
sample position has been chosen to deduce a cross - section ( 2 ) , which
is presented in the bottom right of figure 2 . as reported for other rod - like molecules , a steplike morphology with height levels in the range of 1.82
nm
the obtained value
corresponds to approximately one monolayer of upright standing nnn
molecules .
contrarily , the step size of the boundary , defined by the
islands and the substrate surface , is significantly larger reaching
values in the range of 15 nm . these steps are further characterized
by straight extensions , which suggest the formation of well - defined
crystal facets . scanning force microscopy ( sfm ) images showing the sample
morphology
versus deposition time .
all samples are dominated by needle - like structures .
with increasing growth time , island - like structures start to nucleate
at the needle side walls covering continuously the substrate surface
between the fibers .
exemplary cross sections for both morphologies
are indicated in the bottom part of the figure .
as indicated in part
a , the height of the fiber like structures linearly increases with
growth time reaching values of some 100 nm .
contrarily , the surface
coverage by needles stays approximately constant ( part b ) .
step heights
in the center part of the islands approximately correspond to a monolayer
of upright standing molecules . in a next step ,
x - ray diffraction ( xrd ) has been chosen to
study
the structural properties of the organic crystallites . in order to
obtain sufficient diffraction intensity , a sample with 216 nm high
nnn fibers has been selected .
figure 3a reports
the acquired specular xrd diffraction pattern , which is dominated
by a series of { 00n } diffraction peaks .
these peaks
are characteristic for island - shaped crystal morphologies , built up
by approximately standing nnn molecules , and are consequently abbreviated
by s - orientations ( q001 = 0.324 ) .
arrows in the upper part of figure 3a indicate the positions of ( 00.2n ) diffraction
peaks stemming from the muscovite mica(001 ) substrate .
additionally ,
a diffraction peak arises at qz = 1.36 , which correlates with ( 111 )
orientated nnn crystallites , abbreviated as b orientation .
the orientation
is characteristic for a nearly flat lying molecular configuration
and thus explains the presence of needle - like crystallites as revealed
by sfm analysis .
a more detailed analysis is reported in the supporting information . in order to analyze
the azimuthal alignment of the nnn
crystallites
relative to the muscovite mica(001 ) substrate , xrd - pf measurements
have been performed and are reported in figure 3b . for a profound analysis , xrd - pf have been acquired with a maximum
sensitivity to diffraction intensities stemming from the scattering
at { 202 } and { 201 } netplanes and are depicted in the bottom leftand
right part of figure 3b .
the diffraction
intensities show a distinct azimuthal distribution ,
which underlines a well - defined epitaxial relationship to the muscovite
mica substrate .
the obtained symmetry of the diffraction intensities
further underlines the presence of an terminated muscovite
mica surface , which is characterized by a mirror plane along the [ 110]mica orientation .
the orientation of the substrate has been
determined from diffraction patterns stemming from muscovite mica
( 001 ) and are indicated by black solid circles .
xrd - pf patterns of
the organic crystallites can be constructed by mirror operation from
the top hemisphere , sketched by a gray shaded sector . moreover , the
xrd - pf patterns reveal a 2-fold rotational symmetry , which can be
understood by an approximately equivalent adsorption energy for 180
turned organic crystallites .
consequently , discrimination between
both crystal alignments has been omitted and simulated 2-fold symmetric
diffraction spots are labeled identically .
based on these geometrical
considerations , only the diffraction spots of a single quadrant have
to be analyzed and labeled . ( a ) specular x - ray diffraction ( xrd ) spectrum
of nnn on muscovite
mica(001 ) .
scanning force microscopy images revealed a needle height
of 216 nm for the chosen sample .
the spectrum is dominated by a series
of s ( 001 ) diffractions peaks , which are representative for island - like
morphologies .
( b ) xrd pole figure ( xrd - pf )
analysis of { 202 } and { 201 } diffraction peaks , providing information
about the azimuthal crystal orientation . as indicated by the simulated
pole distribution ( bottom ) ,
all diffraction spots can be explained
by the presence of three differently aligned ( 001 ) crystallites , labeled
as s13 ( red ) .
the mirror symmetry plane of the
muscovite mica ( 001 ) surface is indicated by a horizontal line , which
explains the presence of mirrored s13 * crystallites ( blue ) with a ( 001 )
contact plane .
moreover , xrd - pf reveal a well - defined azimuthal orientation
of b1 ( 111 ) crystallites ( red circles ) .
again mirror symmetric
crystallites b1 * ( 111 ) are indicated by blue symbols .
diffraction
intensities from the muscovite mica substrate are indicated by black
solid filled circles .
diffraction intensities that are characteristic for s - orientated
crystallites are located at = 63 ( 74 ) in the left
( right ) xrd - pf , and their azimuthal distribution can be explained
by the presence of three crystal orientations labeled as s13 .
consistently , both xrd - pf measurements hint the strongest diffraction
intensities originating from s1 crystallites .
diffraction
spots , which can be attributed to b * ( 111)/b
( 111 ) crystallites are expected to appear at 51
for both diffraction geometries and are marked by blue / red filled
circles . as each quadrant reveals the presence of one diffraction
spot , the azimuthal alignment of b orientated fibers
again , mirror symmetric crystals
are labeled as b1 * and are characterized by a ( 111 ) contact plane .
based on the simulated xrd - pf diffraction peaks , the long needle
axis ( lna ) and long molecular axis ( lma ) orientations of b orientated
fibers have been deduced and are presented in figure 4 by solid filled arrows . whereas the lna coincides with the
[ 110 ] orientation , the lma can be approximated by the alignment
of [ 101 ] relative to the muscovite mica substrate .
mirror symmetry
of the muscovite mica substrate leads to the generation of two energetically
equivalent crystallites .
fibers that
are built up by b1 * ( 111 ) orientated crystallites ( blue ) are aligned with their
lna ( lma ) 59.5 ( 49 ) relative to the muscovite
mica substrate s [ 110]mica crystallographic
orientation .
contrarily , their mirror symmetric twins ( b1 ) can be constructed by flipping the b1 * crystallites upside down ( red arrows ) ,
azimuthally aligned with their lna ( lma ) 59.5 ( 49 )
relative to [ 110]mica . in order to verify
the lna alignment , which has been constructed based on xrd - pf measurements
,
optical microscopy has been chosen , and an image of a representative
sample area is depicted in figure 4 .
the sample
morphology is dominated by fibers that are aligned in a v - shaped ,
herringbone fashion . as indicated by large red and blue arrows , which
represent the expected b1 and b1 * lna orientations deduced by xrd - pf analysis
nevertheless , additional needle orientations , which are present in
minor fraction , can be observed and are marked by small red ( b2 ) and blue ( b1 * ) arrows , respectively . in order to gain better statistics ,
a microscopy image of a larger sample area has been chosen to perform
a fast fourier transformation ( fft ) and the obtained pattern is depicted
beside .
the fft is dominated by two stripes , which are characterized
by an enclosing angle that perfectly reflects the lna orientation
of b1 and b*1 crystallites ( indicated by red
and blue arrows ) . long needle axis ( lna ) ( left ) and long molecular axis
( lma ) ( right )
orientations of b1 ( red solid filled arrows ) and mirror
symmetric b1 * ( blue solid filled arrows ) crystallites , deduced by x - ray pole figure
( xrd - pf ) measurements . in the outer ring of the lna polar plot , additionally
the [ 110 ] crystallographic orientations of s13 ( red ) and
it is visualized that
the [ 110 ] crystallographic direction of s1 crystallites
provides the same azimuthal orientation as the lna of b1 fibers .
below , the obtained lna orientations are verified by the
observed sample morphology using optical microscopy . beside b1 and b1
* fibers additionally minor fractions of approximately horizontally
aligned crystallites are observed ( marked by small red and blue arrows ) .
by using fast fourier transformation ( fft )
( depicted beside ) , the
dominant fraction of b1 and b1 * crystal orientations is further underlined . in a next step
in particular , the azimuthal alignment
of their [ 110 ] directions has been deduced and is depicted
in the outer ring of figure 4 ( lna ) . because
s1 and s1 * crystallites represent the major fraction , they are indicated
by large arrows approximately aligned 60 relative to [ 110]mica .
the azimuthal orientation perfectly coincides with the
lna of b1 and b1 * crystallites , which already suggests an epitaxial
relationship of both crystal types . in order to analyze the latter
observation in more detail ,
the sfm image , which
is shown in figure 5 , is dominated by two approximately
vertically aligned fibers . between
fibers
, the presence of an s orientated island can be observed , which
is terminated in the bottom part of the image by a sharp l - shaped
boundary .
the observed boundary shape perfectly correlates with the
expected angle between the and [ 110 ] crystallographic
orientations of an ( 001 ) orientated crystallite .
the generation
of the observed crystal shape can be understood by the formation of
(11n ) and (11n ) side facets , which represent low index planes for n = 1 , 0 , or 1 .
an extracted cross - section of the observed
fiber is presented in
the left part of figure 5 . the observed fiber
is terminated in the right part by a flat plane , which is aligned
parallel to the substrate at a height of approximately 100 nm .
contrarily ,
the left side of the fiber shows a constantly decreasing height level .
the slope of the side facet approximately correlates with a 25 nm
decrease in height along 100 nm of the needle width ( 14 ) .
the lna of b * type crystallites is defined by their [ 110 ] orientation
and consequently all crystallographic planes (11n ) are aligned parallel to it . because the angular tilt of 13.8
between the low index planes ( 110 ) and ( 111 )
perfectly correlates with the observed sfm analysis , the theoretically
expected cross - section of a b1 * fiber has been modeled and is depicted below
the experimental data . although , the observed steep height decrease
at the fiber side walls is below the resolution limit of the sfm ,
a termination of the fibers by ( 001 ) and ( 001 ) facets can be
assumed .
scanning force microscopy ( sfm ) image
showing vertically aligned
fibers and s orientated islands in between after the deposition of
60 min nnn at 80 c substrate temperature .
the island s
boundaries correlate with the geometrical alignment of and [ 110 ]
orientations . the extracted fiber cross - section ( top , left ) can be
explained by the formation of ( 111 ) , ( 110 )
and (001 ) facets . based on the observed crystal shapes , their
crystallographic orientations relative to each other has been deduced
and is visualized by a 3d model below .
the epitaxial relationship
between fibers and islands is consistent with the structural analysis
and can be explained by a nucleation of nnn molecules at the fiber
side walls , also called ledge directed epitaxy .
based on the latter analysis ,
a three - dimensional model of both
crystal types has been generated and is depicted in the bottom part
of figure 5 . as indicated by the xrd - pf analysis ,
which is presented in figure 4 ( lna ) , b1 * and s1 * crystallites shared
the same azimuth for their crystallographic [110 ] orientations .
in that way
, the tilt angle of standing nnn molecules within the s - type
crystallite approximately correlates with the tilt angle of the fiber
( 001 ) low energy plane .
analogous observations were demonstrated for
6 t fibers and are explained by the nucleation of islands at the sidewalls
of already existing needles .
moreover , the latter
picture is perfectly consistent with the sfm analysis presented in
figure 2 , which reports a continuously increasing
island coverage for longer deposition times .
such epitaxial alignment
based on a geometrical fit between nucleating crystallites and already
existing topographic features on the substrate is called
ledge
directed epitaxy . based on the latter
analysis together with xrd - pf data presented in figure 4 ( lna ) , it can be concluded that the minor fraction of s23 crystallites has most likely nucleated at b - type
fibers , which are orientated approximately 30 tilted relative
to [ 110]mica of the muscovite mica substrate .
this conclusion is further consistent with the microscopy image , presented
in figure 4 , revealing the presence of a minor
fraction of such fibers ( below the detection limit of xrd - pf measurements ) ,
which are subsequently labeled by b2 and b2*.
in order to understand the observed growth behavior of b1 and b2 fibers on muscovite mica(001 ) , the left part of
figure 6 depicts a planar nnn molecule in gas
phase .
analogous to 6 t , a planar molecule
is characterized by a mirror plane h , which is aligned in the plane of the naphthalene rings , and a 2-fold
rotational axis , which is aligned normal to h .
, it should be assumed that single nnn molecules tend
to adsorb lying flat on the muscovite mica substrate in order to maximize
their contact area . in that way ,
h is orientated parallel to the substrate , and the molecule becomes
chiral when adsorbed on an arbitrary surface .
molecules that are intrinsically
achiral but obtain a form of 2d chirality when adsorbed on a substrate
surface are also called prochiral .
analogous
to 6 t , two mirror symmetric nnn enantiomers ( sketched
as red and blue molecules ) can adsorb on the muscovite mica surface ,
which can not be brought into congruence by translation and rotation . taking a top view of the molecular stacking at the contact plane
of b - type crystallites , which is depicted in the right panel of figure 6
, reveals that ( 111 ) orientated fibers are built
up by an alternating assembly of only one enantiomer ( red ) and edge - on
nnn molecules .
contrarily , their twin crystallites b * ( 111 )
are built up by the mirrored molecular configuration ( blue ) only .
the latter observation further explains the consistent choice of a
red and blue color code for molecules and crystallites . beside a real
space model for b1 crystallites , which is deduced by xrd - pf
analysis ,
the right part of figure 6 further
includes the proposed geometry of b2 fibers .
based on a
growth model that has been deduced for 6 t fibers , it is assumed that two needle orientations , for example ,
b1 and b2 * can originate from one molecular adsorption site .
the existence
of these two lna orientations is explained by a mirror symmetric molecular
stacking during crystal nucleation .
interestingly , epitaxially grown
6 t on muscovite mica showed a comparable fraction of both stacking
types , which explains the observation of four lna orientations . the
latter phenomenon is further explained by force field simulations
for both crystal types , yielding a similar adsorption energy with
a deviation of some millielectronvolts / molecule .
contrarily , xrd - pf analysis revealed that nnn fibers are
dominantly present only in one configuration , which reduces the observed
lna orientations to two ( see the fft in figure 4 ) .
consequently , it can be stated that both crystal types seem to
significantly differ concerning their adsorption energy , which should
be investigated and underlined by the discussion of force field simulations
within the next paragraphs .
molecular symmetry of a planar nnn in gas phase
and when adsorbed
flat on an arbitrary surface . due to
the presence of a mirror symmetry
plane h parallel to the naphthalene
rings and a 2-fold rotational axis , aligned normal to it , the nnn
molecule in gas phase can be described by the c2h point group .
contrarily , when adsorbed
flat on a substrate surface , nnn can form two mirror symmetric enantiomers
( sketched by red and blue molecules ) , which follow c2 symmetry .
the right panel depicts a real space model
of the discussed crystal orientations ( top view ) .
the molecular alignment
of nnn within the surface unit cell has been deduced from its bulk
structure , oriented with a ( 111)/(111 ) contact
plane for b / b * crystallites .
the orientation of the long molecular
axis ( lma ) or long needle axis ( lna ) is indicated by blue or red arrows .
taking a closer look at the molecular stacking at the contact plane
of b - type crystallites reveals that ( 111 ) orientated crystals are
alternately assembled by red enantiomers and edge - on nnn molecules .
contrarily , their mirror symmetric twins b * only consist of blue molecular
configurations . the real space image of b1 ( b1 * ) and b2 * ( b2 )
the adsorption energy as a function of the long molecular axis
( lma ) orientation ( ) is depicted in the top panel in terms
of a polar plot .
red and blue arrows indicate the molecular orientation
deduced by experiments . at the indicated positions , force field simulations
reveal a broad maximum for the corresponding enantiomer .
contrarily ,
the adsorption site seems less favorable for the mirror symmetric
molecule ( e 20 mev ) . a real space
model that sketches the lateral position for the molecular adsorption
angle at = 57
simulations
reveal a preferred alignment of the terminating naphthalene units
in the surface corrugations of the muscovite mica substrate ( indicated
by horizontal lines ) .
below , an analogous analysis has been done for
a 7 2 molecular stack representative for the contact plane
of a b ( 111 ) crystallite .
simulations reveal a strongly pronounced
adsorption maximum at the experimentally observed adsorption angle
( red arrow ) and a significantly different adsorption energy for a
b * crystal with opposite stacking sequence ( blue arrow , e 300 mev / molecule ) . a real space model of the
optimized adsorption position
is depicted in the right panel . by comparing
the alignment of the nnn molecules with the muscovite mica unit cell
,
an approximately periodic alignment can be recognized along mica/[110]mica for an / terminated
surface . in a first step ,
the optimal adsorption
energy of a single nnn
molecule has been deduced based on force - field simulations by selecting
the most favorable adsorption site for each angle . the angle
characterizes the azimuthal alignment of the lma relative
to mirror symmetry plane of the muscovite mica substrate surface .
the molecules are assumed to adsorb flat on the substrate and the
adsorption energy , ead , is defined as
the difference between the energies of the isolated subsystems and
the energy of the combined system . therefore , maxima in the ead versus curves evidence the favorable
adsorption geometries . to increase the readability ,
ead curves are presented in figure 7 in terms of a polar plot , where ead = e0 ead .
the parameter e0 represents
the adsorption energy at the least favorable angle , yielding
a value of e0 = 2.49 ev for the
isolated molecules .
because nnn molecules can adsorb either in their
left- or right - handed configuration , simulations have been performed
for both enantiomers and are color coded by red- and blue - filled curves .
simulations yield , for both molecular configurations , multiple
adsorption maxima , which are located for the blue marked enantiomer
at max , blue = 42 , 57 , 102 , 161 , and 178.
due to 2-fold rotational symmetry of the nnn molecule , identical values
are obtained for ead(+180 ) .
moreover , optimized adsorption positions for the red molecular type
are found at max , red = max , blue due to mirror symmetry of the substrate surface .
experimentally
obtained adsorption angles are further indicated by a blue ( red ) arrow
at = 49 ( 131 ) .
both adsorption angles correlate
with the broadest maxima obtained by simulations and are importantly
consistent with the simulations of the corresponding enantiomer .
the
fact that beside experimentally observed adsorption geometries force
field calculations also yield additional maxima is attributed to the
usage of empirical potentials , which in some cases may yield the wrong
energetic ordering of competing structure solutions .
nevertheless , it has to be underlined that simulations
indicate a significant less favorable adsorption site for the mirror
symmetric molecular configuration ( 20 mev ) .
in general , adsorption
energies for both molecular configurations significantly differ , which
in further consequence leads to a nonequal distribution or even breakup
of both enantiomers depending on the adsorption angle .
contrarily ,
simulations as well as experimental data that are reported for 6 t indicate a significantly lower energetic splitting
between both molecular configurations , which may result from a higher
symmetry of the molecule .
the latter statement can be understood by
the fact that thiophene molecules with an odd ring number , for example ,
quinquethiophene or septithiophene , are characterized by a mirror
symmetry plane when adsorbed on a surface and consequently do not
show a prochiral character .
contrarily , the asymmetric alignment of
the c c bond between two naphthalene units of nnn inevitably
leads to a prochiral behavior when adsorbed on a substrate surface
and consequently plays an essential role concerning the energetic
separation of both enantiomers at a defined adsorption angle .
the experimentally confirmed adsorption position of nnn molecules
is further depicted in the right part of figure 7 . for both molecules , an adsorption angle =
analogous to calculations for p-6p and 6 t , nnn molecules tend to align their rings in the
surface corrugations , which are indicated by vertical solid lines . in order to study the adsorption energetics of a b - type crystallite
,
a 7 2 molecular stack has been deduced from a ( 111 ) orientated
crystallite .
analogous to the force field simulations of an isolated
molecule , the adsorption energy ead for the stack has been probed depending on the molecular orientation
and the adsorption energy at the least favorable adsorption
angle is given by e0 = 0.41 ev / molecule .
because the curve calculated for the a b * ( 111 )
stack follows the same behavior as discussed for a single molecule
( mirror symmetric ) , only the results for a b contact plane are depicted
in order to increase readability .
interestingly , simulations reveal
that not only the number of energetically favorable adsorption sites
decreases but also the angular acceptance , which becomes visible by
well pronounced peaks .
simulations further indicate the presence of
two adsorption maxima , which are located at = 12 and
48. again , the experimentally obtained adsorption angle
for b1 crystallites is indicated by a red arrow and underlines
a nearly perfect agreement .
moreover , it can be recognized that the
adsorption energy for a b2 crystal ( at + 48 ) becomes
even more unfavorable than that for a single molecule , which is manifested
by a much lower value of ead in
the range of some 100 mev / molecule .
consequently , force field
simulations not only reflect the experimental
observations but also explain the dominant fraction of b1 crystallites by a preferred nucleation of their stacking sequence
in contrast to b2 crystallites
. the observed behavior can
be even better understood by analyzing the real space model of the
simulated adsorption position at = 48. besides
the molecular alignment , also the surface unit cells of the muscovite
mica and b crystal have been indicated .
obviously , the unit vector
[ 110 ] of the nnn crystal stack , which also defines its lna ,
tends to align parallel to one surface unit vector of the muscovite
mica crystal , which is defined by the mica, ( [ 110]mica, ) orientation for an
( ) terminated surface .
the epitaxial growth of ternaphtalene
( nnn ) on muscovite mica(001 ) has been investigated by combining structural
( xrd - pf ) and morphological ( sfm ) methods .
consistently , both methods
reveal the formation of s ( 001 ) orientated nnn island - like structures
which have nucleated at the sidewalls of b ( 111 ) orientated fibers .
it is demonstrated that the latter nnn crystal types tend to align
along two dominant directions , which leads to the formation of a v - shaped
sample fiber morphology .
because the tilt angle of nnn molecules within
s - orientated crystallites correlates with the tilt angle of the fiber
side facets , the island nucleation is explained by ledge directed
epitaxy .
based on this growth model , it can be understood
that both crystal types provide a well - defined azimuthal orientation
relative to the muscovite mica substrate . by use of force field
simulations , the growth of the fibers is further analyzed .
the epitaxial
growth of sexithiophene ( 6 t ) on muscovite mica showed the formation
of four well - defined fiber orientations , which can be explained by
mirror symmetry of the muscovite mica substrate and two differently
stacked 6 t crystallites , which can nucleate at a molecular adsorption
position .
contrarily , experimental investigations
indicate that nnn crystallites tend to stack in a single configuration ,
which explains the dominant formation of only two fiber orientations .
based on force field simulations ,
the latter observation is further
investigated and explained by significantly different adsorption energies
of both crystal types .
it is further demonstrated that the observed
behavior results from an interplay of the molecular adsorption and
lattice match .
|
the
morphology and structure of 2,2:6,2-ternaphthalene
( nnn ) deposited on muscovite mica(001 ) substrates was investigated
by scanning force microscopy ( sfm ) and specular x - ray diffraction
measurements .
consistently , both methods reveal the coexistence of
needle - like structures with a { 111 } contact plane and { 001 } orientated
island - like crystallites , which are built up by almost upright standing
nnn molecules .
both orientations are characterized by a well - defined
azimuthal alignment relative to the substrate surface , which is analyzed
by x - ray diffraction pole figure ( xrd - pf ) measurements .
based on xrd - pf
and sfm analysis , the azimuthal alignment of { 001 } orientated crystallites
is explained by ledge - directed epitaxy along the fibers sidewalls .
these fibers are found to orient along two dominant directions , which
is verified and explained by a doubling of the energetically preferred
molecular adsorption site by mirror symmetry of the substrate surface .
the experimental findings are confirmed by force - field simulations
and are discussed based on a recently reported growth model .
|
Introduction
Experimental Section
Experimental Results
Discussion
|
the task force on child health and maternal health for the united nations millennium development goals reiterated the challenges of achieving the goals in its report who 's got the power ?
transforming health systems for women and children . while the technology and interventions exist to prevent or treat the vast majority of adverse health conditions that affect children and women of reproductive age , many continue to die and even more suffer from ill health [ 2 , 3 ] . the central challenge to achieving these goals is to provide known interventions to the full population and at the right time when most known adverse outcomes can be prevented . despite 15 years of the global safe motherhood
maternal and child health is connected to social , economic , and environmental conditions . in resource - poor countries , aside from pre - existing risks for poor maternal and child health outcomes , global changes have introduced new risks to the health of women and pregnancy , including alcohol and tobacco use , over nutrition , and micronutrient malnutrition [ 5 , 6 ] .
because not all the deficits in achieving adequate prenatal care are likely to be solved in the near future , and global factors that lead to increases in risk can not be reversed , new strategies and policies to improve pregnancy outcome remain a high priority .
one emerging policy is the role of preconception care as a complement to prenatal care .
this report highlights examples of national programs and projects indicating that such approaches are feasible and acceptable to the population .
these case studies on preconception care that were presented at the first national summit on preconception care , atlanta , usa , in 2005 , range from a mature program in hong kong , to emerging programs in korea and belgium , and select program enhancement and recovery efforts in china .
the family planning association of hong kong ( fpahk ) was founded in 1950 with an objective to reduce family size .
the average number of children per woman declined from 4.5 in 1965 to 0.93 by 2004 .
having achieved a low birth rate , a number of sexual and reproductive health programs have been launched with the objective of improving reproductive outcomes .
the pre - pregnancy preparation service was launched in 1998 , in response to public demand for preconception care .
the service integrates medical , counseling , and education services for couples planning conception to achieve the following outcomes :
prevent and treat infections to safeguard the health of both partners and the fetus .
thalassemia is an important hereditary disease in hong kong , because approximately 8% of persons in the local population are heterozygous carriers of mutations for thalassemia .
couples with family history of hereditary diseases are referred for genetic counseling.all women are advised to consume folic acid.help couples understand their health and adjust their lifestyle to optimize pregnancy outcome.identify women who might have difficulty conceiving and to advise them to seek sub - fertility investigations early when indicated . for men , semen analysis
is included in the package.help couples prepare for parenthood.help women with pre - existing medical illness understand the best timing for pregnancy , the effect of their illness on their pregnancy , the effect of pregnancy on their illness , the effect on her offspring , the need to adjust medication , and to avoid drugs with known or uncertain teratogenic effects.help women with gynecological diseases understand the impact of their diseases on conception and pregnancy .
the role of assisted reproductive technology is discussed if appropriate.counsel couples with coital failure to help them consummate marriage . prevent and treat infections to safeguard the health of both partners and the fetus .
thalassemia is an important hereditary disease in hong kong , because approximately 8% of persons in the local population are heterozygous carriers of mutations for thalassemia .
identify women who might have difficulty conceiving and to advise them to seek sub - fertility investigations early when indicated .
help women with pre - existing medical illness understand the best timing for pregnancy , the effect of their illness on their pregnancy , the effect of pregnancy on their illness , the effect on her offspring , the need to adjust medication , and to avoid drugs with known or uncertain teratogenic effects .
help women with gynecological diseases understand the impact of their diseases on conception and pregnancy .
at the first visit , the woman fills in a health assessment checklist that include preexisting medical conditions and medication being taken , adverse gynecological history and history of abnormal pregnancy outcomes , and risk behaviors .
this disc provides detailed information about the objectives of the service ; the physical and genital checkup procedures ; pregnancy preparation , e.g. optimal body mass index , balanced diet , regular exercise , quit smoking and avoiding alcohol ; fertility regulation including contraception , conception and sub - fertility ; and information about the laboratory tests including hiv testing .
, a nurse will explain all the normal reports first and then a physician performs the history , physical examination , and counseling to explain any abnormal results . the basic counseling and assessment
is provided by non - specialists , although women with complicated problems are seen by specialists in obstetrics and gynecology or an internist .
regular case conferencing is conducted to enhance knowledge of the non - specialists and improve their counseling skills .
the service fee is $ 600 hong kong dollars ( usd $ 75 ) per couple which includes laboratory tests and one medical consultation .
this low service fee is achieved through cost sharing with other sexual and reproductive health programs offered by the fpahk .
the continuous flow of clients for the preconception service reflects public support and trust for the service .
as may be seen in many emerging economies , south korea is experiencing decreasing birth rates ( from 16.3 per 1000 live births in 1994 to 9.8 per 1000 live births in 2004 ) and decreasing infant mortality rates ( from 9.9/1ive births in 1993 to 4.6/live births in 2004 ) . due to a high rate of unintended pregnancy ,
an increase in the rate of preterm delivery , a high rate of pregnancy termination following accidental exposure to potential teratogens during pregnancy , and low intake of folic acid , the society of maternal and fetal medicine made a decision to promote and enhance preconception care in south korea .
the goals are to enhance primary prevention of birth defects and preterm birth , increase in intended pregnancy , decrease in unnecessary pregnancy termination , and recovery of a higher birth rate in korea to maintain population replacement levels .
education includes lectures for obstetricians and gynecologists as well as for child - bearing age women attending maternity services .
service delivery is clinic - based preconceptional counseling , medical protocol examination by a physician , and follow - up of women who want to have a baby .
the preconception counseling clinic , based at the korean motherisk program at samsung cheil hospital , an affiliated university hospital , includes teratogen information service for pregnant and breast feeding women , as well as preconception counseling .
the preconception care medical protocol is composed of a physical examination , laboratory tests , and a questionnaire . of
the women served , 40% reported no history of adverse reproductive outcomes , about 30% had history of spontaneous abortion , 13% had maternal disease , 15% had a baby with birth defect , and 2% had family history of genetic disease .
however , careful review of medical and behavioral history indicated that 92% of preconception care attendees have at least one risk factor for adverse outcomes .
patients with specific problems such as a previous child with a birth defect are transferred to the department of maternal and fetal medicine .
also , pre - implantation genetic diagnosis is performed in the department of infertility , a service that predates the introduction of preconception care .
this service resulted in a substantial reduction in pregnancy loss from 91% in 1995 to 19% to 203 among 260 couples with balanced chromosomal translocation .
the korea motherisk program is supported by the samsung chiel hospital and is staffed by three university faculty members and rotating fellows in clinical medicine .
although no consultation fee is charged to the patients , laboratory expenses are paid for by the patients .
depending on family history and other factors and various rationale used for laboratory examinations , the maximum cost of such laboratory examinations can be as high as 150 usd per person .
the current challenge is to market it to all women who plan pregnancy , and to assure that the health care system has the capacity to match the demand and expansion of the service to other parts of the country .
organization for birth and childhood ) , is a belgian governmental organization charged with the protection and promotion of health of women and children . given the low fertility rates and high rates of birth defects ( 2 to 3% of all live births ) in belgium , o.n.e launched a plan to assess the feasibility of implementation of preconception care in the french speaking part of belgium , the region with a population of 5 million people and about 50,000 births per year .
this activity will be implemented through prenatal care , pediatric and gynecology clinics , as well as general practitioners , in partnership with genetic and counselling centers .
an ad - hoc committee was formed to propose strategies , activities , financing options , and evaluation methods , starting with the development of evidence - based guidelines based on belgian epidemiological data .
the committee proposed that the guidelines would be in accordance with the recommendations of the advisory committee of bioethics of belgium concerning genetic tests .
the committee proposed also to organize a campaign targeting both the general public and health care providers to help market preconception care in primary care settings .
the proposed guidelines include various serologic tests , screening of endocrine or genetic diseases , and preventive services including promotion of folic acid supplementation .
the o.n.e . developed a sustainable campaign plan to inform and sensitize the population about the benefits of preconception care , directing their efforts to individuals aged 15 to 45 years , health professionals including general practitioners , gynaecologists , paediatricians , midwives and all medical social workers , members of the family planning centres , and school health promotion centers .
the first step in the social marketing campaign was the creation of the tools including folders , posters , and letters to professionals .
these materials were reviewed by the ad - hoc committee and field tested among a selected group of social workers and the public .
the second step will be an information campaign among men and women of reproductive age using posters , folders , and radio and television advertisements .
medical and social workers will also receive folders and posters for distribution to the target population and a letter to inform them about the purpose and methodology of the campaign .
medical practitioners will then receive the guidelines to help them with information to be shared with patients and the laboratory examinations to be conducted
. the final step will be the evaluation process to assess behavioral changes both within the community and at provider level .
the indicators will include advice received from providers by clients in participating facilities , referral of relevant clients to preconception visits by providers , uptake of preconception visits by clients , and compliance with suggested interventions by clients .
the early phase of implementation of preconception care in primary care settings in belgium was a challenge , as it highlighted the need for a strong partnership of all institutions involved in maternal and child health care .
a three - pronged approach to reducing mother - to - child transmission of hiv has long been advocated by global initiatives to reduce the burden of hiv / aids , but to our knowledge , never been implemented anywhere .
this approach includes reduction of hiv infection among young couples through primary prevention and voluntary hiv counseling and testing ( vct ) , voluntary avoidance of pregnancy by hiv infected couples , and finally antiretroviral treatment of pregnant women who are infected and subsequent management of outcomes in children .
the unique premarital medical examination system prevalent in china and late onset of hiv transmission in china provided an opportunity to pilot implementation of the three - pronged strategy in guangxi province .
babu county ( population 0.93 million ) of hezhou city , guangxi province ( population 48.9 million ) experienced the fastest increase in reported hiv / aids cases in guangxi province between 1998 ( 18 ) and 2002 ( 629 ) since the first case was detected in 1997 .
the epidemic concentrated among the younger age group ( 90% , age 2049 years ) of the population and predominantly rural farmers ( 70% ) .
given the demographics of the affected population and because the condom usage rate was low in this population ( std clients 5% , drug users 5% , sex workers 40% ) , the potential for mother - to - child transmission was evident .
to reduce the impact of hiv on women , children and families , the county health department in 2001 embarked on a three - pronged approach embedded in the mandatory premarital medical examination system .
a feasibility survey in the region had indicated that the majority of the population and public health community were supportive of the concept .
the activities included family and community awareness , preconceptional voluntary hiv counseling and tests for couples ( pvct ) , and vct and antiretroviral therapy to hiv infected pregnant women .
to facilitate pvct and the antenatal vct and antiretroviral treatment component , 40 antenatal and maternal / child care clinicians were trained . to facilitate the broad based interventions ,
942 medical and public health professionals and barefoot doctors were trained on vct and other prevention approaches . to facilitate the community engagement process ,
training seminars using a combination of entertainment and educational films and media slides were conducted in middle schools and high schools for students as well as adults in the catchment 's area .
an one - time pre- and post test ( 8800 respondents ) evaluation using structured questionnaire was conducted in selected locations .
the students were required to take educational materials ( 100,000 copies ) to their homes to educate parents and neighbors as part of their homework requirements .
information on hiv and vct opportunities were displayed on the community information blackboard , which is the usual tool for disseminating information on public health , legislative , and social policies to villagers and updated frequently by community volunteers . in the first quarter after launching the program ( october to december 2002 ) an average of 1099 ( 73% ) of the 1500 eligible couples each month sought pvct consultation , and 52% of them ( or 38% of eligible couples ) accepted hiv testing . by the end of 2003 , the pvct consultation rate among eligible couples increased to 77% and hiv testing rate among them reached 80% ( or 62% of all eligible couples ) .
the main challenge to this approach was the change of legal requirement of premarital medical examination from mandatory to voluntary status in october 2003 .
our pilot efforts indicate that screening for hiv in the preconception period is feasible and acceptable in this rural community .
a combination of testing strategies implemented at various stages before and during pregnancy would help identify all women at risk for hiv - exposed pregnancies .
given that the community is aware of the concept of premarital medical examination , social marketing techniques using family health enhancement as a core benefit , and efforts to regain the conceptual interest of population in preconception care are needed . in october 2003 ,
the longstanding mandatory chinese requirement for couples planning marriage to obtain a premarital health check , a routine physical examination , laboratory tests , and reproductive health education , was removed , and this visit became voluntary .
reported rates of premarital examinations plummeted , and health officials voiced concern that women were not receiving adequate risk assessment and pre - pregnancy education , particularly regarding the benefit of folic acid in preventing neural tube defects . to assess the feasibility of increasing the rate of preconception care uptake , we conducted a pilot social marketing effort in a northern county ( mancheng , in hebei province ) during february and march , 2004 .
we used a three - stage process : 1 ) ascertain the nature of the problem regarding delivery of preconception care through the use of a series of questionnaires and interviews , 2 ) develop a test plan to promote preconception care , and 3 ) evaluate and refine the plan .
overall , 91% of 10,000 questionnaires were completed , and 140 subjects were interviewed , including women of reproductive age , maternal and child health workers , and local government officials .
the major obstacles to preconception care included the following : a ) lack of systematic organization of these services within the health care system ; b ) poor coordination between governmental organizations involved in marriage , family planning and health care ; c ) unclear media messages ; and d ) the perception of the term voluntary by women to mean unnecessary .
the implementation plan included the use of social marketing to solicit government support , coordinate with various government organizations , train maternal child health ( mch ) workers at all levels , and to market the concept of preconception health care to women of childbearing age . to solicit government support , we established a project coordination committee , including high - level officials from the county project office , the county women 's office , the bureaus of health , family planning , and civil administration , and the women 's federation .
in addition , a series of face - to - face interviews were conducted with government officials , workshops were held in the county , and an official document was issued by the county government .
recommendations were developed for improving coordination among government departments and organizations , including development of an education program that can be presented by the mch institute , the marriage registration office , and the family planning office , whenever women have contact with the system .
such recommendations were shared with all stakeholders . following the workshop , 95% of women reported that they would be willing to pay up to rmb 100 ( us$12 ) for preconception health care services , and half indicated that more and better health messages were needed . improving coordination among government agencies , developing training materials targeted to different groups , and promulgating clear , consistent messages concerning the importance of preconception care that are delivered in all settings where women may have contact with the system before pregnancy can create demand for preconception services .
the family planning association of hong kong ( fpahk ) was founded in 1950 with an objective to reduce family size .
the average number of children per woman declined from 4.5 in 1965 to 0.93 by 2004 .
having achieved a low birth rate , a number of sexual and reproductive health programs have been launched with the objective of improving reproductive outcomes .
the pre - pregnancy preparation service was launched in 1998 , in response to public demand for preconception care .
the service integrates medical , counseling , and education services for couples planning conception to achieve the following outcomes :
prevent and treat infections to safeguard the health of both partners and the fetus .
thalassemia is an important hereditary disease in hong kong , because approximately 8% of persons in the local population are heterozygous carriers of mutations for thalassemia .
couples with family history of hereditary diseases are referred for genetic counseling.all women are advised to consume folic acid.help couples understand their health and adjust their lifestyle to optimize pregnancy outcome.identify women who might have difficulty conceiving and to advise them to seek sub - fertility investigations early when indicated . for men , semen analysis
is included in the package.help couples prepare for parenthood.help women with pre - existing medical illness understand the best timing for pregnancy , the effect of their illness on their pregnancy , the effect of pregnancy on their illness , the effect on her offspring , the need to adjust medication , and to avoid drugs with known or uncertain teratogenic effects.help women with gynecological diseases understand the impact of their diseases on conception and pregnancy .
the role of assisted reproductive technology is discussed if appropriate.counsel couples with coital failure to help them consummate marriage . prevent and treat infections to safeguard the health of both partners and the fetus .
thalassemia is an important hereditary disease in hong kong , because approximately 8% of persons in the local population are heterozygous carriers of mutations for thalassemia .
identify women who might have difficulty conceiving and to advise them to seek sub - fertility investigations early when indicated .
help women with pre - existing medical illness understand the best timing for pregnancy , the effect of their illness on their pregnancy , the effect of pregnancy on their illness , the effect on her offspring , the need to adjust medication , and to avoid drugs with known or uncertain teratogenic effects .
help women with gynecological diseases understand the impact of their diseases on conception and pregnancy .
, the woman fills in a health assessment checklist that include preexisting medical conditions and medication being taken , adverse gynecological history and history of abnormal pregnancy outcomes , and risk behaviors .
this disc provides detailed information about the objectives of the service ; the physical and genital checkup procedures ; pregnancy preparation , e.g. optimal body mass index , balanced diet , regular exercise , quit smoking and avoiding alcohol ; fertility regulation including contraception , conception and sub - fertility ; and information about the laboratory tests including hiv testing .
, a nurse will explain all the normal reports first and then a physician performs the history , physical examination , and counseling to explain any abnormal results . the basic counseling and assessment
is provided by non - specialists , although women with complicated problems are seen by specialists in obstetrics and gynecology or an internist .
regular case conferencing is conducted to enhance knowledge of the non - specialists and improve their counseling skills .
the service fee is $ 600 hong kong dollars ( usd $ 75 ) per couple which includes laboratory tests and one medical consultation .
this low service fee is achieved through cost sharing with other sexual and reproductive health programs offered by the fpahk .
the continuous flow of clients for the preconception service reflects public support and trust for the service .
as may be seen in many emerging economies , south korea is experiencing decreasing birth rates ( from 16.3 per 1000 live births in 1994 to 9.8 per 1000 live births in 2004 ) and decreasing infant mortality rates ( from 9.9/1ive births in 1993 to 4.6/live births in 2004 ) . due to a high rate of unintended pregnancy ,
an increase in the rate of preterm delivery , a high rate of pregnancy termination following accidental exposure to potential teratogens during pregnancy , and low intake of folic acid , the society of maternal and fetal medicine made a decision to promote and enhance preconception care in south korea .
the goals are to enhance primary prevention of birth defects and preterm birth , increase in intended pregnancy , decrease in unnecessary pregnancy termination , and recovery of a higher birth rate in korea to maintain population replacement levels .
education includes lectures for obstetricians and gynecologists as well as for child - bearing age women attending maternity services .
service delivery is clinic - based preconceptional counseling , medical protocol examination by a physician , and follow - up of women who want to have a baby .
the preconception counseling clinic , based at the korean motherisk program at samsung cheil hospital , an affiliated university hospital , includes teratogen information service for pregnant and breast feeding women , as well as preconception counseling .
the preconception care medical protocol is composed of a physical examination , laboratory tests , and a questionnaire . of
the women served , 40% reported no history of adverse reproductive outcomes , about 30% had history of spontaneous abortion , 13% had maternal disease , 15% had a baby with birth defect , and 2% had family history of genetic disease . however , careful review of medical and behavioral history indicated that 92% of preconception care attendees have at least one risk factor for adverse outcomes .
patients with specific problems such as a previous child with a birth defect are transferred to the department of maternal and fetal medicine .
also , pre - implantation genetic diagnosis is performed in the department of infertility , a service that predates the introduction of preconception care .
this service resulted in a substantial reduction in pregnancy loss from 91% in 1995 to 19% to 203 among 260 couples with balanced chromosomal translocation .
the korea motherisk program is supported by the samsung chiel hospital and is staffed by three university faculty members and rotating fellows in clinical medicine .
although no consultation fee is charged to the patients , laboratory expenses are paid for by the patients .
depending on family history and other factors and various rationale used for laboratory examinations , the maximum cost of such laboratory examinations can be as high as 150 usd per person . since the establishment of preconception services in 2004
the current challenge is to market it to all women who plan pregnancy , and to assure that the health care system has the capacity to match the demand and expansion of the service to other parts of the country .
the o.n.e ( office de la naissance et de lenfance = organization for birth and childhood ) , is a belgian governmental organization charged with the protection and promotion of health of women and children .
given the low fertility rates and high rates of birth defects ( 2 to 3% of all live births ) in belgium , o.n.e launched a plan to assess the feasibility of implementation of preconception care in the french speaking part of belgium , the region with a population of 5 million people and about 50,000 births per year .
this activity will be implemented through prenatal care , pediatric and gynecology clinics , as well as general practitioners , in partnership with genetic and counselling centers .
an ad - hoc committee was formed to propose strategies , activities , financing options , and evaluation methods , starting with the development of evidence - based guidelines based on belgian epidemiological data .
the committee proposed that the guidelines would be in accordance with the recommendations of the advisory committee of bioethics of belgium concerning genetic tests .
the committee proposed also to organize a campaign targeting both the general public and health care providers to help market preconception care in primary care settings .
the proposed guidelines include various serologic tests , screening of endocrine or genetic diseases , and preventive services including promotion of folic acid supplementation .
the o.n.e . developed a sustainable campaign plan to inform and sensitize the population about the benefits of preconception care , directing their efforts to individuals aged 15 to 45 years , health professionals including general practitioners , gynaecologists , paediatricians , midwives and all medical social workers , members of the family planning centres , and school health promotion centers .
the first step in the social marketing campaign was the creation of the tools including folders , posters , and letters to professionals .
these materials were reviewed by the ad - hoc committee and field tested among a selected group of social workers and the public .
the second step will be an information campaign among men and women of reproductive age using posters , folders , and radio and television advertisements .
medical and social workers will also receive folders and posters for distribution to the target population and a letter to inform them about the purpose and methodology of the campaign .
medical practitioners will then receive the guidelines to help them with information to be shared with patients and the laboratory examinations to be conducted
. the final step will be the evaluation process to assess behavioral changes both within the community and at provider level .
the indicators will include advice received from providers by clients in participating facilities , referral of relevant clients to preconception visits by providers , uptake of preconception visits by clients , and compliance with suggested interventions by clients .
the early phase of implementation of preconception care in primary care settings in belgium was a challenge , as it highlighted the need for a strong partnership of all institutions involved in maternal and child health care .
a three - pronged approach to reducing mother - to - child transmission of hiv has long been advocated by global initiatives to reduce the burden of hiv / aids , but to our knowledge , never been implemented anywhere .
this approach includes reduction of hiv infection among young couples through primary prevention and voluntary hiv counseling and testing ( vct ) , voluntary avoidance of pregnancy by hiv infected couples , and finally antiretroviral treatment of pregnant women who are infected and subsequent management of outcomes in children .
the unique premarital medical examination system prevalent in china and late onset of hiv transmission in china provided an opportunity to pilot implementation of the three - pronged strategy in guangxi province .
pilot experience in babu county in guangxi province is outlined here . babu county ( population 0.93 million ) of hezhou city , guangxi province ( population 48.9 million ) experienced the fastest increase in reported hiv / aids cases in guangxi province between 1998 ( 18 ) and 2002 ( 629 ) since the first case was detected in 1997 .
the epidemic concentrated among the younger age group ( 90% , age 2049 years ) of the population and predominantly rural farmers ( 70% ) .
given the demographics of the affected population and because the condom usage rate was low in this population ( std clients 5% , drug users 5% , sex workers 40% ) , the potential for mother - to - child transmission was evident .
to reduce the impact of hiv on women , children and families , the county health department in 2001 embarked on a three - pronged approach embedded in the mandatory premarital medical examination system . a feasibility survey in the region
had indicated that the majority of the population and public health community were supportive of the concept .
the activities included family and community awareness , preconceptional voluntary hiv counseling and tests for couples ( pvct ) , and vct and antiretroviral therapy to hiv infected pregnant women . to facilitate pvct and the antenatal vct and antiretroviral treatment component ,
40 antenatal and maternal / child care clinicians were trained . to facilitate the broad based interventions ,
942 medical and public health professionals and barefoot doctors were trained on vct and other prevention approaches . to facilitate the community engagement process ,
training seminars using a combination of entertainment and educational films and media slides were conducted in middle schools and high schools for students as well as adults in the catchment 's area .
an one - time pre- and post test ( 8800 respondents ) evaluation using structured questionnaire was conducted in selected locations .
the students were required to take educational materials ( 100,000 copies ) to their homes to educate parents and neighbors as part of their homework requirements .
information on hiv and vct opportunities were displayed on the community information blackboard , which is the usual tool for disseminating information on public health , legislative , and social policies to villagers and updated frequently by community volunteers . in the first quarter after launching the program ( october to december 2002 ) an average of 1099 ( 73% ) of the 1500 eligible couples each month sought pvct consultation , and 52% of them ( or 38% of eligible couples ) accepted hiv testing . by the end of 2003 , the pvct consultation rate among eligible couples increased to 77% and hiv testing rate among them reached 80% ( or 62% of all eligible couples ) .
the main challenge to this approach was the change of legal requirement of premarital medical examination from mandatory to voluntary status in october 2003 .
our pilot efforts indicate that screening for hiv in the preconception period is feasible and acceptable in this rural community .
a combination of testing strategies implemented at various stages before and during pregnancy would help identify all women at risk for hiv - exposed pregnancies .
given that the community is aware of the concept of premarital medical examination , social marketing techniques using family health enhancement as a core benefit , and efforts to regain the conceptual interest of population in preconception care are needed .
in october 2003 , the longstanding mandatory chinese requirement for couples planning marriage to obtain a premarital health check , a routine physical examination , laboratory tests , and reproductive health education , was removed , and this visit became voluntary .
reported rates of premarital examinations plummeted , and health officials voiced concern that women were not receiving adequate risk assessment and pre - pregnancy education , particularly regarding the benefit of folic acid in preventing neural tube defects . to assess the feasibility of increasing the rate of preconception care uptake , we conducted a pilot social marketing effort in a northern county ( mancheng , in hebei province ) during february and march , 2004 .
we used a three - stage process : 1 ) ascertain the nature of the problem regarding delivery of preconception care through the use of a series of questionnaires and interviews , 2 ) develop a test plan to promote preconception care , and 3 ) evaluate and refine the plan .
overall , 91% of 10,000 questionnaires were completed , and 140 subjects were interviewed , including women of reproductive age , maternal and child health workers , and local government officials .
the major obstacles to preconception care included the following : a ) lack of systematic organization of these services within the health care system ; b ) poor coordination between governmental organizations involved in marriage , family planning and health care ; c ) unclear media messages ; and d ) the perception of the term voluntary by women to mean unnecessary .
the implementation plan included the use of social marketing to solicit government support , coordinate with various government organizations , train maternal child health ( mch ) workers at all levels , and to market the concept of preconception health care to women of childbearing age . to solicit government support , we established a project coordination committee , including high - level officials from the county project office , the county women 's office , the bureaus of health , family planning , and civil administration , and the women 's federation .
in addition , a series of face - to - face interviews were conducted with government officials , workshops were held in the county , and an official document was issued by the county government .
recommendations were developed for improving coordination among government departments and organizations , including development of an education program that can be presented by the mch institute , the marriage registration office , and the family planning office , whenever women have contact with the system .
following the workshop , 95% of women reported that they would be willing to pay up to rmb 100 ( us$12 ) for preconception health care services , and half indicated that more and better health messages were needed . improving coordination among government agencies , developing training materials targeted to different groups , and promulgating clear , consistent messages concerning the importance of preconception care that are delivered in all settings where women may have contact with the system before pregnancy can create demand for preconception services .
as illustrated by the six examples highlighted above , it is evident that preconception care is not just a conceptual debate but a primary approach used to address various health issues and emerging national challenges . as noted so cogently by atrash et al .
a healthy baby and a healthy mother are valued hopes and dreams of families and cultural heritages across the world .
further , in the countries such as south korea and hong kong , and others in similar demographic transition , this
although adequate prenatal , obstetric , and primary care services can reduce the infant and maternal mortality in high - mortality - developing countries , such countries are not free from emerging risks to maternal and child outcomes .
tobacco and alcohol use rates among women in many developing countries have increased . however , as more women than before have access to education and information , are employed , have personal income and decision making power , and delay pregnancy , there are many opportunities to inform them about the need for preconception care and a healthy reproductive life . indeed , even a perfectly organized preconception care system is not likely to address other deficits in the reproductive health care system or in an inadequate prenatal care system in developing countries ; neither will it lead to rapid positive changes in related indicators .
similar to the arguments in 1990s for and against hiv voluntary counseling services amidst the lack of availability of antiretroviral treatment , or against the attempts to market cell phones where land phones services are inadequate , advocating preconception care as a component of maternal child care services will remain controversial . as such , establishment of preconception care services
in fact , introduction to preconception care can complement the efforts to improve prenatal care uptake .
consistent with the conceptual framework of the 2005 bangkok charter on health promotion , preconception care should be seen as a program for the future , a development agenda that is aimed at overall health of the family .
making preconception care available can have transgenerational impact on some women ; those who are aware of such opportunities and can access such services . our responsibility is to provide that opportunity .
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globally , maternal and child health faces diverse challenges depending on the status of the development of the country .
some countries have introduced or explored preconception care for various reasons .
falling birth rates and increasing knowledge about risk factors for adverse pregnancy outcomes led to the introduction of preconception care in hong kong in 1998 , and south korea in 2004 . in hong kong , comprehensive preconception care including laboratory tests
are provided to over 4000 women each year at a cost of $ 75 per person . in korea ,
about 60% of the women served have known medical risk history , and the challenge is to expand the program capacity to all women who plan pregnancy , and conducting social marketing .
belgium has established an ad hoc - committee to develop a comprehensive social marketing and professional training strategy for pilot testing preconception care models in the french speaking part of belgium , an area that represents 5 million people and 50,000 births per year using prenatal care and pediatric clinics , gynecological departments , and the genetic centers . in china ,
guangxi province piloted preconceptional hiv testing and counseling among couples who sought the then mandatory premarital medical examination as a component of the three - pronged approach to reduce mother to child transmission of hiv .
hiv testing rates among couples increased from 38% to 62% over one year period . in october 2003 ,
china changed the legal requirement of premarital medical examination from mandatory to voluntary .
this change was interpreted by most women that the premarital health examination was unnecessary and overall premarital health examination rates dropped .
social marketing efforts piloted in 2004 indicated that 95% of women were willing to pay up to rmb 100 ( us$12 ) for preconception health care services .
these case studies illustrate programmatic feasibility of preconception care services to address maternal and child health and other public health challenges in developed and emerging economies .
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Introduction
Hong Kong: A done deal
South Korea: An evolving initiative
Belgium: Baby steps
China: Preconception care as an HIV/AIDS intervention model
China: Regaining the old glory
Conclusions
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a basic set of proteins and mrnas are differentially expressed among cell types , temporally and spatially , generating a vast assortment of cell phenotypes and/or activation states within a single tissue .
outlining this protein / mrna portrait is thus crucial for understanding not only the uniqueness characterizing cells , but especially their distinguished functions .
this becomes of major relevance when the balance between cell - intrinsic properties and identity cues received and provided by each cell to its neighboring cells then shapes the cell - to - cell cross talk during physiopathological conditions . in the cns , neuroinflammation is the typical consequence of the interchange among different cell types , particularly neurons , astrocytes , oligodendrocytes and microglia , of a variety of cues as neurotransmitters , cytokines , chemokines , toxic metabolites that condition the final protein / mrna profiles of cells , their activation states and functional outcomes [ 2 , 3 ] .
since neuroinflammation accompanies a large variety of neurodegenerative diseases , there is increasing interest in determining how the different cell phenotypes and cellular interconnectivity might contribute to reduce inflammation and reverse neurodegeneration .
microglia actively participate to the context - dependent , neuroprotective / neurotoxic molecular network that is triggered during neuroinflammation . among the molecular cues having a key role in this process ,
extracellular nucleotides are major responsible for intercellular communication and propagation of inflammatory stimuli [ 57 ] .
this occurs by specific binding to various receptor subtypes , termed ionotropic p2x and metabotropic p2y , which are simultaneously localized on several different cell phenotypes . among these
, the p2y12 receptor subtype belonging to the gi class of g protein - coupled receptors is activated by adp .
two transcript variants apparently encoding the same protein isoform have been identified so far for p2ry12 gene , but the determinants for cell specificity of p2y12 protein expression are still unknown .
p2y12 is found on the surface mainly , but not exclusively , of blood platelets , where it acts as blood clotting regulator and target for the treatment of thromboembolisms [ 9 , 10 ] . in the nervous system ,
the tissue- and cellular - selective expression of p2y12 exhibits a pattern throughout white and gray matter consistent with astrocyte expression , although it has not been found colocalization between p2y12 and gfap - positive astrocytes in rat brain cortex and nucleus accumbens , despite the abundant presence of p2y12 mrna .
moreover , we previously established in vivo the expression of p2y12 in oligodendrocytes and myelin sheaths of rat cerebral cortex , subcortical areas , and periventricular white matter . this localization is confirmed throughout the corticospinal tract , therefore suggesting high conserved tissue - homogeneity and phenotype - specificity , and a hypothesized role in myelination [ 1215 ] .
p2y12 is finally observed in brain and spinal cord resident microglia , where it affects activation , chemotaxis [ 1619 ] and neuropathic pain , but it is not observed , for example , in peripheral macrophages in spleen [ 18 , 20 ] .
p2y12 expression in primary microglia is variable with postnatal development and shows sexually dimorphic behavior . through the use of all the available p2y12-immunoreactive antibodies recognizing the c - terminus or the second intracellular loop of the receptor , the aim of the present work is to provide comparative evidence about p2y12 cell specificity in microglia versus oligodendrocytes particularly from the healthy and diseased cns under neuroinflammatory conditions as those sustained during amyotrophic lateral sclerosis ( als ) and multiple sclerosis ( ms ) .
adult b6.cg-tg ( sod1-g93a)1gur / j mice expressing high copy number of mutant human sod1 with a gly93ala substitution ( sod1-g93a ) were originally obtained from jackson laboratories ( bar harbor , me , usa ) and housed in our indoor animal facility as described in apolloni and collaborators , .
the animals were euthanized , according to the guidelines for preclinical testing and colony management .
also neonatal wistar and adult lewis rats ( from charles river laboratories , lc , italy ) were housed in our indoor animal facility .
animal procedures were performed according to european guidelines for the use of animals in research ( 86/609/cee ) and the requirements of italian laws ( d.l .
ethical procedures were approved by animal welfare office , department of public health and veterinary , nutrition and food safety , general management of animal care and veterinary drugs of the italian ministry of health .
all efforts were made to minimize animal suffering and number of animals necessary to produce reliable results .
microglia primary cultures prepared from mouse cortex as previously described were about 98% pure , as verified by immunofluorescence with cd11b ( for microglia ) , glial fibrillary acidic protein ( gfap , for astrocytes ) , neuronal nuclei ( neun , for neurons ) , and smi94 ( for oligodendrocytes ) .
purified cultures of oligodendrocytes were prepared from forebrain of postnatal day 1 - 2 wistar rats , according to minor modifications from a previously described method . after removing the meninges ,
cortices were minced , digested , and dissociated by passage through 70 m nylon cell strainer ( bd biosciences , europe ) .
cells were plated in dulbecco 's modified eagle 's medium ( dmem ) supplemented with 20% heat - inactivated fetal bovine serum ( fbs ) , 4 mm glutamine , 1 mm sodium pyruvate , 50 u / ml penicillin , 50 g / ml streptomycin , and 100 g / ml gentamicin in t75 poly - d - lysine - coated flasks , at about 10 million cells / flask .
the cultures were maintained at 37c in a 5% co2 and 95% air atmosphere for 14 days .
mixed glial cultures were then shaken at 200 rpm at 37c for 1 h and 98% pure microglia collected from the supernatant of each flask , as verified by immunofluorescence with gfap , neun , neural / glial antigen 2 ( ng2 , for oligodendroglial precursor cells ) , myelin basic protein ( mbp , for mature oligodendrocytes ) , and cd11b .
after further shaking at 200 rpm and 37c for 1518 h , the detached cell suspension was finally incubated for 1 h at 37c for differential adhesion of contaminating cells .
the non - adherent cells were filtered through 40 m nylon cell strainer ( bd biosciences ) , spun for 10 min at 100 g , resuspended in oligodendroglial precursor cells medium ( basal chemically defined medium : dmem , 4 mm l - glutamine , 1 mm sodium pyruvate , 0.1% bovine serum albumin ( bsa ) , 50 g ml apo - transferrin , 5 g ml insulin , 30 nm sodium selenite , 10 nm d - biotin , and 10 nm hydrocortisone ) containing 10 ng ml platelet derived growth factor - aa ( pdgf - aa ) and 10 ng ml basic fibroblast growth factor ( bfgf ) , and seeded at the density of 1 10 cells / cm into poly d , l - ornithine - coated plates .
the cells were then induced to differentiate into mature oligodendrocytes when the basal chemically defined medium was added with 15 nm triiodothyronine , 10 ng ml ciliary neurotrophic factor ( cntf ) and 0,05 mg 10 ml n - acetyl - l - cysteine ( nac ) , for 47 days .
a 98% pure population of oligodendrocytes was thus obtained , as verified by immunofluorescence with ng2 , mbp , gfap , neun , and cd11b antibodies .
organotypic cerebellar slice cultures were prepared with modifications from previously published protocols [ 26 , 27 ] .
briefly , cerebella were excised from neonatal wistar rat ( 810 days old ) , cut on a mc ilwain tissue chopper ( 400 m ) and parasagittal slices separated into cold hank 's balanced salt solution ( hbss ) .
two to three slices were plated on each millicell cm culture inserts ( millipore , italy ) and kept in organotypic maintenance medium ( 50% basal medium eagle - bme , 25% hbss , 25% heat - inactivated horse serum , supplemented with 5 mg / ml glucose , 1 mm glutamine , 50 u / ml penicillin , and 50 g / ml streptomycin ) at 37c in a 5% co2 and 95% air atmosphere .
the medium was changed every 2 days and double immunofluorescence was performed in free floating after 710 days in vitro .
organotypic cultures were washed three times with pbs , fixed with 4% paraformaldehyde for 1 h , rinsed , and blocked for 1 h in phosphate buffered saline ( pbs)/10% normal donkey serum ( nds)/0.4% triton x-100 .
primary antibodies were incubated for 24 h in pbs/2% nds/0.4% triton x-100 ( table 1 ) .
the secondary antibodies used for double labelling are cy3-conjugated donkey anti - rabbit igg ( 1 : 100 , jackson immunoresearch , europe ) or alexa fluor 488-affinipure donkey anti - mouse igg ( 1 : 200 , jackson immunoresearch ) . after rinsing ,
sections were mounted , covered with fluoromount medium ( sigma - aldrich , milan , italy ) and a coverslip , and analyzed by confocal microscopy .
microglia and oligodendrocytes were washed three times with pbs , fixed with 4% paraformaldehyde for 10 min ( oligodendrocytes ) or 20 min ( microglia ) , washed , permeabilized with 0.050.1% triton x-100 for 10 min , rinsed , and blocked for 30 min in pbs/1% bsa .
microglia were stained for about 3 h at 37c in 1% pbs / bsa with 5 g / ml cy2-phalloidin ( sigma - aldrich ) , in combination with primary antibodies against p2y12 receptor , as reported in table 1 .
the secondary antibodies used for double labelling are cy3-conjugated donkey anti - rabbit igg ( 1 : 100 , jackson immunoresearch ) or alexa fluor 488-affinipure donkey anti - mouse igg ( 1 : 200 , jackson immunoresearch ) . cells were extensively washed and stained with the nucleic acid blue dye , hoechst 33342 ( 1 : 1000 ) .
after rinsing , cells were covered with fluoromount medium ( sigma - aldrich ) and a coverslip and analyzed by confocal microscopy .
animals were anaesthetized by intraperitoneal injection of chloral hydrate ( 500 mg / kg ) and transcardially perfused with pbs followed by 4% paraformaldehyde at ph 7.4 .
tissue samples ( mice spinal cord and rat brain ) were then postfixed for 1 - 2 days , and cryoprotected in 30% sucrose in pbs at 4c .
mice spinal cords ( l3l5 ) were cut at 30 m thickness with a frozen microtome .
sections were mounted on slide glasses and allowed to air - dry ( 1 - 2 h ) .
a rectangle was then drawn around the sections with a pap pen ( sigma - aldrich ) .
rat brains were cut at 40 m thickness using a cryostat microtome and sections were processed in free - floating .
double immunofluorescence analysis was performed after blocking in pbs containing 10% nds and 0.3% triton x-100 for 1 h at room temperature .
sections were incubated with different combinations of primary antibodies ( table 1 ) , in pbs , 0.3% triton x-100 and 2% nds , for 2448 h at 4c .
finally , sections were washed with pbs and incubated with appropriate fluorescent - conjugated secondary antibodies for 3 h at room temperature . after rinsing
, sections were covered with fluoromount medium ( sigma - aldrich ) and a coverslip and analyzed by confocal microscopy .
tissues supplied by uk multiple sclerosis tissue bank at imperial college , london , were collected postmortem with fully informed consent from both donors and close relatives .
procedures for retrieval , processing , and storage have gained ethical approval from all appropriate committees .
the brain tissues analyzed were from 7 neuropathologically confirmed cases of secondary progressive ms ( spms ) .
analysis was performed also on samples from patients who died due to nonneurological diseases ( healthy ) .
cerebral hemispheres were fixed with 4% paraformaldehyde for about two weeks , coronally sliced , and blocked .
individual blocks were cryoprotected in 30% sucrose for one week and frozen by immersion in isopentane precooled on a bed of dry ice .
cryostat sections ( 3040 m thick ) were either stained with luxol fast blue and cresyl fast violet ( kluver - barrera staining ) , in order to detect white matter lesions and their cellularity , or subjected to immunohistochemistry for mbp , in order to distinguish grey matter lesions .
sections were processed in free - floating for double immunofluorescence studies , as described in amadio and collaborators .
immunofluorescence was analyzed by means of a confocal laser scanning microscope ( zeiss , lsm700 , germany ) equipped with four laser lines : 405 , 488 , 561 , and 639 nm .
the brightness and contrast of the digital images were adjusted using the zen software ( zeiss ) .
human p2y12 complete cdna was obtained by reverse transcription with enhanced avian rt - pcr kit ( sigma - aldrich ) from total human brain rna ( life technologies , paisley , uk ) .
the obtained cdna was then cloned into the xhoi and xbai sites of the eukaryotic expression vector cs2 + mt to provide n - terminal 6x c - myc epitope - tagged mammalian expression plasmids , which has been validated by dna sequencing .
oligos used for amplification were as follows : forward 5gcctcgagatgcaagccgtcgacaacctc3 and reverse 5gctctagagtttacattggagtctcttc designed on human p2y12 mrna ( nm_022788.3 ) .
human embryonic kidney 293 cells ( hek 293 ) and sloan - kettering neuroblastoma sh - sy5y clone ( sh - sy5y ) cells were grown in dmem supplemented with 10% fbs , 100 unit / ml penicillin , and 100 g / ml streptomycin at 37c in atmosphere containing 5% co2 .
one day before transfection , hek293 or sh - sy5y cells were plated and transfection of p2y12-cs2 + mt or cs2 + mt empty vector was performed with lipofectamine 2000 ( life technologies ) , according to manufacturer instructions .
primary rat microglial and oligodendrocyte cells were lysed with trizol ( life technologies ) and total rna was extracted following the manufacturer 's instructions .
after dnase treatment ( qiagen , hilden , germany ) , equal amount of total rna ( 1 g ) was subjected to retrotranscription by high capacity rna - to - cdna kit ( life technologies ) and 50 ng of each cdna were amplified with rat p2y12 specific primers ( f : 5-gattgataaccattgacc-3 ; r : 5-ggtgagaatcatgttagg-3 ) .
amplification products ( 10 l of 20 ) were electrophoresed on 2% agarose gel containing ethidium bromide ( 1 g / ml , sigma aldrich ) , photographed under uv light using kodak image station 440cf , with molecular imaging software 4.0.1 . in order to isolate total protein extracts , microglia and oligodendrocytes were harvested with ice - cold ripa buffer ( pbs , 1% nonidet p-40 , 0.5% sodium deoxycholate , and 0.1% sds ) , added with protease inhibitor cocktail ( sigma aldrich ) .
lysates were kept for 30 min on ice and then centrifuged for 10 min at 14,000 rpm , at 4c .
snap - frozen blocks from cases ms114 , ms125 , and ms163 supplied by uk multiple sclerosis tissue bank at imperial college in london were homogenized in ripa buffer , added with protease inhibitor cocktail , incubated on ice for 1 h , and centrifuged at 14,000 rpm for 10 min at 4c . rat brain and mouse brain and spinal cord were homogenized and sonicated in ripa buffer , added with protease inhibitor cocktail , kept for 1 h on ice , and centrifuged at 4c for 10 min at 13,000 rpm .
supernatants were collected and analyzed for protein content by bradford colorimetric assay ( biorad , milan , italy ) .
analysis of protein components was performed by polyacrylamide gel ( sds - page ) separation ( biorad ) and transfer onto nitrocellulose membranes ( amersham biosciences , cologno monzese , it ) .
after saturation , blots were probed overnight at 4c , with the specified antibody ( table 1 ) , and finally incubated for 1 h with hrp - conjugated secondary antibodies and detected on x - ray film ( aurogene , rome , italy ) , using ecl advance western blotting detection kit ( amersham biosciences ) .
p2y12 protein was detected with molecular mass comprised between 40 kda ( as predicted by amino acid sequence ) and 49 - 50 kda ( as predicted by the manufacturer data sheet ) .
analysis was performed with the statistical software package medcalc ( medcalc software , mariakerke , belgium ) .
in order to provide wide ranging comparative analysis of p2y12 expression particularly in microglia and oligodendrocytes under neuroinflammatory conditions , we performed immunofluorescence and confocal analysis of receptor expression in primary cortical and organotypic cerebellar cultures , in tissue slices from rat striatum and cerebellum , in spinal cord sections from symptomatic and end stage sod1-g93a als mouse model , finally in autoptic cortical tissue from progressive ms donors .
p2y12 receptor is formed by two transcript variants that give rise to identical proteins with 342 amino acids , a secondary structure constituted by seven hydrophobic transmembrane domains connected by three extracellular and three intracellular loops , with four extracellular cysteine residues most likely contributing to the nucleotide binding site ( figure 1(a ) ) .
the commercially available p2y12 antibodies that we mostly used in our work are raised against the second intracellular loop , and precisely amino acids 125142 ( here named intra1 , intra2 , and intra fl ; see red circle in figure 1(a ) ) , and against the c - terminus , here named c - ter ( see green oval in figure 1(a ) ) ( see also table 1 ) . in order to validate the use of these different antibodies for p2y12 receptor , we compared them on recombinant p2y12 receptor protein obtained by cloning the complete cdna of the human receptor into the eukaryotic expression vector cs2 + mt , to provide the expression of a n - terminal c - myc tagged fusion protein .
after transfection into sh - sy5y and hek293 cell lines , we analyzed total protein extracts by sds - page and western blotting using c - ter , intra1 , and intra2 antibodies .
although with different intensities , all the antibodies recognize the myc - p2y12 protein band at the predicted molecular mass of 50 kda .
these results confirm the specificity of the used antibodies toward denatured recombinant p2y12 receptor ( figure 1(b ) ) . in order to verify the expression of p2y12 mrna in rat primary oligodendrocytes ( ol ) and microglia ( rmg )
, rt - pcr was performed using specific primers designed on receptor sequence . as shown in figure 1(d ) , rt - pcr on both rmg and ol reveals the presence of the predicted p2y12 mrna band .
next , we validated the antibodies with protein extracts from dissociated primary cultures or tissues from different species .
p2y12 protein is specifically recognized in extracts from human cerebral cortex snap frozen tissue , rat and mouse brain , mouse primary microglia ( mmg ) and ol ( figure 1(c ) ) .
no signals are obtained when the immunoreactions are performed in the presence of p2y12 neutralizing peptides , when available from manufacturer ( data not shown ) . in order to establish cell specificity of p2y12 receptor expression
, we performed comparative immunofluorescence and confocal analysis in primary dissociated cortical microglia and oligodendrocytes ( figures 2(a ) and 2(b ) ) , as well as organotypic cerebellar cultures ( figure 2(c ) ) .
p2y12 is strongly recognized by both c - terminus- and second intracellular loop - recognizing antibodies ( red , c - ter , upper left inset ; intra fl , upper right inset ; intra1 , lower right inset and intra2 , lower left inset ) , specifically distinguishing the very heterogeneous morphological features of mouse primary microglia , as shown by double fluorescence and confocal analysis performed with phalloidin ( green ) , a marker for filamentous actin ( figure 2(a ) ) .
fan - like cells ( insets ) , elongated rod - like cell bodies with short and tiny branches , asymmetrical hairy cells with miniature processes or lamellipodia are simultaneously observed ( c - ter phalloidin hoechst , merged ) .
likewise , all the p2y12 antibodies ( intra2 , intra1 , c - ter , and intra fl ) recognize the multibranched morphology of rat mature oligodendrocytes ( figure 2(b ) , ol , insets , red ) in primary cultures , as confirmed by double immunofluorescence and confocal analysis with mbp antibody ( ol , intra1-mbp , yellow merged image ) .
in addition , both intra2 and c - ter antibodies distinguish the ng2-positive rat oligodendrocyte precursor cells ( opc , merged insets ) .
p2y12 immunoreactivity is confirmed in the ex - vivo system of organotypic rat cerebellar cultures ( figure 2(c ) ) .
however , differently from mouse microglia and rat oligodendrocyte primary cultures , the second intracellular loop- ( red , intra1 ) and c - terminus - recognizing ( red , c - ter ) antibodies surprisingly immunoreact with different cell phenotypes when the integrity and cytoarchitecture of the tissue is preserved , as in organotypic cultures .
while intra1 antibody ( red ) exclusively labels mbp - positive fibers ( green ) and highlights myelin structures , c - ter antibody does not immunoreact with myelinated fibers , being present on cells likely resembling microglia ( insets ) , as also confirmed by colocalization with microglial marker cd11b ( data not shown ) .
results similar to those found with intra1 were also obtained with intra2 antibodies , lots an01/02/04 ( data not shown ) .
we next verified if the cell type - specific presence of p2y12 receptor observed in organotypic culture , either in myelin structures or in microglia , respectively , by the use of the second intracellular loop-(intra1 ) or the c - terminus - recognizing ( c - ter ) antibody , is also confirmed in rat cerebellar and striatal slice tissues ( figure 3 ) .
all the antibodies raised against the second intracellular loop ( red , intra1 , intra2 , intra fl ) identify the abundant presence of p2y12 receptor only on myelinated fibers from both cerebellum ( figures 3(a ) , 3(b ) , and 3(c ) ) and striatum ( figures 3(d ) , 3(e ) , and 3(f ) ) , as confirmed by colocalization of signals obtained with intra fl and mbp antibodies in cerebellum ( + mbp , inset c2 , merged , yellow ) and striatum ( data not shown ) ; colocalization of signals obtained with intra1 and intra2 with mbp antibodies in cerebellum and striatum ( data not shown ) ; immunoreactivity of intra1 , intra2 , and intra fl antibodies for structures identical to those observed in cerebellum ( data not shown ) and striatum with mbp antibody ( see for instance mbp , inset e1 , green ) ; identification by second intracellular loop p2y12 antibodies of structures totally different from both bergmann glia and astrocytes recognized by specific gfap antibody in cerebellum ( inset b1 , green ) , and from microglia identified by specific cd11b antibody in cerebellum ( + cd11b , inset c1 , merged , green ) and striatum ( + cd11b , inset f1 , merged , green ) . of notice ,
all the 2nd intracellular loop antibodies are able to describe the specific cytoarchitecture of both cerebellum , where radiant and sparse fibers clearly characterize the lobuli , and of striatum , where white matter is instead organized in distinct bundles . as in organotypic cultures ,
the c - terminus antibody ( red , c - ter ) instead recognizes only microglia in rat cerebellum ( figures 3(g ) , 3(h ) , and 3(i ) , and insets g1 , h1 , i1 ) , striatum ( figures 3(j ) , 3(k ) , and 3(l ) ) and cerebral cortex slices ( data not shown ) , with a signal more uniformly distributed throughout the whole tissue . by comparing the cd11b and c - ter immunolabelling in the striatum
, we furthermore observe that the mutual intensity of signals is cell - selective within the microglia population , with some cells exclusively positive for c - ter ( arrow ) , and others instead showing different grades of cd11b - c - ter colocalization ( see for instance orange , yellow and greenish cells in the merged field ) .
regrettably , double immunofluorescence with c - ter and iba1 microglia marker is not practicable , since both antibodies are raised in the same species .
however , no colocalization of signals is ever shown between mbp ( green ) and c - ter antibodies ( + mbp , inset j1 , merged ) . in order to verify
if p2y12 recognized by c - ter antibody exclusively in microglia from rat brain slices could be used as specific microglia marker during neuroinflammation , we validated its use in a typical neuroinflammatory disease such as als and for the first time characterized the presence of p2y12 receptor in sod1-g93a mouse model ( figure 4 ) . by immunofluorescence and confocal analysis on lumbar spinal cord sections ( l3l5 ) of wild - type ( wt ) mice , we first compared the immunoreactive signals obtained with c - ter and specific microglia markers cd11b ( red , recognizing ramified microglia ) and cd68 ( red , recognizing roundish activated microglia ) . in wt mice
, c - ter ( green ) is abundantly and strongly immunoreacting with the microglia population and colocalizing with the majority of cd11b - positive cells , in both dorsal ( dh ) and ventral ( vh ) horns of spinal cord ( figure 4(a ) , left panel , merged yellow signal ) .
all cd11b - positive cells share immunoreactivity for c - ter , as proved by the absence of red cd11b signal .
conversely , not all c - ter - positive cells immunoreact also with cd11b antibody , as proved by the presence of some green c - ter signals .
in addition , we never observe colocalization with the rare activated cd68-positive ( red ) microglia cells present in wt healthy tissue ( figure 4(a ) , right panel , merged and inset ) . to test if c - ter antibody can further recognize microglia activation during the progression of als , we next examined sod1-g93a spinal cord sections at two different stages of the disease , that is , 20 weeks , a phase when the disease accelerates , and end stage , that is , 23 weeks , when the animals reach the score of 1 accordingly to a behavioral score system . at both stages ,
sod1-g93a mice show a significant increase in cd68 immunostaining not only when compared to wt mice ( figure 4(a ) ) , but also in vh with respect to dh , and this is even more evident at end stage with respect to 20 weeks ( red , figure 4(b ) ) .
remarkably , the immunoreactive signal of c - ter ( green , figure 4(b ) ) decreases during disease progression and the effect is pronounced especially in vh , where motor neuron loss , tissue damage , and microglia activation are known to be increased . particularly at 20 weeks
, c - ter - expressing microglia are still ramified in dh , where few cd68-positive cells are present , but start to disappear in vh , where instead cd68-positive cells are increased . at end stage
, c - ter staining decreases in dh with respect to 20 weeks and disappears almost completely in vh , concomitantly with a robust increase in cd68 staining . a parallel decrease in c - ter and increase of cd68 immunoreactivities
is also confirmed by western blot analysis performed on sod1-g93a lumbar spinal cord homogenates at end stage ( figure 4(c ) ) . as in rat organotypic cerebellar cultures ( figure 2(b ) ) and rat cerebellar , striatal , cortical , or mouse spinal cord tissue slices
( figures 3 and 4 ) , a similar pattern of p2y12 receptor expression is shown in human frontal cortex autoptic tissue by using c - ter antibody that highlights only microglia uniformly distributed throughout the entire healthy tissue ( red ) , but not mbp - positive myelinated structures ( green ) , as detected by the absence of overlapping immunofluorescent signals ( figures 5(a ) , 5(b ) , and 5(c ) , merged and insets a1 , c1 ) . on the contrary , intra1 ( red , figures 5(d ) , 5(e ) , and 5(f ) ) , intra2 , and intra fl ( data not shown ) antibodies recognize exclusively myelinated fibers in tissue from healthy ( insets d1 , e1 , and f1 ) and progressive ms donors ( figures 5(d ) , 5(e ) , and 5(f ) ) , as it is found with rat tissue ( figures 2 and 3 ) .
moreover , we confirm a decrease of p2y12 receptor signal in proximity to the demyelinating lesion , as detected by loss of mbp - positive fibers ( see asterisks ) .
no colocalization of signals is shown with mhc ii - positive microglia ( + mhc ii , inset d3 , merged ) .
importantly , c - ter , intra1 ( red , insets a - f2 ) and intra2 , and intra fl ( data not shown ) antibodies all recognize the presence of p2y12 receptor in integrin ii/3-positive platelets ( green ) contained in the blood vessels of the analyzed tissues . as observed in als mouse spinal cord where p2y12 receptor detected by c - ter antibody is shown to temporally and regionally decrease in microglia as a function of increasing inflammatory damage ( figure 4 ) , we notice that microglia gradually lose immunoreactivity for c - ter antibody ( figure 6 ) in proximity to the demyelinating active cortical lesions of ms expressing augmented positivity for mhc ii [ 3335 ] .
moreover , by comparing mhc ii and c - ter signals , we recognize four areas ( a d ) inside and around the lesion , where microglia express different amount of these proteins . in zone
a , at the edge of the lesion , we observe a predominance of c - ter immunoreactivity ( red ) compared to that of mhc ii ( green ) , as depicted in the merged field by a major occurrence of red signal . in zone b ,
closer to the lesion , we notice a prevalence of active mhc ii - positive green cells , but still the presence of few red and yellow signals .
c and d , apparently in the core or outside the lesion , respectively , where microglia express either almost exclusively mhc ii protein ( c ) or p2y12 receptor on ramified microglia ( d ) .
the interchange among different cell types of molecular cues that condition the cell specificity and the protein profile of each cell characterizes the morphological and functional heterogeneity in particular of microglia within various cns regions , developmental stages [ 37 , 38 ] and , even more , states of activation during pathological conditions . in the case , for instance , of als , the release of signals from motor neurons apparently denotes one of the earliest phase of the disease , with microglia behaving as an m2 phenotype producing neuroprotective factors to repair motor neurons and preventing them against further injury . as disease rises , motor neurons start releasing alarm signals
that in turn convert microglia from beneficial m2 to cytotoxic m1 phenotype , with consequent release of proinflammatory cytokines .
for instance , m1 markers are abundantly expressed in normal appearing white matter and throughout active demyelinating ms lesions by activated microglia and macrophages , although in human active ms lesions microglia show an intermediate activation status .
in addition , m2 microglia appear fundamental to guide oligodendrocyte remyelination in mice , and a switch from m1- to m2-dominant response occurs in microglia and peripherally derived macrophages when remyelination starts . only therapeutic procedures
that both down - regulate the harmful responses and up - regulate the beneficial responses may hopefully slow pathological progression and provide meaningful hope for treatment . at the same time , the identification of clear markers involved in the m2/m1 microglia transition becomes mandatory for presymptomatic diagnosis , monitoring of disease progression , and efficacy of therapies . under this perspective , and consistently with previous findings establishing the role of purinergic receptors in the pathogenesis of both als and ms [ 14 , 43 ] , our present work serves this aim , by highlighting the gradual loss of p2y12 immunoreactivity as an early marker of neuroinflammation and microglia metamorphosis .
we have indeed demonstrated here that p2y12 receptor protein identified in primary cultures of both microglia ( figure 2(a ) ) [ 18 , 44 ] and oligodendrocytes ( figure 2(b ) ) by different but p2y12-selective antibodies can be instead recognized in branched microglia exclusively by the use of c - ter antibody ( figures 36 ) .
this occurs only when the integrity and cytoarchitecture of the tissue is typically preserved in the presence of the least experimental manipulations , that is , in organotypic cultures ( figure 2(c ) ) and tissues slices for instance from rat striatum and cerebellum ( figure 3 ) , mouse spinal cord ( figure 4 ) , and human cerebral cortex ( figures 5 and 6 ) .
a similar difference in primary cultured cell versus tissue distribution of a protein was previously demonstrated with large - conductance calcium - activated potassium channel expression , in vascular endothelium .
a first implication emerging from these results is that a reliable evidence about selective p2y12 expression in cells of healthy or neuroinflammatory states is genuine only when cell connectivity and tissue architecture are fully preserved .
we have indeed shown this , by proving that the antibodies used for p2y12 , recognizing either the c - terminus or the second intracellular loop of the receptor ( figure 1 and table 1 ) and immunolabelling , respectively , microglia or myelinated fibers in the cns , are all still able to immunoreact for instance with platelets ( figure 5 ) , where the receptor was originally described to be present and to have a role in the processes of activation , aggregation [ 4649 ] , primary hemostasis , and arterial thrombosis [ 5055 ] . a possible explanation for the microglia versus oligodendrocyte selectivity of the p2y12 antibodies might be that gi - coupling , and/or quaternary structure , post - transcriptional modifications , and subcellular localization of p2y12 that remain strictly preserved in platelets , are instead divergent in microglia with respect to oligodendrocytes / myelinated fibers . in this case , a cell - specific network of p2y12 oligomeric interactions and/or a distinct subcellular partitioning might simply mask the recognition sites of the different antibodies on p2y12 protein in different cell types . to support this hypothesis
, we know that in platelets p2y12 indeed resides in subcellular lipid raft structures and its partitioning out from rafts causes for instance inactivation and that also the presence of another purinergic receptor , the ionotropic p2x3 , in lipid rafts has cell - specific properties shared in cerebellar granule neurons and total brain tissue but not in neuroblastoma cells and dorsal root ganglia and that the specific antagonist clopidogrel inhibits p2y12 by breaking down the homoligomeric complex to single monomers and finally , that hetero - oligomerization of p2y12 is demonstrated with p2y1 , p2y2 , p2y13 , and with adenosine a1 , a2a receptors in different cellular backgrounds .
all these features might very well explain also why it has not been found colocalization between p2y12 and gfap - positive astrocytes in rat brain cortex and nucleus accumbens , despite the abundant presence of p2y12 mrna and , moreover , why p2y12 is specifically observed in brain and spinal cord resident microglia but is not observed , for example , in peripheral macrophages in spleen [ 18 , 20 ] .
a second implication that emerges from our results is that the morphological metamorphosis that microglia undergo under neuroinflammatory conditions as those triggered during als and ms , can be remarkably highlighted by the progressive reduction of p2y12 immunostaining obtained with c - ter antibody that reacts , also in this case , exclusively with multibranched microglia still present in the tissue ( figures 4 and 6 ) .
this closely reflects the expression of p2y12 that is robust in the resting / surveillant branched state but dramatically decreased after morphological transition and activation of microglia .
our observations are also in line with the central role played by p2y12 in branched microglia membrane ruffling and inspection of the environment . on the other hand , they depict a morphological / functional state of microglia that only partially overlaps with cd11b and mhc ii immunoreactivities , which are furthermore known to increase during activation but instead highly contrasts with cd68 immunostaining that is totally absent in ramified microglia . in parallel to our results , these last antibodies actually accentuate the progressive transition of microglia from a lesser ramified shape to a significantly more activated amoeboid phenotype ( figures 4 , 6 , and 7 ) , thus suggesting the dual use of c - ter and cd68 antibodies as markers , respectively , for branched resting / surveillant versus roundish / activated microglia . a reduction of c - ter - p2y12 immunostaining that is concomitant to an increase , for instance , of mhc ii / cd11b / cd68 immunoreactivity , could thus become a feasible approach to detect an increasing neuroinflammatory condition .
indeed , p2y12 is considered an essential component and primary site at which nucleotides such as adp act to promote directional microglia movement or chemotaxis at early stages of cns injury . in particular ,
microglia from mice lacking p2y12 exhibit normal baseline motility but are unable to polarize and to elicit directional branch extension and migration toward nucleotides in vitro , or sites of cortical damage in vivo .
these notions are consistent with our results that fail to describe c - ter - p2y12 immunoreactivity on roundish phagocytizing , or polarized migratory microglia .
however , we still do not know if reduction / absence of p2y12 c - ter - immunolabelling on activated microglia might be a cause or a consequence of morphological / functional transition or might simply reflect a cell - selective hindrance and lack of access to the immunogenic sites by the antibody .
anyhow , we can assert that the distinctive recognition of multibranched microglia renders the c - ter antibody a novel and useful tool to discriminate among microglia morphological states , thus making p2y12 receptor a selective and early marker for the ramified phase .
in parallel to this , we have also proven that all the several antibodies raised against the second intracellular loop of p2y12 ( intra1 , intra2 , and intra fl ) can likely be employed as markers for the presence of ms lesions .
although with different intensities , they not only recognize the receptor specifically on myelinated fibers of organotypic cultures ( figure 2(c ) ) , tissues slices from rat striatum or cerebellum ( figure 3 ) and human cerebral cortex , but also furthermore highlight the reduction of p2y12 signal that occurs for instance in ms tissue ( figure 5 ) in correlation to the extent of demyelination found in all types of grey matter cortical plaques ( i iii ) and subcortical white matter . in brief , we have shown here that the presence of p2y12 receptor can be simultaneously identified by the c - terminus and the second intracellular loop antibodies .
when this occurs , a condition of intact myelinated fibers and branched / surveillant microglia is represented at once that perhaps signifies a
for instance in ms , a decrease in the second intracellular loop immunoreactions accompanied by an increase of c - terminus immunoreactivity will possibly depict the loss of myelin and replacement by ramified microglia that often occur in an inactive plaque . on the contrary , a decrease in c - terminus immunolabelling in the abundant presence of second intracellular loop - positive myelinated fibers would indicate an early active plaque where m1/m2 microglia reactivity starts to take place .
by comparative analysis of all the available p2y12-immunoreactive antibodies recognizing the c - terminus or the second intracellular loop of the receptor , we have established here that , under experimental conditions of well - preserved cytoarchitecture and tissue integrity , p2y12 receptor expressed by both ramified microglia or oligodendrocytes / myelinated fibers might serve a dual function as specific marker , respectively , of branched / surveillant microglia as well as demyelinating lesions .
we believe that p2y12 identification and modulation might potentially acquire an important predictive value under neuroinflammatory conditions , as those found in als and ms .
p2y12 is likely to deserve a key role in the verge of a neuroinflammatory breakdown .
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in the cns , neuroinflammation occurring during pathologies as amyotrophic lateral sclerosis ( als ) and multiple sclerosis ( ms ) is the consequence of an intricate interplay orchestrated by various cell phenotypes . among the molecular cues having a role in this process , extracellular nucleotides are responsible for intercellular communication and propagation of inflammatory stimuli .
this occurs by binding to several receptor subtypes , defined p2x / p2y , which are widespread in different tissues and simultaneously localized on multiple cells .
for instance , the metabotropic p2y12 subtype is found in the cns on microglia , affecting activation and chemotaxis , on oligodendrocytes , possessing a hypothesized role in myelination , and on astrocytes . by comparative analysis ,
we have established here that p2y12 receptor immunolabelled by antibodies against c - terminus or second intracellular loop , is , respectively , distributed and modulated under neuroinflammatory conditions on ramified microglia or myelinated fibers , in primary organotypic cerebellar cultures , tissue slices from rat striatum and cerebellum , spinal cord sections from symptomatic / end stage sod1-g93a als mice , and finally autoptic cortical tissue from progressive ms donors .
we suggest that modulation of p2y12 expression might play a dual role as analytic marker of branched / surveillant microglia and demyelinating lesions , thus potentially acquiring a predictive value under neuroinflammatory conditions as those found in als and ms .
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1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusion
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putting a halt to the rising trend in overweight prevalence has high priority in public health policy all over the developed world .
prevention , preferably starting at an early age , is considered as the key strategy to achieve this goal , and considerable effort is put into the design of preventive measures . whereas we know in general terms that overweight is the result of an unbalance between energy intake and energy expenditure , development of effective preventive measures requires a much more specific understanding . specific dietary habits and ( in)activities associated with overweight risk in specific age groups need to be identified and their quantitative contribution to the overweight prevalence in the population needs to be assessed .
in addition , we need to know whether the importance of specific behaviors for the development of overweight differs between sub groups in the population .
it is well established that children from families with a low socio - economic status and children with overweight parents are at increased risk to develop overweight [ 39 ] .
it is therefore particularly important to assess the role of behavioral risk factors specifically in these children , since they are the priority target groups for interventions to prevent and reduce overweight .
the aim of this study was to identify specific behavioral risk factors for overweight in young children and to assess their quantitative contribution to the prevalence of overweight in the general population and in high risk sub groups . in a large population - based birth cohort , we prospectively investigated associations between dietary habits , screen time and physical activity when the children were 5 and 7 years old and overweight at the age of 8 years .
we also assessed the role of these behavioral factors separately in high risk sub groups : children of mothers with overweight and children of mothers with low education .
we estimated the reduction of overweight prevalence that could be achieved if the prevalence of risk factors would be reduced .
the study population consisted of children who participated in the dutch prevention and incidence of asthma and mite allergy ( piama ) birth cohort study .
the baseline study population consisted of 4146 pregnant women , recruited from the general population in 1996 - 1997 .
postal questionnaires , including questions on the child 's lifestyle and health , were sent to the parents during pregnancy , at the child 's ages of 3 and 12 months , and yearly thereafter up to the age of 8 years . at the age of 8 years , the children were invited for a medical examination which included measurement of weight and height .
the study protocol was approved by the medical ethics committees of the participating institutes , and all parents gave written informed consent . of the baseline population of 4146 pregnant women , 183 ( 5% )
were lost to follow - up before any data on the child had been collected .
parental completed questionnaires from age 3 months to 8 years were available for 3934 , 3817 , 3694 , 3563 , 3518 , 3473 , 3373 , and 3269 children , respectively .
when the medical examination of 8-year - old children was planned , 3668 children ( 93% of 3963 ) were still in the study , 3522 children were invited and 2214 participated ( 63% of those invited ) and had their weight and height measured .
146 of the 3668 children were not invited for the medical examination , although they were still participating in the study . in the majority of cases
the reason for this was that their families had moved and were now living too far away from the study centers where the medical examination was conducted .
complete data on overweight and risk factors were available for 1871 children ( 47% of the 3963 children in the study at birth ) . during the medical examination of the 8-year - old children ,
weight was measured to 0.1 kg and height to 0.1 cm by trained research staff using calibrated measuring equipment . from the weight and height measurements
bmi ( body mass index : weight ( kg)/height ( m ) ) was calculated .
overweight and obesity were defined according to age and gender - specific international standards that use cutoff points equivalent to the 25 kg / m and 30 kg / m cut - offs that are commonly used for adults .
the term overweight is used for the total group of children who were overweight , including those who were obese . for physical activity , 3 indicators were selected : time spent walking or cycling to school ( 3 categories ) , membership of a sports club ( yes / no ) , and time spent playing outside ( 3 categories ) . screen time ( 4 categories ) included time spent watching television , video 's , or at the computer .
for dietary intake , 3 indicators were used : fast food consumption , snack consumption , and soft drink consumption .
data on these exposures were obtained from the questionnaires administered at the ages of 5 and 7 years .
these questionnaires contained a food frequency questionnaire with 40 different items and 5 answering categories per item ( ranging from never in the last month to on 6 - 7 days per week ) , which was used to construct the dietary exposure variables . for fast food consumption
a score was constructed based on the consumption frequencies of chips / french fries and of foods like hamburgers and hot dogs .
snack consumption was based on the reported consumption frequencies of 7 different foods / food groups including pie / cake , muffins , candy bars , sweets , chocolate , biscuits , and crisps .
soft drink consumption was based on the reported frequencies of consumption of 4 types of soft drinks .
each of these variables was based on a number of different items , which differed with respect to their energy content . to be able to add up
the different items , total weekly energy intake from each item was calculated based on the reported frequencies , assuming average portion sizes and average energy content per portion .
data on maternal education and maternal weight and height were obtained from the questionnaire completed when the child was 1 year old .
maternal bmi was calculated from reported weight and height and overweight and was defined as a bmi 25 kg / m .
the associations between the exposure variables and overweight at the age of 8 years were analysed by logistic regression , adjusted for gender and birth weight .
exposure variables were constructed using the data collected at both the age of 5 and at the age of 7 years in order to obtain a more stable estimate of exposure during the years preceding the measurement of weight and height . walking / cycling to school , playing outside and screen time
were recorded in categories , numbered 1 , 2 , and 3 for walking / cycling to school , 1 , 2 , and 3 for playing outside , and 1 , 2 , 3 , and 4 for screen time ( see table 1 ) .
the data collected at the ages of 5 and 7 years , were combined by taking the mean of the numbers of the categories reported at the ages of 5 and 7 years respectively .
this resulted in ordinal variables with 5 categories for walking / cycling to school ( ranging from never at ages 5 and 7 to > 1/2 an hour per day at both ages ) , 5 categories for playing outside ( ranging from < 1 x per week at ages 5 and 7 to > 3 x per week at both ages ) and 7 categories for screen time ( ranging from < 1/2 an hour per day at ages 5 and 7 to > 2 hours per day at both ages ) . after having checked for nonlinearity
fast food , snack and soft drink consumption at age 57 ( the mean of the consumption scores at age 5 and at age 7 ) , were included in the regression analyses as linear variables , but are shown in categories ( tertiles ) in table 1 .
overweight ( including obesity ) was used as the outcome measure in the regression analyses .
the number of obese children was too small for analyses with fully adjusted regression models , and obesity was therefore not used as outcome measure in these analyses . in previous studies in the piama cohort , breast feeding was investigated in relation to overweight risk and was found to be associated with reduced overweight risk [ 12 , 13 ] . although breastfeeding is not the subject of this study , results on breast feeding are shown in the tables for completeness . in additional analyses ,
the associations of physical activity and diet with overweight were also assessed separately for the exposures at age 5 and at age 7 .
thirteen percent of all the data potentially in the study ( 3963 participants 20 variables = 79260 ) were missing and 2092 children ( 53% ) had a missing value on at least 1 of the variables .
if data are not missing completely at random ( mcar ) , complete case analysis may lead to biased results [ 14 , 15 ] . in our dataset
several variables ( including maternal education and breast feeding ) were associated with the probability of a subject having one or more missing values .
missing data were multiple times imputed , using the multivariate imputation by chained equations
( mice ) procedure [ 16 , 17 ] , that runs under the statistical program r version 2.5.0 . for the multiple imputation we used all the data that were used in the analyses , as well as data on the children 's weight and height that were measured and reported by the parents in the yearly questionnaires .
. maternal education and maternal overweight could be confounders of the associations studied but could also be factors in causal pathways .
in addition , the analyses were repeated , stratified for maternal education , maternal overweight and gender .
differences between the associations observed in the different sub groups were tested by inclusion of interaction terms in the regression models . for the behavioral risk factors that were found
to be statistically significantly associated with overweight , adjusted population attributable risk percentages ( par% ) were calculated , using the mantel - haenszel approach , as described by benichou .
table 1 shows characteristics of the study population and the prevalence of overweight and obesity in different sub groups .
children with incomplete data on either risk factors or on measured weight and height at 8 years were compared to the children with complete data , with respect to a number of characteristics assessed during the first year of the study ( data not shown ) .
children with incomplete data had , compared to children with complete data , a relatively high prevalence of low maternal education ( 27.5% versus 19.4% ) and of no breast feeding ( 19.7% versus 15.9% ) .
as expected , in the imputed data the prevalence of these characteristics was somewhat higher ( 23.6% and 17.9% , see table 1 ) than in the children with complete data .
the prevalence of maternal overweight was similar in children with incomplete data and in children with complete data ( 24.1% and 26.1% , resp . ) .
the prevalence of overweight in the children was almost the same in the imputed data and in the complete cases ( 13.8% and 14.4% , resp . ) and the prevalence of obesity was the same in both datasets ( 2.7% ) .
table 2 shows the associations between diet , physical activity , screen time and overweight . in the analyses adjusted for gender and birth weight only ( model 1 ) , fast food consumption and screen time ,
sports club membership , active transport to school , playing outside , snack consumption , and soft drink consumption were not associated with overweight . when all risk factors ( including breast feeding ) were included in the same regression model ( model 2 ) ,
the associations with overweight became somewhat weaker for most factors , indicating some association between the individual risk factors .
the association of fast food consumption with overweight lost statistical significance in this analysis whereas the association with screen time remained statistically significant .
surprisingly , an inverse association between snack consumption and overweight was observed . in additional analyses ,
the analyses shown in table 2 were repeated with the behavioral risk factors measured at age 5 and ( separately ) with those factors measured at age 7 instead of the variables combining the exposures measured at age 5 and age 7 . for the physical activity indicators and for screen time , results of these analyses were similar to those shown in table 2 , showing associations between screen time and overweight ( aor ( 95% ci ) at age 5 : 1.35 ( 1.191.53 ) and aor ( 95% ci ) at age 7 : 1.22 ( 1.081.39 ) ) but not between the physical activity indicators and overweight .
snack consumption at age 5 was not associated with overweight at the age of 8 years ( aor ( 95% ci ) : 0.87 ( 0.661.14 ) ) , but there was a significant inverse association between snack consumption at age 7 and overweight at the age of 8 years ( aor ( 95% ci ) 0.70 ( 0.540.91 ) ) .
low maternal education and especially maternal overweight were strong predictors of childhood overweight ( see table 2 , model 1 ) .
the association between low maternal education and overweight weakened substantially in the model including the behavioral risk factors ( an almost 25% change in the regression coefficient from 0.50 to 0.38 ) , indicating that these specific factors explain at least part of the excess overweight prevalence in children of mothers with low education ( see table 2 , model 3 ) .
the association between maternal overweight and overweight in the child weakened only marginally in the model including behavioral risk factors ( 5% change in regression coefficient from 1.07 to 1.02 ) , indicating that the association between maternal overweight and overweight of the child is only to a limited extent mediated by the behavioral factors studied ( see table 2 , model 4 ) .
the analyses were repeated after stratification for low maternal education , maternal overweight and gender , respectively . in the sub groups with low maternal education and with maternal overweight
children of mothers with a low level education had a higher prevalence of high fast food and snack intakes , of > 2 hours screen time per day and of not being member of a sport club than children of more highly educated mothers ( see table 3 ) .
children of overweight mothers had a higher prevalence of high fast food intake and of > 2 hours screen time per day ( at the age of 5 , but not at 7 years ) than children of normal weight mothers ( see table 3 ) .
girls had a slightly higher prevalence of overweight and slightly less screen time than boys ( data not shown ) .
the results suggest that the associations between screen time and overweight were weaker in children of mothers with overweight or low education than in children of mothers with a normal weight or higher level of education ( table 4 ) .
interactions between the exposure variables and low maternal education and maternal overweight were not statistically significant , however . for boys and girls these associations were very similar ( data not shown ) .
of the behavioral risk factors studied , only screen time was statistically significantly associated with overweight . based on the prevalence and adjusted odds ratios for screen time > 1 hr per day ( see table 5 ) , we estimated how much screen time contributed to the total prevalence of overweight .
table 5 shows the adjusted population attributable risk percentage ( par% ) for a screen time of more than 1 hour per day at age 5 and/or age 7 as compared to less than 1 hour at both ages . of the total overweight prevalence in the low risk sub groups
an estimated 17% could be attributed to screen time of > 1 hour per day as compared to 10% in the high risk sub groups .
this means that , if all children would reduce their screen time to less than 1 hour per day , the following reductions in overweight prevalence could be achieved : from 18.3% to 16.4% in children of mothers with low education ; from 12.4% to 10.3% in children of mothers with higher education from 25.0% to 22.4% in children of overweight mothers and from 10.0% to 8.3% in children of normal weight mothers .
all analyses were conducted in a data set containing only complete cases as well as in the imputed data .
the odds ratios ( 95% ci ) for screen time , for example , were 1.47 ( 1.241.75 ) and 1.41 ( 1.171.70 ) in model 1 and model 2 , respectively , in the complete case analysis , compared to 1.45 ( 1.261.66 ) and 1.39 ( 1.211.61 ) in the analysis in the imputed data set .
of 8 different potential behavioral risk factors , only screen time showed consistent and significant associations with overweight . in high risk sub groups of the population ,
screen time was higher than in low risk sub groups , but the associations between screen time and overweight were weaker ( although not significantly weaker ) in the high risk sub groups .
important strengths of the study were the prospective design , the large study population , the repeated measurements ( at ages 5 and 7 ) of different behavioral risk factors and the availability of measured ( rather than parental reported ) data on weight and height.however , a number of limitations have to be considered .
thirteen percent of our data were missing and 53% of the study population had a missing value on 1 or more of the variables used in the analyses .
we used multiple imputation to deal with missing data in our study in order to avoid bias due to selective missing of data and to make more efficient use of the available data .
results of the complete case analysis and the analyses in the imputed data sets were similar .
information on the behavioral risk factors was obtained from parental completed questionnaires and we therefore need to address the possibility of misreporting .
the questions asked ranged from simple matter - of - fact items ( like sports club membership yes / no ) to items that are more difficult to observe and recall accurately ( like the consumption frequency of a range of different food items ) . we expect that recalling or reporting problems will not have substantially influenced the data on sports club membership or transport to school and that any misreporting on these items will not have been systematically associated with overweight development .
the data on foods and drinks consumption may well have been influenced by reporting bias .
many of the foods included in the dietary variables have a bad reputation in relation to overweight , and selective underreporting by parents of children who were developing overweight may have occurred .
another problem with respect to the diet variables is that data were available on the consumption frequencies of food items , but not on portion sizes .
estimates of the intakes of fast food , snacks and soft drinks and the ranking of children according to their intakes were therefore based on reported consumption frequencies only , and the possibility of misclassification of food intake can not be excluded .
membership of a sports club and active transportation to school were not associated with overweight in this population .
the reason that we did not observe the hypothesized associations might be that in young children the duration and intensity of these activities are usually too low to have a sizeable impact on total activity .
if this would be confirmed in further studies , it may have potentially important implications for the allocation of budgets for the prevention of overweight in this age group . in older children and adolescents active transport to school and sports club membership
we found some evidence for an inverse relation between playing outside and overweight , but the associations were weak and not statistically significant .
children who played outside 1 x per week seemed to have a higher overweight risk , but such children were a small minority in the study population .
screen time was consistently and significantly associated with overweight and the association was largely independent of other lifestyle factors .
a dose - response relationship was observed when it was included as a categorical variable in the regression analysis .
our results thus suggest that , among the indicators of active and sedentary behavior that we studied , screen time is the most important risk factor for overweight in the age group studied .
this result is in agreement with the conclusion of a recent study in which the evidence was summarized for six different strategies to prevent or treat pediatric overweight .
our findings on screen time and overweight risk are also in line with the results of a comprehensive meta - analysis on the subject , which observed a statistically significant relationship between tv viewing and body fatness among children and youth .
we estimated that a reduction in overweight prevalence of up to 2 percentage points could be achieved if all children would reduce their screen time to less than 1 hour per day .
maximum achievable reduction of prevalence ( marp ) of 1.5 percentage points that was estimated for watching television > 1 hr / day in a study on 5 - 6 year old german children .
consumption of soft drinks and fast food at ages 57 was not associated with overweight whereas an unexpected inverse association between snack consumption and overweight was observed .
snack consumption at the age of 5 years was not associated with overweight at age 8 , but snack consumption at age 7 was inversely associated with overweight at age 8 . as indicated previously , these results may have been influenced by selective underreporting of snack consumption by parents of children who were becoming overweight . besides selective underreporting , reverse causation may also have played a role , in the sense that parents of children who were becoming overweight really have reduced their children 's consumption of specific foods and drinks .
the results on fast food , snack and soft drink intakes seem to indicate that in particular the items included in our snack , variable are the kind of foods that come to parents ' minds when they think of trying to limit excessive weight gain in their child .
the selective underreporting and reverse causation that are likely to be present in our dietary data , make it impossible to judge from our results how diet influences the development of overweight .
we hypothesize that these mechanisms might play a role also in other epidemiological studies on diet and overweight . whereas , on theoretical grounds , it is hard to imagine that food intake is unrelated to weight status , observational studies have generally not produced consistent evidence for the expected associations [ 20 , 2327 ] .
our results do not provide an answer to the question whether consumption of soft drinks , fast food , and snacks causes overweight but seem to indicate that parents may see reduction of snack consumption as a method to counteract the development of overweight .
further studies into the strategies that parents themselves use to influence their children 's weight gain might provide insights that could be useful for the design of preventive strategies .
low maternal education and maternal overweight were found to be strongly associated with overweight in the child .
the association between low maternal education and overweight weakened substantially when the behavioral risk factors were included in the model , indicating that these factors explain part of the association .
the behavioral risk factors had little influence on the association between maternal overweight and overweight in the child , indicating that other factors , possibly genetic factors , play a more important role in this association .
we repeated the regression analyses in sub groups stratified by maternal weight status and maternal education . whereas in the high risk sub groups , screen time was higher than in low risk sub groups , the associations between screen time and overweight were weaker ( although not statistically significantly ) in these groups , and the population attributable risk percentage was lower than in the low risk sub groups . as the children of mothers with overweight and/or low education are the children at highest risk to develop overweight , this observation is potentially important for the development of preventive strategies and needs to be clarified in future observational as well as intervention studies .
reduction of screen time should be part of interventions to prevent or reduce overweight in young children and could result in a reduction of overweight prevalence in the order of 2 percentage points in both high and low risk sub groups .
our results also suggest however that interventions aimed to promote sports club membership , active transport to school , and playing outside may have little impact on the prevalence of overweight in this age group .
the possibility that , due to reverse causation , associations between food intake and overweight can not be assessed in observational studies , needs further study .
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objective . to prospectively identify behavioral risk factors for childhood overweight and to assess their relevance in high risk sub groups ( children of mothers with overweight or low education )
.
methods . in the piama birth cohort ( n = 3963 ) ,
questionnaire data were obtained at ages 5 and 7 on screen time , walking or cycling to school , playing outside , sports club membership , fast food consumption , snack consumption and soft drink consumption .
weight and height were measured at age 8 years .
results .
screen time , but none of the other hypothesized behavioral factors , was associated with overweight ( aor 1.4 ( ci : 1.21.6 ) ) . the adjusted population attributable risk fraction for screen time > 1 hr / day was 10% in the high risk and 17% in the low risk sub groups .
conclusion .
reduction of screen time to < 1 hr / day could result in a reduction of overweight prevalence in the order of 2 percentage points in both high and low risks sub groups .
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1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusions
|
the protective effects of vagus nerve during ischemia / reperfusion ( i / r ) have been suggested ( 1 ) .
vagus nerve stimulation decreases the inflammation and the injuries resulted from i / r through affecting neutrophils while vagotomy increases these processes ( 1 ) .
moreover , it has been observed that stimulation of vagus nerve can cause protective effects on gastric injury through releasing prostaglandins , nitric oxide ( no ) and calcitonin gene related peptide ( cgrp ) ( 2 ) .
it is probable that vagus nerve exerts its gastric protective effects through releasing the aforementioned mediators .
also , it has been reported that cholinergic activity of the efferent vagus nerve can participate in immunity modulation ( 3 ) .
another study has shown that nicotine in monocytes not only decreases the production of pre - inflammatory cytokines , but also changes the response to il-10 , an anti - inflammatory cytokine ( 4 , 5 ) .
cho et al have shown that alternative stimulation of cervical vagus nerve increases intra - gastric pressure and also causes bleeding ulcers in mucosal glands of stomach , and these effects can be prevented by atropine administration or sub - diaphragmatic vagotomy ( 6 ) .
it has been reported that gastric vagotomy is effective in treating gastric and duodenal ulcers ( 7 ) .
melatonin as a neurohormone is basically produced in the pineal gland and also in epiphysis , gastrointestinal ( gi ) mucosa and other organs .
melatonin has several physiological activities such as controlling circadian rhythm , sleep induction , regulation of seasonal reproduction , and improvement of immunity .
the most important effect of melatonin is an anti - oxidant effect that protects living organisms against oxidative stress ( 11 ) . in several studies ,
the ability of melatonin in decreasing molecular injury has been shown during i / r .
melatonin prevents myocardial infarction , necrotic cell death and renal abnormality after i / r ( 8 , 12 ) , reduces brain edema in ischemia ( 12 ) , and prevents acute gastric ulcers resulted from stress .
these effects can be done through direct reactive oxygen species / reactive nitrogen species ( ros / rns ) detoxification , increase in anti - oxidative enzymes , and the prevention of electron leakage in mitochondrial inner membrane ( 11 , 13 ) .
melatonin has a role in expression and function of nicotinic acetylcholine receptors and increases the efficacy of beta bungarotoxin - sensitive acetylcholine receptors ( 14 ) .
it has been recognized that intra - cerebral injection of melatonin is effective in prevention of acid and pepsin secretion through cholinergic activity ( 15 ) .
it has been identified that 5ht3 and 5ht2 receptors are involved in stimulatory effects of melatonin in releasing pancreatic enzymes ( 11 ) .
protective effects of melatonin are not only due to antioxidant activity , but it also activates capsaicin - sensitive afferent fibers ( 1 , 16 ) .
melatonin may have a potential impact on the treatment of peptic ulcer via significant increase in ghrelin expression ( 17 ) .
melatonin increases the release of pancreatic amylase and proteins through vagus nerve ( 18 ) .
the effects of melatonin on pancreatic enzymes are reversed by vagotomy and administration of capsaicin ( 18 , 19 ) .
intra - lumen administration of melatonin increases plasma cholecystokinin ( cck ) and antioxidants ( 11 , 19 , 20 ) .
it has been shown that sensory nerve fibers are involved in protective effects of melatonin in the healing of acute gastric injury and peptic ulcer ( 16 ) .
it has been reported that vagus nerve increases the secretion of bicarbonate from duodenum mucosa through an increase in melatonin secretion ( 21 ) .
vagal pathways are the most important regulatory pathways in the gastrointestinal tract which is one of the most important sources of melatonin production . in previous studies ,
both protective and harmful effects of the stimulation of vagus nerve in digestive system have been reported .
the role of sensory neurons in protective effects of melatonin in the healing of acute gastric injury and peptic ulcer has been reported .
there is no study about the combined effects of melatonin administration and intervention of vagus nerve during gastric i / r injury .
probably , vagus nerve and melatonin have interactions in their protective effects during gastric i / r events
so , the present study was performed to examine this interaction during gastric i / r .
this study was performed in 42 male wistar rats weighing 180 - 220 g. animals were kept at room temperature 20 - 22 c and 12 hr-12 hr light - dark cycle .
animals had free access to water and food and were randomly divided into 6 groups of 7 rats .
rats were fasted for 12 hr prior to the experiment but had access to water .
experimental groups were : 1 ) base+i / r+vehicle ; 2 ) base+i / r+melatonin ; 3 ) vagotomy+i / r+vehicle ; 4 ) vagotomy+i / r+melatonin ; 5)vagus nerve stimulation+i / r+vehicle ; 6)vagus nerve stimulation+i / r+ melatonin . base , means condition that neither vagus nerve was stimulated nor vagotomy was performed .
animals underwent tracheostomy and were cannulated ( in order to prevent probable airway occultation ) under anesthesia induced by intraperitoneal pentobarbital ( 50 mg / kg ) . after local shaving , a small incision was made in cervical area and branches of cervical vagus nerve were carefully dissected from carotid artery and cut ( 1 ) .
after dissection of vagus nerve , distal end was covered with mineral oil and stimulated with bipolar electrode of a stimulator using 10 volt / msec pulses at the frequencies of 0.625 , 1.25 , 2.5 , 5 or 7.5hz for 30 sec .
stimulations were performed with 2-min intervals ( 23 ) . in order to ensure the stimulation of vagus nerve , electrocardiogram
gastric i / r injury was induced by dissecting celiac artery from the surrounding tissues in rats anesthetized with 50 mg / kg pentobarbital , 30 minutes after dissection of vagus nerve .
then , celiac artery was closed with a microvascular clamp and returned to its place and the area was sutured using 4 - 0 silk .
occlusion was continued for 30 min ( 18 ) followed by a 3-h circulation ( reperfusion ) .
melatonin ( 10 mg / kg ) was injected intraperitoneally before reperfusion in groups 5 , 6 and 7 ( 18 , 25 ) .
melatonin was dissolved in 1% ethanol , diluted with 0.9% saline , and injected at a final volume of 0.3 ml ( 18 ) .
one part was kept in 10% formalin solution for histopathological assessment and the rest was kept at -70 c for evaluation of oxidant and antioxidant factors ( 18 , 25 ) .
mda level was measured according to hiroshi ohkawa method via reaction with thiobarbituric acid and color formation .
optical absorbance was determined at 532 nm and mda level was reported as nmol / mg protein ( 26 ) .
glutathione peroxides activity was determined by the method described by valentine and paglia method ( 20 ) , and enzyme activity was reported as u / mg protein .
superoxide dismutase activity was determined using ransod kit ( randox , uk ) and enzyme activity was reported as u / mg protein ( 19 ) .
catalase activity was determined by the method described by mari and reported as u / mg protein ( 20 ) .
the h & e stained sections were examined under low power ( 40x ) to identify the areas of neutrophil aggregates within all tissue blocks .
the number of neutrophils was calculated in a semiquantitative manner using the mean value of 20 non - overlapping high power fields ( hpf ; magnification of 400x ; 0.08 mm ) by using a 40x objective and a square grid mounted in a 10x microscopic eyepiece .
comparisons among different groups were made by one - way and two - way anova followed by post hoc tukey s test .
animals underwent tracheostomy and were cannulated ( in order to prevent probable airway occultation ) under anesthesia induced by intraperitoneal pentobarbital ( 50 mg / kg ) .
after local shaving , a small incision was made in cervical area and branches of cervical vagus nerve were carefully dissected from carotid artery and cut ( 1 ) .
after dissection of vagus nerve , distal end was covered with mineral oil and stimulated with bipolar electrode of a stimulator using 10 volt / msec pulses at the frequencies of 0.625 , 1.25 , 2.5 , 5 or 7.5hz for 30 sec .
stimulations were performed with 2-min intervals ( 23 ) . in order to ensure the stimulation of vagus nerve , electrocardiogram
gastric i / r injury was induced by dissecting celiac artery from the surrounding tissues in rats anesthetized with 50 mg / kg pentobarbital , 30 minutes after dissection of vagus nerve .
then , celiac artery was closed with a microvascular clamp and returned to its place and the area was sutured using 4 - 0 silk .
occlusion was continued for 30 min ( 18 ) followed by a 3-h circulation ( reperfusion ) .
melatonin ( 10 mg / kg ) was injected intraperitoneally before reperfusion in groups 5 , 6 and 7 ( 18 , 25 ) .
melatonin was dissolved in 1% ethanol , diluted with 0.9% saline , and injected at a final volume of 0.3 ml ( 18 ) .
one part was kept in 10% formalin solution for histopathological assessment and the rest was kept at -70 c for evaluation of oxidant and antioxidant factors ( 18 , 25 ) .
mda level was measured according to hiroshi ohkawa method via reaction with thiobarbituric acid and color formation .
optical absorbance was determined at 532 nm and mda level was reported as nmol / mg protein ( 26 ) .
glutathione peroxides activity was determined by the method described by valentine and paglia method ( 20 ) , and enzyme activity was reported as u / mg protein .
superoxide dismutase activity was determined using ransod kit ( randox , uk ) and enzyme activity was reported as u / mg protein ( 19 ) .
catalase activity was determined by the method described by mari and reported as u / mg protein ( 20 ) .
the h & e stained sections were examined under low power ( 40x ) to identify the areas of neutrophil aggregates within all tissue blocks .
the number of neutrophils was calculated in a semiquantitative manner using the mean value of 20 non - overlapping high power fields ( hpf ; magnification of 400x ; 0.08 mm ) by using a 40x objective and a square grid mounted in a 10x microscopic eyepiece .
comparisons among different groups were made by one - way and two - way anova followed by post hoc tukey s test .
mean number of neutrophils in base+ i / r+ melatonin group ( 23.80.37 ) was lower than that in base+ i / r+ vehicle group ( 27.20.37 ) ( p<0.01 ) . also , this parameter in vagus stimulation + i / r+ vehicle group ( 450.32 ) was higher than that in base+ i / r+ vehicle and vagotomy+ i / r+ vehicle groups ( 27.20.36 ) ( p<0.001 ) .
/ r+melatonin group ( 20.6 0.4 ) in comparison to vagus stimulation+i / r+vehicle group ( p<0.001 ) .
/ r+ melatonin group as compared to base+i / r+ melatonin group ( p<0.01 ) .
a significant increase in neutrophils count was shown in vagotomy+i / r+melatonin group ( 260.32 ) as compared to base+i / r+melatonin group ( p<0.05 ) . the number of neutrophils in gastric tissue of the study groups ( n=7 ) .
# # # p<0.001 : vagus stimulation+i / r+veh group vs. vagotomy+i / r+veh group .
base : condition that neither vagus was stimulated nor vagotomy was performed ; i / r : ischemia / reperfusion ; mel : melatonin ; veh : vehicle mda level in the study groups is presented in figure 2 .
as it is seen , mda level in gastric tissue was lower in base+i / r+melatonin group ( 4.090.15 nmol / mg protein ) in comparison with base+i / r+vehicle group ( 5.530.11 nmol / mg protein ) ( p<0.05 ) .
moreover , a significant increase in mda level was shown in vagus stimulation+i/-r+vehicle group ( 7.310.25 nmol / mg protein ) in comparison with base+i / r+vehicle and vagotomy+i/- r+vehicle ( 5.570.14 nmol / mg protein ) groups ( p<0.01 ) .
/ r+vehicle group ( p<0.001 ) . also , mda level was lower in vagus stimulation+i
/ r+melatonin group ( 2.48 0.06 nmol / mg protein ) in comparison with base+i / r+melatonin group ( p<0.01 ) .
mda level was higher in vagotomy+i / r+melatonin group ( 5.75 0.63 nmol / mg protein ) in comparison with base+ i / r+melatonin group ( p<0.05 ) .
* p<0.05 : base+i / r+ mel group vs. base + i / r+ veh group . # # p<0.01 : vagus stimulation+i / r+veh group vs. vagotomy+i / r+veh group .
a p<0.01 : vagus stimulation+i / r+mel group vs. base + i / r+mel group .
base : condition that neither vagus was stimulated nor vagotomy was performed ; i / r : ischemia / reperfusion ; mel : melatonin ; veh : vehicle catalase activity in the study groups are shown in figure 3 .
catalase activity was increased in base+i / r+melatonin group ( 0.060.003 u / mg protein ) in comparison with base+i / r+vehicle group ( 0.040.003 u / mg protein ) ( p<0.001 ) .
a significant increase in catalase activity was shown in vagus stimulation+ i / r+vehicle group ( 0.07 0.004 u / mg protein ) in comparison with base+i / r+vehicle and vagotomy+i / r+vehicle groups ( 0.0420.002 u / mg protein ) ( p<0.001 ) .
/ r+melatonin group ( 0.070.004 u / mg protein ) in comparison with base+i / r+melatonin group ( p<0.05 ) .
catalase activity was lower in vagotomy+i / r+melatonin group ( 0.040.003 u / mg protein ) in comparison with base+i / r+melatonin group ( p<0.05 ) .
catalase activity ( u / mg protein ) in gastric tissue of the study groups ( n=7).***p<0.001 : base+i / r+mel group vs. base+i / r+veh group . #
base : condition that neither vagus was stimulated nor vagotomy was performed ; i / r : ischemia / reperfusion ; mel : melatonin ; veh : vehicle figure 4 shows glutathione peroxidase activity in the studied groups .
a significant increase in gpx activity was shown in gastric tissue of basal+i / r+melatonin group ( 10.310.37 ) ( p<0.001 ) in comparison with that in basal+i / r+vehicle group ( 4.420.57 ) .
/ r+vehicle group ( 7.120.31 ) as compared to that observed in basal+i / r+vehicle and vagotomy+i / r+vehicle groups ( 4.430.38 ) ( p<0.001 ) . also , a significant increase in gpx activity was indicated in vagus stimulated+i
this parameter was significantly lower in vagotomy+ i / r+melatonin group ( 4.610.4 ) as compared to that in basal+i / r+melatonin group ( p<0.01 ) .
level of gpx activity ( u / mg protein ) in gastric tissue of the study groups ( n=7 ) * * * p<0.001 : base+i / r+mel group vs. base+i / r+veh group . #
b p<0.001 : vagotomy + i / r+ mel group vs. base+i / r+mel group .
base : condition that neither vagus was stimulated nor vagotomy was performed ; i / r : ischemia / reperfusion ; mel : melatonin ; veh : vehicle sod activity ( u / mg protein ) in gastric tissue of studied groups has been shown in figure 5 .
as it is seen , this parameter in basal+i / r+melatonin group ( 0.310.004 ) was significantly higher ( p<0.05 ) than that in basal+i / r+vehicle group ( 0.230.016 ) . a significant decrease in sod activity
/ r+ vehicle group ( 0.310.004 ) in comparison with that in vagotomy+i / r+vehicle group ( 0.360.003 ) ( p<0.05 ) , but it was higher than that in basal+ i / r+vehicle group .
this parameter in vagotomy+i / r+melatonin group ( 0.170.04 ) was lower than that in basal+i/-r+melatonin group ( p<0.05 ) .
superoxide dismutase activity ( u / mg protein ) in gastric tissue of the study groups ( n=7 ) * p<0.05 : base+i / r+mel group vs. base+i / r+veh group .
base : condition that neither vagus was stimulated nor vagotomy was performed ; i / r : ischemia / reperfusion ; mel : melatonin ; veh : vehicle
mean number of neutrophils in base+ i / r+ melatonin group ( 23.80.37 ) was lower than that in base+ i / r+ vehicle group ( 27.20.37 ) ( p<0.01 ) . also , this parameter in vagus stimulation + i / r+ vehicle group ( 450.32 ) was higher than that in base+ i / r+ vehicle and vagotomy+ i / r+ vehicle groups ( 27.20.36 ) ( p<0.001 ) .
/ r+melatonin group ( 20.6 0.4 ) in comparison to vagus stimulation+i / r+vehicle group ( p<0.001 ) .
/ r+ melatonin group as compared to base+i / r+ melatonin group ( p<0.01 ) .
a significant increase in neutrophils count was shown in vagotomy+i / r+melatonin group ( 260.32 ) as compared to base+i / r+melatonin group ( p<0.05 ) . the number of neutrophils in gastric tissue of the study groups ( n=7 ) .
# # # p<0.001 : vagus stimulation+i / r+veh group vs. vagotomy+i / r+veh group .
base : condition that neither vagus was stimulated nor vagotomy was performed ; i / r : ischemia / reperfusion ; mel : melatonin ; veh : vehicle
as it is seen , mda level in gastric tissue was lower in base+i / r+melatonin group ( 4.090.15 nmol / mg protein ) in comparison with base+i / r+vehicle group ( 5.530.11 nmol / mg protein ) ( p<0.05 ) .
moreover , a significant increase in mda level was shown in vagus stimulation+i/-r+vehicle group ( 7.310.25 nmol / mg protein ) in comparison with base+i / r+vehicle and vagotomy+i/- r+vehicle ( 5.570.14 nmol / mg protein ) groups ( p<0.01 ) .
/ r+vehicle group ( p<0.001 ) . also , mda level was lower in vagus stimulation+i
/ r+melatonin group ( 2.48 0.06 nmol / mg protein ) in comparison with base+i / r+melatonin group ( p<0.01 ) .
mda level was higher in vagotomy+i / r+melatonin group ( 5.75 0.63 nmol / mg protein ) in comparison with base+ i / r+melatonin group ( p<0.05 ) .
* p<0.05 : base+i / r+ mel group vs. base + i / r+ veh group . #
a p<0.01 : vagus stimulation+i / r+mel group vs. base + i / r+mel group .
base : condition that neither vagus was stimulated nor vagotomy was performed ; i / r : ischemia / reperfusion ; mel : melatonin ; veh : vehicle
catalase activity was increased in base+i / r+melatonin group ( 0.060.003 u / mg protein ) in comparison with base+i / r+vehicle group ( 0.040.003 u / mg protein ) ( p<0.001 ) .
a significant increase in catalase activity was shown in vagus stimulation+ i / r+vehicle group ( 0.07 0.004 u / mg protein ) in comparison with base+i / r+vehicle and vagotomy+i / r+vehicle groups ( 0.0420.002 u / mg protein ) ( p<0.001 ) .
/ r+melatonin group ( 0.070.004 u / mg protein ) in comparison with base+i / r+melatonin group ( p<0.05 ) .
catalase activity was lower in vagotomy+i / r+melatonin group ( 0.040.003 u / mg protein ) in comparison with base+i / r+melatonin group ( p<0.05 ) .
catalase activity ( u / mg protein ) in gastric tissue of the study groups ( n=7).***p<0.001 : base+i / r+mel group vs. base+i / r+veh group .
# # # p<0.001 : vagus stimulation+i / r+veh group vs. vagotomy+i / r+veh group .
base : condition that neither vagus was stimulated nor vagotomy was performed ; i / r : ischemia / reperfusion ; mel : melatonin ; veh : vehicle
a significant increase in gpx activity was shown in gastric tissue of basal+i / r+melatonin group ( 10.310.37 ) ( p<0.001 ) in comparison with that in basal+i / r+vehicle group ( 4.420.57 ) .
/ r+vehicle group ( 7.120.31 ) as compared to that observed in basal+i / r+vehicle and vagotomy+i / r+vehicle groups ( 4.430.38 ) ( p<0.001 ) . also , a significant increase in gpx activity was indicated in vagus stimulated+i
this parameter was significantly lower in vagotomy+ i / r+melatonin group ( 4.610.4 ) as compared to that in basal+i / r+melatonin group ( p<0.01 ) .
level of gpx activity ( u / mg protein ) in gastric tissue of the study groups ( n=7 ) * * * p<0.001 : base+i / r+mel group vs. base+i / r+veh group . # # p<0.01 : vagus stimulation+i / r+veh group vs. vagotomy+i / r+veh group .
b p<0.001 : vagotomy + i / r+ mel group vs. base+i / r+mel group .
base : condition that neither vagus was stimulated nor vagotomy was performed ; i / r : ischemia / reperfusion ; mel : melatonin ; veh : vehicle
sod activity ( u / mg protein ) in gastric tissue of studied groups has been shown in figure 5 .
as it is seen , this parameter in basal+i / r+melatonin group ( 0.310.004 ) was significantly higher ( p<0.05 ) than that in basal+i / r+vehicle group ( 0.230.016 ) . a significant decrease in sod activity
/ r+ vehicle group ( 0.310.004 ) in comparison with that in vagotomy+i / r+vehicle group ( 0.360.003 ) ( p<0.05 ) , but it was higher than that in basal+ i / r+vehicle group .
this parameter in vagotomy+i / r+melatonin group ( 0.170.04 ) was lower than that in basal+i/-r+melatonin group ( p<0.05 ) .
superoxide dismutase activity ( u / mg protein ) in gastric tissue of the study groups ( n=7 ) * p<0.05 : base+i / r+mel group vs. base+i / r+veh group . # # p<0.01 : vagus stimulation+i / r+veh group vs. vagotomy+i / r+veh group .
base : condition that neither vagus was stimulated nor vagotomy was performed ; i / r : ischemia / reperfusion ; mel : melatonin ; veh : vehicle
the present study was performed to investigate the protective effect of interaction between melatonin and vagus nerve during gastric i / r .
the results of this study showed that melatonin administration decreased neutrophils infiltration and mda level and increased sod , cat and gpx activities in gastric tissue . also , vagus stimulation increased neutrophils infiltration and mda level in gastric tissue while melatonin administration along with vagus stimulation decreased the effect of vagus stimulation in gastric tissue .
finally , vagotomy prevented some of the protective effects of melatonin during gastric i / r . according to the results of this study
, melatonin can reduce gastritis during i / r , probably through decreasing the activity of oxidant enzymes and increasing the activity of anti - oxidant enzymes such as sod , cat and gpx .
it has also been demonstrated that melatonin decreases mad level ( 27 - 29 ) .
other probable mechanisms underlying the protective effect of melatonin include an increase in gastric microcirculation ( 1 ) , a reduction in acid and pepsin secretions ( 15 ) , a reduction in production of inflammatory cytokines ( 16 ) and an increase in the expression of anti - oxidant gene ( 17 , 30 - 32 ) .
moreover , in our study , a stimulation of vagus nerve increased gastritis which was associated with an increase in mda level while vagotomy after i / r was ineffective .
it is probable that the stimulation of vagus nerve following gastric i / r aggravated the condition and increased the activity of oxidant enzymes .
since vagotomy in basal condition did not have any effect , it seems that the control of gastritis and the activity of oxidant and anti - oxidant enzymes are not under the control of vagus nerve in basal conditions .
it has been demonstrated that the stimulation of vagus nerve increases output of gastric juice and acid ( 33 ) , and decreases the activity of anti - oxidant enzymes like sod ( 33 ) . on the other hand , vagotomy showed healing effects on duodenal ulcers ( 7 ) similar to the effect of atropine ( 34 ) . in a study ,
diaphragmatic vagotomy did not change the activity of catalase in the intestine ( 35 ) which was similar to the result of the present study .
the results of some studies are not consistent with our data . for example , it has been identified that vagal afferent fibers decrease gastrointestinal tract inflammation through nicotinic receptors leading to a decrease in inflammatory cytokines ( 16 ) .
cholinergic agonists such as neostigmine decrease superoxide anion and inflammation in i / r ( 36 ) .
elevation of acetylcholine transferase activity and decrease in acetylcholine esterase inhibitors increase gpx ( 35 , 37 ) .
treatment with acetylcholine esterase inhibitors increases catalase half - life ( 38 ) . in the present study , melatonin administration affected the outcomes in i / r+vagus stimulation group .
melatonin decreased gastritis and mda level , and increased sod and gpx activity , but not in vagotomy group .
these findings show that protective effects of melatonin are probably mediated through vagus nerve . also , interaction between melatonin and vagus stimulation is suggested since these show a synergic effect in relation with anti - oxidant factors . regarding this probable interaction ,
it is suggested that melatonin exerts its gastric protective effect through stimulation of gi tract neurons leading to cgrp release ( 1 ) .
intra - cerebral injection of melatonin inhibits acid and pepsin secretions through vagus cholinergic activity ( 15 ) .
melatonin increases the release of amylase and pancreas proteins through vagus nerve ( 16 ) .
effect of melatonin administration accompanied with vagotomy has not suggested in the release of amylase(18 ) .
studies have shown that intra - lumen administration of melatonin or its precursor induces the release of pancreatic enzymes , while this effect is not shown in isolated pancreas . moreover ,
these effects of melatonin are reversed by vagotomy and capsaicin administration ( 11 , 18 , 19 ) .
it has been reported that melatonin effect is mediated indirectly through the release of cck followed by vagovagal reflex ( 11 , 19 , 39 ) .
it has been shown that intra - lumen administration of melatonin increases plasma cck level ( 11 , 19 ) and cck acts through stimulation of vagal afferent fibers in gi tract ( 40 , 41 ) .
it has been reported that vagotomy in combination with melatonin administration inhibits the release of pancreas proteins ( 7 , 11 , 16 ) .
it has been reported that melatonin increases the expression and activity of ach receptors , and synergistic effects are induced during vagus stimulation ( 42 ) . according to a study , injection of phenylephrine causes melatonin release from mucosa enterochromaffin cells in the presence of normal vagus nerve and sympathetic paths ( 39 ) .
melatonin increases the release of bicarbonate from gastric mucosa ( 39 ) and decreases the release of inflammatory cytokines of gi through increasing vagus activity ( 16 ) .
it is suggested that melatonin is neuroprotective in gastric i / r probably by decreasing gastritis and mda and increasing the activities of cat , sod and gpx .
these effects of melatonin are probably mediated by vagus nerve . moreover , in gastric i / r , melatonin can reverse harmful effects of vagus stimulation .
it is suggested that further studies are required to find the mechanisms involved in this interaction .
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objectives : vagal pathways in gastrointestinal tract are the most important pathways that regulate ischemia / reperfusion ( i / r ) .
gastrointestinal tract is one of the important sources of melatonin production .
the aim of this study was to investigate probable protective effect of the interaction between vagus nerve and melatonin after i / r.materials and methods : this study was performed in male rats that were divided into six groups .
cervical vagus nerve was cut bilaterally after induction of i / r and the right one was stimulated by stimulator .
melatonin or vehicle was injected intraperitoneally .
the stomach was removed for histopathological and biochemical investigations.results:a significant decrease in infiltration of gastric neutrophils and malondialdehyde ( mda ) level after i / r was induced by melatonin and was disappeared after vagotomy .
the stimulation of vagus nerve significantly enhanced these effects of melatonin .
however , a stimulation of vagus nerve alone increased neutrophils infiltration and mda level .
melatonin significantly increased the activities of catalase , glutathione peroxidase ( gpx ) , superoxide dismutases ( sod ) .
unlike stimulation of vagus nerve , vagotomy decreased these effects of melatonin.conclusion:according to these results , it is probable that protective effects of melatonin after i / r may be mediated by vagus nerve . therefore , there is an interaction between melatonin and vagus nerve in their protective effects .
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Introduction
Materials and Methods
Vagus nerve stimulation
Ischemia/reperfusion
Measuring malondialdehyde (MDA) level, and glutathione peroxides (GPX), catalase and superoxide dismutase (SOD) activities
Infiltration of neutrophils
Statistical analysis
Results
Infiltration of neutrophils
MDA level
Catalase activity
Glutathione peroxidase (GPX) activity
Superoxide dismutase (SOD) activity
Discussion
Conclusion
Conflict interest
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a stratified , multistage probability cluster sampling design was used in this survey ( 13 ) . based on socioeconomic characteristics
, in the first stage of sampling , 22 counties were randomly selected from each of the 6 regions in china . in the second stage ,
three townships were randomly selected from each of the selected counties . from each of the townships ,
2 residential villages were randomly selected ; and 90 households were then randomly sampled from each village for physical examination .
one - third of the households were selected to participate in the dietary survey and blood draw .
for the present study , we used residents who were living in rural areas and were born between october 1 , 1952 , and september 30 , 1964 , as our analytic population . to minimize misclassification of the exposure periods , subjects who were born between october 1 , 1958 , and
september 30 , 1959 , and between october 1 , 1961 , and september 30 , 1962 , were excluded since the exact dates of the start and the end of the chinese famine were not available and not the same across regions .
flow chart on the sampling method in each region * of the 2002 china national nutrition and health survey . *
the mainland of china is classified into 6 regions defined by the chinese bureau of statistics according to their socioeconomic development .
they are metropolis , general city , type i rural site , type ii rural site , type iii rural site , and type iv rural site .
subjects were categorized into five exposure cohorts : nonexposed cohort , fetal - exposed cohort , early childhood exposed cohort , mid childhood exposed cohort , and late childhood exposed cohort .
subjects who were born between october 1 , 1962 , and september 30 , 1964 , were classified as the nonexposed cohort ; and subjects who were born between october 1 , 1959 , and september 30 , 1961 , were classified as fetal - exposed cohort .
subjects who were born between october 1 , 1952 , and september 30 , 1958 , were grouped by every 2 years and were classified into one of the three childhood - exposed cohorts .
mean ages for subjects in nonexposed cohort , fetal - exposed cohort , early childhood exposed cohort , mid childhood exposed cohort , and late childhood
exposed cohort were 39 , 42 , 45 , 47 , and 49 years , respectively .
the chinese famine affected the entire mainland of china , but the severity varied across regions due to different weather conditions , population density , and local policies regarding food shortage ( 7 ) . as previously described , we used the excess death rate of each province to determine the severity of the famine ( 7 ) .
the excess death rate was calculated as the percentage change in mortality rate from the mean level in 19561958 to the highest value during the period 19591961 ( 7 ) .
an excess death rate of 50% was used as the threshold : regions that had an equal or higher rate than this cutoff were categorized as severely affected famine areas , and otherwise as less severely affected famine areas .
we split all five cohorts into severely affected famine areas and less severely affected famine areas .
this enabled us to test the hypothesis that the famine effect is stronger in the severely affected famine areas than that in the less severely affected famine areas and to consider both birth cohort effects and regional differences .
all subjects were invited for blood collection after an 10 to 14 h overnight fast .
the plasma was separated by centrifugation at 3,200 rpm for 1015 min within 1 h of collection , and kept at room temperature without sunshine .
fasting plasma glucose ( fpg ) concentration was measured using the glucose oxidize enzymatic method within 3 h of plasma preparation .
every tenth sample was measured twice ( the correlation coefficient of duplicate measurements was 0.98 ) .
we used criteria proposed by the who expert committee on diabetes mellitus ( 14 ) .
mmol / l , including impaired fasting glucose , impaired glucose tolerance , and type 2 diabetes .
in addition , subjects who had been previously diagnosed with type 2 diabetes were added as cases of hyperglycemia and type 2 diabetes .
dietary patterns , economic status , and bmi measured in 2002 were used as measures of the nutritional environment in adulthood and to examine the mismatch between fetal nutrition and adult nutrition .
the method for assessing dietary patterns has been described in detail elsewhere ( 15 ) .
briefly , four dietary patterns were derived through cluster analysis , which were labeled as green water , yellow earth , new affluence , and western adopter . green water and yellow earth
patterns represent the traditional chinese diets in the south and the north , respectively , whereas the other two represent westernized dietary patterns . in this study
, we combined the clusters of green water and yellow earth as the traditional dietary pattern , and we combined the clusters of new affluence and western adopter as the affluent / western pattern .
current economic status was assessed by the mean annual income in the year prior to the 2002 cnnhs , which was treated as a dichotomous variable .
the mean level of the current sample ( 2,000 chinese yuan per person per year ) was used as a cutoff point for economic status .
we used the criteria recommended for chinese adults and classified subjects as overweight if bmi 24 kg / m , or otherwise normal ( 16 ) .
the protocol of the 2002 cnnhs was approved by the ethical committee of the national institute for nutrition and food safety , chinese center for disease control and prevention . signed consent forms were obtained from all participants .
we performed survey analyses with sas 9.2 for windows ( sas institute , cary , nc ) to estimate statistics for this complex , multistage - designed survey sample .
survey weights were derived from the 2000 china national population census and associated administrative data .
mean fpg differences between the exposed cohorts and the nonexposed cohort were tested by generalized least squares estimation ( 17 ) .
risks of hyperglycemia and type 2 diabetes among fetal and childhood - exposed subjects , compared with nonexposed subject , were examined with the method of maximum likelihood by using the survey logistic regression model . interaction between famine exposure cohort ( fetal- or childhood - exposed vs. nonexposed ) and area ( severely affected and less severely affected ) was tested by adding a multiplicative factor in the survey logistic regression model .
analyses were adjusted for sex , family history of diabetes , educational level , current smoking , alcohol use , and physical activity level , all assessed in 2002 . to explore whether the associations between fetal exposure to severe famine and hyperglycemia were affected by an improved nutritional environment in later life , we subsequently stratified the analyses by dietary patterns , economic status and bmi in adulthood .
the odds ratio of hyperglycemia in the fetal - exposed cohort compared with the nonexposed cohort was calculated within each category of the stratified factor . to distinguish severely and less severely affected famine areas more appropriately , we performed sensitivity analysis by using a more stringent cutoff point , i.e. , we used an excess death rate 100% to define the severity of famine .
in addition , we performed analyses by using the cohort born during october 1 , 1962 , to september 30 , 1968 , as a nonexposed cohort for association analyses , or by excluding participants with a family history of diabetes .
subjects were categorized into five exposure cohorts : nonexposed cohort , fetal - exposed cohort , early childhood exposed cohort , mid childhood exposed cohort , and late childhood exposed cohort .
subjects who were born between october 1 , 1962 , and september 30 , 1964 , were classified as the nonexposed cohort ; and subjects who were born between october 1 , 1959 , and september 30 , 1961 , were classified as fetal - exposed cohort .
subjects who were born between october 1 , 1952 , and september 30 , 1958 , were grouped by every 2 years and were classified into one of the three childhood - exposed cohorts .
mean ages for subjects in nonexposed cohort , fetal - exposed cohort , early childhood exposed cohort , mid childhood exposed cohort , and late childhood exposed cohort were 39 , 42 , 45 , 47 , and 49 years , respectively .
the chinese famine affected the entire mainland of china , but the severity varied across regions due to different weather conditions , population density , and local policies regarding food shortage ( 7 ) . as previously described , we used the excess death rate of each province to determine the severity of the famine ( 7 ) .
the excess death rate was calculated as the percentage change in mortality rate from the mean level in 19561958 to the highest value during the period 19591961 ( 7 )
. an excess death rate of 50% was used as the threshold : regions that had an equal or higher rate than this cutoff were categorized as severely affected famine areas , and otherwise as less severely affected famine areas .
we split all five cohorts into severely affected famine areas and less severely affected famine areas .
this enabled us to test the hypothesis that the famine effect is stronger in the severely affected famine areas than that in the less severely affected famine areas and to consider both birth cohort effects and regional differences .
all subjects were invited for blood collection after an 10 to 14 h overnight fast .
the plasma was separated by centrifugation at 3,200 rpm for 1015 min within 1 h of collection , and kept at room temperature without sunshine .
fasting plasma glucose ( fpg ) concentration was measured using the glucose oxidize enzymatic method within 3 h of plasma preparation .
every tenth sample was measured twice ( the correlation coefficient of duplicate measurements was 0.98 ) .
we used criteria proposed by the who expert committee on diabetes mellitus ( 14 ) .
mmol / l , including impaired fasting glucose , impaired glucose tolerance , and type 2 diabetes .
in addition , subjects who had been previously diagnosed with type 2 diabetes were added as cases of hyperglycemia and type 2 diabetes .
dietary patterns , economic status , and bmi measured in 2002 were used as measures of the nutritional environment in adulthood and to examine the mismatch between fetal nutrition and adult nutrition .
the method for assessing dietary patterns has been described in detail elsewhere ( 15 ) .
briefly , four dietary patterns were derived through cluster analysis , which were labeled as green water , yellow earth , new affluence , and western adopter . green water and yellow earth
patterns represent the traditional chinese diets in the south and the north , respectively , whereas the other two represent westernized dietary patterns . in this study , we combined the clusters of green water and yellow earth as the traditional dietary pattern , and we combined the clusters of new affluence and western adopter as the affluent / western pattern .
current economic status was assessed by the mean annual income in the year prior to the 2002 cnnhs , which was treated as a dichotomous variable .
the mean level of the current sample ( 2,000 chinese yuan per person per year ) was used as a cutoff point for economic status .
we used the criteria recommended for chinese adults and classified subjects as overweight if bmi 24 kg / m , or otherwise normal ( 16 ) .
the protocol of the 2002 cnnhs was approved by the ethical committee of the national institute for nutrition and food safety , chinese center for disease control and prevention . signed consent forms were obtained from all participants .
we performed survey analyses with sas 9.2 for windows ( sas institute , cary , nc ) to estimate statistics for this complex , multistage - designed survey sample .
survey weights were derived from the 2000 china national population census and associated administrative data .
mean fpg differences between the exposed cohorts and the nonexposed cohort were tested by generalized least squares estimation ( 17 ) .
risks of hyperglycemia and type 2 diabetes among fetal and childhood - exposed subjects , compared with nonexposed subject , were examined with the method of maximum likelihood by using the survey logistic regression model .
interaction between famine exposure cohort ( fetal- or childhood - exposed vs. nonexposed ) and area ( severely affected and less severely affected ) was tested by adding a multiplicative factor in the survey logistic regression model .
analyses were adjusted for sex , family history of diabetes , educational level , current smoking , alcohol use , and physical activity level , all assessed in 2002 . to explore whether the associations between fetal exposure to severe famine and hyperglycemia were affected by an improved nutritional environment in later life , we subsequently stratified the analyses by dietary patterns , economic status and bmi in adulthood .
the odds ratio of hyperglycemia in the fetal - exposed cohort compared with the nonexposed cohort was calculated within each category of the stratified factor . to distinguish severely and less severely affected famine areas more appropriately , we performed sensitivity analysis by using a more stringent cutoff point , i.e. , we used an excess death rate 100% to define the severity of famine .
in addition , we performed analyses by using the cohort born during october 1 , 1962 , to september 30 , 1968 , as a nonexposed cohort for association analyses , or by excluding participants with a family history of diabetes .
basic characteristics of the study population are shown in table 1 . in our main study population
( n = 7,874 ) , 1,005 ( 12.8% ) subjects had been exposed to the chinese famine during fetal life , and 4,915 ( 62.4% ) subjects had been exposed during childhood . as compared with the nonexposed individuals , fetal - exposed subjects were 0.9 cm shorter as adults , and childhood - exposed subjects were 1.5 cm shorter ( table 1 ) .
the prevalence of hyperglycemia among adults in the nonexposed , fetal - exposed , early childhood , mid childhood , and late childhood exposed birth cohorts was 2.4% , 5.7% , 3.9% , 3.4% , and 5.9% , respectively .
basic characteristics of study population according to chinese famine exposure * * data are adjusted means ( se ) .
adjusted factors included sex , educational level , family history of diabetes ( only for glucose ) , current smoking , alcohol use , and physical activity level .
compared with the nonexposed cohort , p < 0.05 . in severely affected famine areas ,
fpg concentration was significantly higher in the fetal - exposed cohort than in the nonexposed cohort with a mean difference of 0.20
no significant difference was observed in the less severely affected famine areas ( p for interaction = 0.001 , table 2 ) .
compared with nonexposed subjects , fpg was higher in the late childhood exposed cohort in both the severely affected famine areas and less severely affected famine areas .
a significant interaction between the exposed cohort and areas was found only for the fetal - exposed cohort ( table 2 ) .
concentrations of fasting plasma glucose and prevalence rates of hyperglycemia and type 2 diabetes by birth cohort and severity of the chinese famine area * data are adjusted means ( se ) for fasting plasma glucose , sex standard prevalence , and odds ratio for hyperglycemia and diabetes .
adjusted factors included sex , education level , family history of diabetes , and current smoking , alcohol use , and physical activity level .
subjects exposed to famine during fetal life in severely affected famine areas had a higher prevalence of hyperglycemia than the nonexposed cohort .
the odds ratios were significantly different between the severe and less severe famine areas ( table 2 ) , suggesting a stronger famine effect in the severely affected famine areas . compared with the nonexposed cohort , subjects in the late childhood exposed cohort had a higher risk of hyperglycemia in both severely and less severely affected famine areas , but the odds ratios were not significantly different between the severe and less severe famine areas ( table 2 ) .
a significantly higher prevalence of type 2 diabetes was observed among subjects exposed in late childhood as compared with the nonexposed cohort ( table 1 ) .
however , table 2 shows that after stratification of this group by severity of famine exposure , no significant difference of type 2 diabetes risk was observed anymore between different famine cohorts .
stratified analyses by dietary pattern , economic status , and bmi for severely affected famine areas are shown in fig .
figure 2a1 shows that the prevalence of hyperglycemia was highest ( 18.9% ) in subjects in the fetal - exposed cohort and who consumed an affluent / western diet .
as compared with the relatively nonexposed cohort , the odds ratio of hyperglycemia in the fetal - exposed cohort was 7.63 ( 95% ci : 2.4124.1 , p = 0.0005 ) for those who had an affluent / western dietary pattern , and 2.34 ( 95% ci : 0.826.70 , p = 0.112 ) for those with a traditional dietary pattern .
prevalence of hyperglycemia among birth cohorts according to early life famine exposure and later life dietary patterns ( a1 and b1 ) , socioeconomic status ( a2 and b2 ) , and bmi ( a3 and b3 ) in severely ( column a ) and less severely affected famine areas ( column b ) .
figure 2a2 shows that as compared with nonexposed subjects , the odds ratio of hyperglycemia in the fetal - exposed cohort was 6.20 ( 95% ci : 2.0818.5 , p = 0.001 ) in subjects with a higher adult economic status , and 1.68 ( 95% ci : 0.505.71 , p = 0.404 ) in subjects with a lower adult economic status .
figure 2a3 shows that overweight subjects in the fetal - exposed cohort had the highest prevalence of hyperglycemia ( 13.9% ) .
however , the risks of hyperglycemia were largely comparable in these two groups ; the odds ratio of hyperglycemia in the fetal - exposed cohort was 3.71 ( 95% ci : 1.1312.2 , p = 0.031 ) in overweight subjects and 4.37 ( 95% ci : 1.1516.5 , p = 0.030 ) in normal weight subjects , respectively , compared with the nonexposed cohort .
similar analyses were performed in subjects exposed to less severely affected famine areas during fetal life and childhood ( fig .
2 , right column , graphs b1 , b2 , and b3 ) , but did not show consistent associations . when we defined the severely affected famine areas as those with an excess death rate 100% , the prevalence of hyperglycemia among the fetal - exposed cohort in severely affected famine areas increased to 8.1% , but this did not change the associations between fetal exposure to famine and risk of hyperglycemia in adulthood .
in addition , neither using subjects who were born between october 1 , 1962 , and september 30 , 1968 , as a nonexposed cohort nor excluding subjects with a family history of diabetes materially changed the associations ( table 3 )
. prevalence rate of hyperglycemia by birth cohorts and severity of famine areas : sensitivity analyses all odds ratios used the nonexposed cohort as the reference cohort . adjusted factors include sex , educational level , family history of diabetes , and current smoking , alcohol use , and physical activity level in 2002 .
in this study of a large sample of chinese adults , we found a significant association between severe famine exposure during the fetal period and an increased risk of hyperglycemia in adulthood .
this association was stronger in subjects with a western dietary pattern or higher economic status in adulthood .
several mechanisms might explain the associations between fetal famine exposure and risk of diabetes in later life .
exposure to extreme starvation in rats led to poor development of pancreatic -cell mass and function and insulin resistance , which might persist in later life ( 18 ) .
a poor intrauterine environment may also reduce skeletal muscle development ( 19 ) , which may subsequently lead to insulin resistance in peripheral tissues ( 20 ) .
it has also been suggested that stress suffering from fetal famine exposure could change the setpoint of the hypothalamic - pituitary - adrenal ( hpa ) axis , which could result in long - term changes in secretion of neuroendocrine mediators of the stress response , and predispose to cardiovascular and metabolic disease in later life ( 21,22 ) . to our knowledge ,
thus far three studies have assessed the associations of exposure to famine with measures of glucose intolerance .
these studies were performed in the netherlands ( the dutch famine study ) ( 4,5 ) , russia ( the leningrad siege study ) ( 6 ) , and china ( our chinese famine study ) .
the dutch famine study reported higher 2-h glucose and insulin levels among subjects who were exposed to famine during fetal life ( 4,5 ) , but this association was not observed in the leningrad siege study ( 6).the inconsistent results might be due to differences in postnatal environmental life exposures . although the dutch population rapidly developed into a wealthy and rich population after the famine , the leningrad people remained relatively poor . in our study , we observed that fetal exposure to the severe chinese famine increases the risk of hyperglycemia in adulthood , which was exacerbated by an unhealthy adult diet and higher economic status .
our results support the hypothesis that exposure to a nutritionally rich environment modifies the association between fetal famine exposure and disease in later life ( 1,20,23 ) .
the association between fetal famine exposure and hyperglycemia was stronger in participants with an affluent / western dietary pattern .
these subjects were , to a large extent , less poor and more highly educated ( 15 ) , and they have benefited most from dramatically enhanced economic opportunities and have broken away from traditional chinese food patterns ( 15 ) .
their diet is characterized by a high intake of meat , eggs , dairy , sugary beverages , edible oils , and a low vegetable use ( 15 ) . apparently , this nutrition
rich environment did not match the fetal starvation environment that people of fetal exposed cohort experienced , which in turn increased the risk of hyperglycemia in later life ( 1,20,23 ) .
our study used annual mean income as the cutoff to categorize economic status ( 2,000 chinese yuan / person / year ) .
subjects in the lower economic group might consume mostly traditional plant foods with little meat .
therefore , the discrepancy between the nutritional environment in adulthood and fetal undernutrition conditions may be less evident for those with a higher economic status . in other words
, there was probably greater mismatch between in utero and adulthood environments in the higher economic group , which triggered an increased prevalence of hyperglycemia in the fetal - exposed cohort .
similar results were described in the dutch famine study ( 4 ) , showing that 2-h glucose concentrations were especially high among people exposed to the famine during fetal life and who became obese in later life . however , the relative risk of hyperglycemia in overweight subjects was not different from that in normal weight subjects
. this may be partly due to the increased prevalence of hyperglycemia in the nonexposed cohort in overweight subset .
these results therefore indicate that both improving fetal nutritional environmental and controlling bmi in later life are important for prevention of a disturbed glucose metabolism .
childhood nutritional status , particularly during infancy , is another key factor in influencing the propensity to develop disease in adulthood ( 23 ) .
animal studies have shown that postnatal caloric restriction might hamper -cell development ( 24 ) and might disturb glucose metabolism in later life in rats ( 25 ) .
our study found significantly increased fpg in the early childhood exposed cohort in the severely affected famine areas , but no significant differences in fpg in the less severely affected famine areas .
we also observed a higher risk of hyperglycemia among subjects exposed in late childhood in both severely and less severely affected famine areas .
these results suggest that famine exposure during childhood may increase the risk of hyperglycemia in later life .
however , we can not exclude a potential cohort effect , such as aging ( 26 ) .
similar risks of hyperglycemia among subjects exposed during childhood in both famine - exposed areas and nonfamine - exposed areas suggest rather a cohort ( older age ) effect than a famine effect .
however , since almost all rural regions in china were affected by the famine during 19591961 , no valid nonfamine - exposed cohort comprising subjects born in the same time period was available .
thus , the association between childhood exposure to famine and risk of hyperglycemia needs to be studied in more detail .
first , we assumed that the residents we investigated at the time of the survey were born in the same province and in a similar rural area .
however , severe restrictions on migration and relocation in china made our sample quite stable .
migration with permanent resident permission still needed to be approved by authorities on a case - by - case basis in china .
according to the 2000 china national population census , 2.68% of the rural population lived in provinces other than the provinces of their birthplaces ( 27 ) .
our study sample was based on the residence registration system ; only subjects with permanent resident permission in local areas were involved in our study .
therefore , we do not expect that intraprovince migration leading to measurement error in the coding of birth place is a major concern in our results ( 12 ) .
second , subjects in our fetal - exposed cohort may have actually experienced severe famine during both the fetal period and the infancy period because the famine lasted approximately 3 years .
it was therefore difficult to distinguish whether the fetal period or the infancy period was more important .
however , the early childhood cohort also included subjects exposed to famine in infancy , which did not have a substantial influence on the risk of hyperglycemia .
thus , our results indicate that the fetal period should be considered as the primary critical period .
third , our subjects who experienced severe famine in the fetal period were in their early 40s in 2002 , and the cases of type 2 diabetes were few .
the small numbers may partly explain why we did not observed significant associations with the risk of type 2 diabetes .
we used the excess death rate as an indirect measure of famine exposure . with this method
, we could not distinguish death due to famine from death due to unfavorable weather conditions or infections .
we also did not have reliable information about individual food availability during the famine period .
therefore , from our data , we can not conclude that the higher risk of hyperglycemia among subjects exposed to famine is exclusively due to malnutrition in early life . however , nutrition deficiency was highly prevalent during the chinese famine .
china 's grain output declined by 15% in 1959 and in the following 2 years , and its food supply plunged further to 70% of its 1958 level ( 8) . as almost all foods were delivered through communal kitchens at that time , no social groups were spared from the effects of the famine ( 9 ) .
however , since the famine effect on glucose intolerance did not depend on birth size in the dutch famine study ( 4 ) , we do not consider the lack of information about individual birth outcomes as a major limitation . in conclusion
, we found that exposure to severe famine in fetal life increased the risk of hyperglycemia in adulthood .
the mismatched nutrition postnatal environment represented by a western dietary pattern and improved economic status further increased susceptibility to hyperglycemia in those who experienced fetal exposure to famine . together with previous studies
, our study emphasizes that early life environment is critical for the risk of hyperglycemia in adult life .
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objectiveearly developmental adaptations in response to undernutrition may play an essential role in susceptibility to type 2 diabetes , particularly for those experiencing a mismatched rich nutritional environment in later life .
we examined the associations of exposure to the chinese famine ( 19591961 ) during fetal life and childhood with the risk of hyperglycemia and type 2 diabetes in adulthood.research design and methodswe used the data for 7,874 rural adults born between 1954 and 1964 in selected communities from the cross - sectional 2002 china national nutrition and health survey .
hyperglycemia was defined as fasting plasma glucose 6.1 mmol / l and/or 2-h plasma glucose 7.8
mmol / l and/or a previous clinical diagnosis of type 2 diabetes.resultsprevalences of hyperglycemia among adults in nonexposed , fetal exposed , early - childhood , mid - childhood , and late - childhood exposed cohorts were 2.4% , 5.7% , 3.9% , 3.4% , and 5.9% , respectively . in severely affected famine areas ,
fetal - exposed subjects had an increased risk of hyperglycemia compared with nonexposed subjects ( odds ratio = 3.92 ; 95% ci : 1.649.39 ; p = 0.002 ) ; this difference was not observed in less severely affected famine areas ( odds ratio = 0.57 ; 95% ci : 0.251.31 ; p = 0.185 ) .
the odds ratios were significantly different between groups from the severe and less severe famine areas ( p for interaction = 0.001 ) . in severely affected famine areas , fetal - exposed subjects who followed an affluent / western dietary pattern ( odds ratios = 7.63 ; 95% ci : 2.4124.1 ; p = 0.0005 ) or who had a higher economic status in later life experienced a substantially elevated risk of hyperglycemia ( odds ratios = 6.20 ; 95% ci : 2.0818.5 ; p = 0.001).conclusionsfetal exposure to the severe chinese famine increases the risk of hyperglycemia in adulthood .
this association appears to be exacerbated by a nutritionally rich environment in later life .
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RESEARCH DESIGN AND METHODS
Famine cohorts and areas.
Assessments of blood glucose and type 2 diabetes.
Stratification factors.
Statistical analyses.
RESULTS
DISCUSSION
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the circadian system organizes the different biological functions in 24 h , such as sleep / activity , temperature , heart rate , glucose level , cortisol production , and oxidative stress . in mammals ,
this system is organized by a central clock localized in the suprachiasmatic nucleus of the hypothalamus ( scn ) and in a series of peripheral oscillators such as the liver , lung , adrenal gland , fibroblast cells , and others tissues [ 2 , 4 ] .
the peripheral oscillators are synchronized every day via nervous or humoral signals , and the most important humoral signal is the melatonin hormone , secreted by the pineal gland during the dark hours , and its impairment is associated with different disorders such as insomnia , cardiovascular disease , and cancer .
the molecular clock is organized by transcriptional / translational feedback loop of clock genes named clock , bmal1 , per13 , and cry1 - 2 . at the molecular level ,
the complex clock - bmal1 stimulates the expression of negative regulators per13 and cry1 - 2 , and their protein inhibits the effect of the heterodimer clock / bmal1 [ 6 , 7 ] .
moreover , the molecular clock has different modulators which give fine tuning of output signals such as rev - erb , a negative regulator of bmal-1 expression [ 79 ] , sirt1 , a regulator of clock - mediated acetylase activity [ 10 , 11 ] , and pgc1 , a stimulator of bmal1 expression [ 12 , 13 ] ( see figure 1 ) .
this system provides an output signal to genes such as hexokinase , dbp [ 1517 ] , vegf , steroidogenic enzymes star , 3-hsd , and wee-1 , giving a circadian oscillation of physiological functions such as metabolism , angiogenesis , cortisol production [ 2 , 19 ] , and cellular proliferation .
the molecular clock can be modified by environmental changes and our modern lifestyle , resulting in physiological alteration and risk of pathologies ; for example , during the monkey 's pregnancy , the light exposition during night hours induces a lower body temperature and absence of circadian rhythm of temperature in the newborn . in humans ,
different reports showed that the alterations of the circadian system increased the risks of cancer [ 2123 ] , preeclampsia , diabetes [ 3 , 25 ] , and mood disorder .
curiously , a large quantity of polymorphisms has been detected in clock genes , which can be influencing the development of diseases through different physiological systems .
this review will primarily focus on the hypothesis which states that the variation of genetic components of the circadian system , similar to environmental changes , can affect several physiological systems and produce an elevation of the risk of developing a disease . in recent years
, there has been a significant increase in available information from polymorphic variations and epigenetic modification over clock genes and their risk of diseases .
the report of health from the united states ( 2014 ) showed about 10% of population have a poor health associated with pathologies such as obesity ( 35.5% ) , hypercholesterolemia ( 30% ) , diabetes ( 32% ) , and cancer ( 6.4% ) . curiously , despite its low frequency in population , cancer is the second cause of death in the united states .
the above pathologies add the infertility , which affects about 10.9% of women in the united states and 17% of women in other developed countries . during short - stay in the hospital ,
mood disorder and psychosis are the principal causer of hospital stay during 2014 and represent the third cause of morbidity worldwide .
this present research reports the last single nucleotide polymorphism ( snp ) associated with an elevated risk of developing principal pathologies worldwide such as mood disorder , infertility , cancer , metabolism and diabetes and addictions , and a description of usefulness for a more detailed study in other pathologies .
in mammals , the circadian system is a supraphysiological system that regulates biological functions every 24 h such as glucose homeostasis , temperature , blood pressure , tone in the coronary artery , and heart rate .
the bmal1/clock complex target genes are related to the circadian expression of metabolic pathways as was observed by hatanaka et al . through high resolution
the author detected a circadian expression of ( i ) glucose metabolism - related genes such as glut2 , por , pck1 , and gys2 and ( ii ) cholesterol metabolism - related genes such as cyp2a4 , cyp2a5 , cyp4a14 , cyp7a1 , and cyp2c55 ; and considering the very important role these systems have in metabolism , any alteration of them can have negative health effects .
today there are several metabolic problems affecting the population , such as obesity , hyperglycemia , dyslipidemia , and hypertension .
the cooccurrence of three or more metabolic disorders , including obesity , is defined as metabolic syndrome , showing a prevalence in the united states during 2009 - 2010 of about 22% of adults ( 21.4626.15 ) . the insulin resistance or type
2 diabetes is one of the most important metabolic diseases currently affecting about 366 million people , causing more than 3.8 million deaths , and showing a rise in the number of affected people as observed in the united states population during 19992010 .
several factors could be increasing the risk of death by diabetes , such as dyslipidemia , hypertension , and obesity , all of which are driving to an elevated risk of cardiovascular disease .
a variant of this pathology can also develop during the pregnancy ( gestational diabetes mellitus ) and is characterized by glucose intolerance in the mother and adverse consequences for her offspring , such as an increase in blood pressure , body mass index ( bmi ) , and body fat and a decrease in hdl levels . at the molecular level ,
both diseases have in common the impairment of signal transduction of insulin receptors such as pi3kinase / akt and ras / map kinases [ 42 , 45 , 46 ] , leading to metabolic dysregulations such as inhibitions of glycogen synthesis , impaired translocation of glut4 to plasmatic membrane , or antilipolytic effects of insulin in white adipose tissue [ 39 , 46 ] .
meta - analyses from 448 articles reporting shift work and health consequence have detected a strong relationship between shift work , a potent chronodisruptor , and diabetes mellitus type 2 ( odd ratio about 1.42 times higher than day workers ) .
however , this is not the only chronodisruption which we are exposed too . in animal models exposed to light / dark patterns similar to shift work , the length of the working day and changes of feeding times induce a modification of the liver weight and plasmatic glucose and a modification of the circadian profile for glucose , insulin , and triglyceride .
these findings add to the observation in the circadian disruption in mutant animals , showing alterations of cholesterol metabolism , abolition of the circadian production of glycerol , free fatty acids , and impaired expression of rate - limiting lipolytic enzymes such as lipase , all of which add to increased weight gain , adipocyte hypertrophy , high level of glucose , glucose intolerance , and hypersecretion of insulin [ 4952 ] .
polymorphic variations of clock genes can be incrementing the risks of developing a disease similar to chronodisruption by environmental changes as it has been detected in human metabolic disorders ( see table 1 ) .
for example , the genotyping of clock genes in 346 greek pregnant women and their risk of diabetes was performed , detecting that the polymorphisms of bmal1 rs7950226 and rs11022775 are associated with gestational diabetes mellitus ( p = 0.025 , or = 1.46 and p = 4.455e 06 , or = 2.64 , resp . ) , while the study of the haplotype analysis of rs7950226/rs11022775 showed a major frequency in women with gestational diabetes mellitus ( p = 0.0069 , or = 6.96 ) .
similarly , a study performed in subjects from the united kingdom and pakistan reported that cry1 and cry2 polymorphisms rs2292912 and rs12315175 , respectively , are associated with diabetes ( p = 0.015 and 0.008 , resp . ) . moreover
, the variant rs12315175 for cry2 has a tendency to elevate the risk at about 5% compared to the variation of cry1 ( or = 1.05 and 0.95 , resp . ) .
also , the authors did not find associations between diabetes and bmal1 polymorphisms rs7950226 and rs11022775 , as occurs in gestational diabetes mellitus .
genotyping of 19,000 adults from northern sweden for variants rs8192440 for cry1 and rs11605924 for cry2 is associated with higher level of glucose concentrations at 2 hours ( p = 0.06 and p = 0.005 , resp . ) .
another study genotyping 1304 individuals from 424 british families , containing at least one patient with diabetes type 2 ( diabetes in families study collection ) , demonstrates the relation between bmal1 polymorphisms rs7950226 and rs11022775 and diabetes ( p = 0.002 ) , reinforcing what was previously described .
similar relations are observed for bmal2 polymorphism rs7958822 in obese men and women ( or 2.2 and 2.7 , resp . ) and the deletion / insertion of 54 base pair sequences of five repeat alleles on per3 gene ( rs57875989 ) [ 57 , 58 ] .
in contrast , the per2 polymorphism rs7602358 is associated with a protection from type 2 diabetes in the uk population , which suggests that not all polymorphisms are negative for health . at the level of dyslipidemia
, an interesting correlation has been detected between small dense ldl level and polymorphism of clock genes . in individuals carrying the polymorphism
associated with the clock gene ( rs1801260 ) , the genotype tt or tc showed a major level of small dense ldl , which leads to increased triglycerides and increased risk of cardiovascular and obesity diseases , similar to the metabolic syndrome . likewise , genotyping in the japanese population identified clock gene polymorphism rs1801260 associated with higher odds ratio ( or ; 1.5 ) of type 2 diabetes ; in contrast , a multicenter study detected the clock polymorphism rs4580704 is associated with prevention of diabetes and cardiovascular disease .
the haplotypes of rs10002541 and rs4864546 from clock ( cg and tg variations ) are associated with abdominal obesity in chinese population ( or 0.74 and 1.70 , resp . ) and polymorphism rs4864546 is associated with low level of hdl / apolipoprotein a1 ratio in spain .
moreover , clock polymorphisms rs12649507 and rs3749474 are associated with higher intake of polyunsaturated fatty acid and fat intake , which can modify the body mass index ( bmi ) .
this is an antecedent which , bearing in mind the ideas above , suggests the genetic components of the circadian system as a critical factor in the development of metabolic diseases .
in humans , infertility affects about 9% of couples , and about one - third of infertility cases associated with idiopathic male infertility are multifactorial , with 50% due to genetic abnormalities .
the circadian system is important during reproduction and development [ 89 , 90 ] ; for example , it is involved in the timing of the lh surge [ 90 , 91 ] , stimulation of ovulation [ 90 , 92 ] , and regulating the level of steroidogenic acute regulatory protein ( star ) expression and is critical for cholesterol translocation inside mitochondria and sperm count .
the genetic factors of clock genes are implicated in the pathogenesis of infertility ( see table 2 ) , as it occurs in male partners of infertile couples .
the genotyping of male partners detected a correlation between single nucleotide polymorphism of clock rs11932595 , rs6811520 , and 6850524 , with infertility ( p < 0.05 and or ranged between 1.4 and 1.9 ) .
similarly , the analysis of bmal1 polymorphism rs4757144 in slovenian and serbian caucasian men showed a significant correlation with infertility ( p = 0.047 ) , suggesting that clock genes clock and bmal1 contribute to successful fertilization .
mood or affective disorders are important causes of morbidity , where we can highlight depressive pathologies and hypomaniac and maniac disorders as major diseases affecting the population [ 29 , 66 ] , and it can be highlighted that depressive pathologies are the third largest source of morbidity in the world .
curiously , a potent correlation has been detected between mood disorders , sleep / activity , and the circadian system suggesting that the circadian system can be modulating the neuronal activity . during pregnancy ,
the prevalence of mental illnesses in women is about 8% , suggesting an imbalance between physiology of sleep and/or the circadian system .
this can be observed in the onset of a mental disease as has been observed in the impaired circadian production of melatonin during the pregnancy of depressed women ( approximately 34 weeks of gestation ) , which shows an advance onset time of melatonin production of about 40 minutes and a minor production during the dark hours [ 97 , 98 ] .
moreover , workers who do shift work ( circadian disruption ) showed decreased alertness , cognitive functions , mood , social and work activities , and health [ 26 , 99 ] , which are associated with an impaired circadian system via melatonin suppression , all of which leads to the appearance of a mood disorder .
for this reason , we can say that the circadian system may contribute to the risk of developing a mood disorder and the genetic component of clock genes can be involved in the development of mood pathologies ( see table 2 ) .
at the genetic component level of the circadian system , a study of 744 people who carry polymorphisms rs2291739 and rs11171856 from tim , a member of the clock gene family which interacts with per1 - 2 proteins [ 100 , 101 ] , showed that they have an elevated risk of developing mood disorders ranging between 19 and 23% ( or 1.19 and 1.23 , resp . ) .
moreover , gene variants of positive regulator clock have also been associated with mood disorders .
people who carry the variants rs1801260 and rs11932595 showed a major risk of developing a bipolar disorder ranging between 45 and 37% , respectively , in comparison to a patients without polymorphism ( or 1.45 and 1.37 , resp . ) . in the same study
, it was also observed that the polymorphisms rs2291739 and rs11171856 from tim gene are associated with unipolar disease , with a risk ranging between 37 and 40% in comparison to a patient without polymorphism ( or 1.37 and 1.40 , resp . ) .
/ rs11022779g / rs1122780 t ( haplotype ) are associated with mood disorders and bipolar disease [ 29 , 66 ] .
it has also been seen that the bipolar pathologies are associated with other genetic variations of bmal1 gene ( rs4757144 , rs1982350 , and rs1481892 ) and the negative regulator per3 ( rs2859387 ) .
curiously , a study performed an indian families reported the bmal1 polymorphisms rs2279287 are associated with seasonal affective disorder .
in addition , the gene variant of cry2 gene ( rs4132063 ) and deletion / insertion of 54 base pair sequences on per3 gene ( rs57875989 ) may also be contributing to mood disorders via increased risk of developing bipolar disease .
for example , a study in south india showed the prevalence of five repeat homozygotes from rs57875989 is associated with bipolar disease ( or 1.72 ) but not with schizophrenia . at the level of depressive pathologies , a study performed in china compared 485 subjects ( control ) to 105 patients suffering from a depression disorder .
the genotyping for clock genes showed that the variants of cry1 ( rs2287161 ) and cry2 ( rs10838524 ) are correlated to the depression disease with an odds ratio of 1.75 ( p = 0.012 ) , which suggests that a patient with the allele has 1.75 times more risk of developing depression .
moreover the authors detected a single nucleotide polymorphism for tef gene , the variant rs738499 , is associated with 2.22 times more risk of development of depression ( odds ratio of 2.22 .
finally , in a study performed in young adults , the risk of excessive intake of alcohol is minor when the polymorphism of per2 gene rs56013859 is present , suggesting a possible protector role for allelic variation of clock genes in addictions .
in fact , this suggests that some polymorphisms are not negative for our health and it is necessary to study them in greater depth .
cellular proliferation is a critical event for the survival and restitution of tissues of all living things and the circadian system is capable of delivering temporary information to the cell cycle [ 102104 ] .
however , an uncontrolled cellular proliferation and an excessive tissue growth are observed by an altered cellular cycle during cancer , a pathology that showed a higher incidence in patients exposed to an impaired circadian system such as a shiftwork [ 106 , 107 ] . the cell cycle is a finely regulated process from a cell that is capable of generating multiple cells through a series of cell divisions , including four critical and successive steps named g1 phase ( growth phase 1 ) , s phase ( synthesis ) , g2 ( growth phase 2 ) , and m phase ( mitosis ) . during the cell cycle , cyclin - dependent kinases ( cdk )
are critical for the transition between different stages , for example , cdc2 plus cyclin b protein kinase form the complex cdk1 which regulates g2/m transition [ 108 , 109 ] . during dna replication
, there are a number of controls or checkpoints that are critical in the cell cycle when dna damage is detected : the activation of the rad protein , which induces the action of cds 1 proteins , is triggered , as well as chk1 which are involved in cell cycle arrest via action of wee-1 and mik-1 proteins .
however , the efficiency of this process changes depending on the time of the day when the lesion occurred .
a lesion in the liver , occurring during the last light hours , induced a massive entry to m phase compared to a lesion which occurs early in the morning , which shows that the hour of surgery and the circadian system are critical for liver regeneration .
similarly , oral mucosa is a highly proliferative tissue and it showed a circadian expression of clock genes bmal1 , per1 , and cry1 and thymidylate synthase activity , critical for dna synthesis during phase s. curiously , the peak of per1 mrna precedes the peak of thymidylate synthase activity , which suggests that the circadian system modulates the cellular proliferation in mucosa .
wee-1 gene regulates cellular proliferation , and its promoter has three conserved sequence cacgtg ( e - box ) critical for the circadian expression of wee-1 .
its protein is capable of inactivating the cdc2-cyclin b complex via phosphorylation which inhibits transition between g2/m .
curiously , knock - down of bmal1 in carcinoma cells of the colon ( c26 cells ) , fibroblast cells ( l929 cell ) , and intestine epithelial cells ( iecs ) produces cellular proliferation in vitro and increments the size of tumor cells injected subcutaneously , via the inhibition of apoptosis and the reduction of the time transition between g2/m , reduction of p53 and wee-1 expression .
moreover , the knock - out for clock genes bmal1 y per2 in mice previously exposed to gamma radiation caused in mice hyperplasic growth and development of lymphoma , hepatic carcinomas , ovary tumors , and osteosarcomas via reduction of p53 expression .
a precedent that reinforces the idea that clock genes can be modulators of cellular cycle via wee-1 and p53 proteins . at the genetic component level
, different studies reveal the importance of the polymorphic variant of clock genes ( see table 3 ) and how the chrono - type modulates the risk of cancer , such as observed in breast cancer , in which the risk is more elevated in premenopausal women ( or , 2.43 and 2.55 ; resp . ) .
similarly , women showed evening or night preference such as shown in the case of a study performed on norwegian nurses working in night shift which revealed two polymorphisms associated with breast cancer .
the risk is incremented when women spend more time working during night hours , but this risk is higher when women have the alleles for bmal1 rs2290035 ( or 1.91 ) , rs969485 ( or 1.64 ) , and rs3903529 ( or 2.77 ) or variant rs3750420 ( or 1.6 ) of roreb gene .
similarly , an incremented risk is associated with breast cancer in women from france when they have the allele rs11932595 in clock gene ( or = 0.74 ) or in connecticut ( usa ) when they have the polymorphisms rs7698022 ( or 1.34 ) and rs1048004 ( or , 1.43 ) .
curiously , when norwegian nurses are exposed to shift work during four nights , they showed three times more risk ( or 2.75 ) of developing breast cancer when they were carrying clock polymorphism rs11133373 , which suggests that the disruption of endogenous circadian rhythm by polymorphism associated with clock genes increases the risk of cancer .
moreover , clock gene expression is induced in breast tissue from patients with breast cancer ; this expression is associated with hypomethylation of clock gene .
the silencing of clock gene lowered when women carry polymorphisms associated with cancer such as rs10448004 and rs7698022 , which suggests that clock gene is a critical protagonist of cancer development . at level of colorectal cancer
, a strong correlation is detected between cancer and the genetic component of the circadian system .
for example , a polymorphism in the clock gene ( rs1801260 ) showed a major prevalence in a cancer patient ( p < 0.0001 , or = 1.78 for c - allele ) compared to a control patient .
similarly , a screening performed in south carolina , usa , showed that a variation by deletion / insertion of a 54 base pair sequence on per3 gene ( rs57875989 ) is associated with a higher risk of colorectal adenoma formation with odds ratio within 2.15.1 .
moreover , a population study performed on the residents of king county , washington ( usa ) , detected six different types of polymorphism , which are significantly associated with the risk and aggressiveness of prostate cancer .
these genetics variants are rs1012477 for per3 ( or 1.3 ) , rs7602358 for per2 ( or 1.24 ) , and rs2289591 in per1 ( or 1.7 ) .
similarly , a genotyping of a patient with prostate cancer showed a strong correlation between cry1 polymorphisms rs7297614 , rs1921126 , and rs12315175 and fatal prostate cancer ( or mean within 1.52 ) and a study conducted in china showed that the cry2 variant rs1401417 and the deletion / insertion of 54 base pair sequences on per3 gene ( rs57875989 ) were associated with a major risk of developing prostate cancer ( or ; 1.7 and 1.3 , resp . ) . a study
performed in brazilian patients with pulmonary cancer showed a strong correlation of per3 polymorphism rs228644 and the risk of cancer ( or 1.99 ) .
moreover , the authors reported the ancestral haplotypes for per3 rs228729 , rs228727 , rs707467 , rs228644 , and rs10462020 are associated with a higher cancer frequency ; similarly , a meta - analysis performed by literature search showed that the variant insertion / deletion of per3 rs57875989 is associated with an increase of cancer susceptibility in about 17% or 70% . in a similar way
, a soft risk is associated with breast cancer in premenopausal women from india , which suggests there is a relation between per3 and cellular proliferation .
the frequency analysis of 1,538 breast cancer cases and 1,605 controls in china for clock gene variants showed the strong associations between three snps in circadian clock genes and the risk of developing breast cancer .
the variants for cry1 rs1056560 are correlated to cancer , which elevate the risk in about 11% ( or 1.11 ) ; per2 rs934945 in about 15% ( or 1.15 ) ; and clock rs3805151 in about 35% ( or 1.35 ) .
in contrast , tim protection for breast cancer development is detected in patients that carry the c - allele of rs7302060 ( or , 0.54 ) .
the g allele of rs2291738 and the c - allele of rs7302060 are associated with reduced risk of breast cancer among estrogen receptor ( ) or progesterone receptor ( ) positive breast cancer cases ( or , 0.46 and 0.36 , resp . ) .
the exogenous expression of human clock in cell lines of colorectal carcinoma induces the cellular proliferation in about 28% .
in contrast , knock - down of endogenous clock gene expression inhibits the cell proliferation in about 34% . moreover
, exogenous clock inhibits the apoptosis ( 42% reduction ) via inhibition on apoptosis associated proteins expression of bax and bid and the increase of phosphorylation of akt . in vivo experiments by xenograft transplant of colorectal carcinoma cell line transduced with clock
increase the tumor volume and tumor weight in about 61% and a 91% , respectively .
however , the pharmacological inhibition of cry in human breast cancer by treatment with pharmacological agent ks15 inhibits the proliferation and cell viability by stimulation of wee-1 expression and stimulates the activity of heterodimer complex bmal1:clock [ 112 , 113 ] .
curiously , two polymorphisms for clock gene cry2 , rs11038689 and rs1401417 , have a protective action over the mammary cancer , which suggests that not all polymorphisms associated with clock genes are negative for our health .
non - hodgkin 's lymphoma is characterized by lymphoproliferation and advanced clinical stages , invasion to other tissues , and death .
the estimated deaths from non - hodgkin 's lymphoma in the united states amounted to 19,790 during 2015 .
analysis of cry2 variants showed the polymorphisms rs11038689 , rs7123390 , and rs1401417 increase the risk of lymphoma ( or , 2.34 ; 2.40 and 2.97 , resp . ) .
moreover , a minor association of clock gene variants and glioma is detected for per1 rs2585405 , clock rs11133391 , and cry1 rs12315175 ( or , 1.16 ; 1.08 and 1.02 , resp . ) .
genotyping of han chinese patients diagnosed with primary hepatocellular carcinoma showed an association between single snps of per3 rs228669 and cry1 rs3809236 with odds ratio of 1.41 and 1.26 , respectively .
these precedents reinforce the idea that clock genes can be modulators of cellular cycle and that modulates the risk of cancer .
the circadian system is a supraphysiological system which modulates different physiological systems , and any alteration of this can have a negative impact on human health .
different chronodisruptors have been described in literature such as light / dark pattern and inhibition of melatonin production as occurs in shifts work .
however , other factors can be contributing on a minor scale , such as mealtimes or a genetic component .
the relevance of the genetic variation of clock genes and how it can interact with the environment is unknown .
but it has been described that the genetic component in the population predisposes the development of different pathologies such as diabetes , dyslipidemias , obesity , mood disorders , and addiction , all of which suggest the importance of this system to our health .
however , it is also necessary to say that the genetic component could be protecting our health such as in the case of polymorphisms associated with per2 and cry2 genes in diabetes , alcohol intake , and cancer . these findings will need to be implemented and evaluated at the genetic interaction level and also the way in which the environment factors trigger the expression of these pathologies will be examined .
finally , prospective studies are necessary to assess the predictive potential of these markers and to implement early treatment with consequent cost reduction for the health system .
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the circadian system is a supraphysiological system that modulates different biological functions such as metabolism , sleep - wake , cellular proliferation , and body temperature .
different chronodisruptors have been identified , such as shift work , feeding time , long days , and stress .
the environmental changes and our modern lifestyle can alter the circadian system and increase the risk of developing pathologies such as cancer , preeclampsia , diabetes , and mood disorder .
this system is organized by transcriptional / tranductional feedback loops of clock genes clock , bmal1 , per13 , and cry1 - 2 .
how molecular components of the clock are able to influence the development of diseases and their risk relation with genetic components of polymorphism of clock genes is unknown .
this research describes different genetic variations in the population and how these are associated with risk of cancer , metabolic diseases such as diabetes , obesity , and dyslipidemias , and also mood disorders such as depression , bipolar disease , excessive alcohol intake , and infertility .
finally , these findings will need to be implemented and evaluated at the level of genetic interaction and how the environment factors trigger the expression of these pathologies will be examined .
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1. Introduction
2. Clock Genes and Metabolic Disorder
3. Clock Genes and Infertility
4. Clock Genes, Mood Disorder, and Others
5. Clock Genes and Cancer
6. Conclusion
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connectivity is key to understanding activity in neural systems ( sporns et al . , 2000 ) .
network connectivity in science and in engineering fields as diverse as mechanics , communication technology , public health , geography and town planning , is studied mathematically using the concepts of graph theory ( bollobas , 1998 ) .
recently , graph theory is being applied to brain connectivity ( sporns and zwi , 2004 ; bullmore and sporns , 2009 ) and its pathologies in alzheimer 's disease ( stam , 2004 ; stam et al . , 2007 ) , brain tumors ( bartolomei et al . , 2006 ) , epilepsy ( ponten et al . , 2007 ) and , in particular , schizophrenia ( bleuler , 1911/1950 ; friston and frith , 1995 ; andreasen , 1999 ; micheloyannis et al . , 2006 ;
applying graph - theoretic concepts to the brain sheds new light on the basic principles of integration and segregation underlying adaptive cognitive processes , and on their disruption in maladaptive states .
schizophrenia has been understood as a cognitive disorder ( bleuler , 1911/1950 ) based on the breakdown of large - scale cortico - cerebellar - thalamic - cortical ( andreasen , 1999 ) or prefronto - temporal circuits ( friston and frith , 1995 ; goldman - rakic and selemon , 1997 ) , or more generally the inability to integrate neural processes in different brain areas , a syndrome termed dysconnectivity ( stephan et al .
this condition may pare down , in particular , the input to pyramidal cells of the dorsolateral prefrontal and temporal cortex ( garey et al .
these cells are glutamatergic and receive projections from the thalamus and widespread cortical areas , and hence are likely to be involved in higher - level cognition .
reduced connectivity may thus lead to fragmentation , a loss of coherence in cognitive activity .
graph theory enables us to model the loss of connectivity in simulated neuronal networks and predict the time course of fragmentation . on the face of it ,
percolation plays an important role in the evolution , growth , and maintenance of a large variety of natural , technological , and social systems ( ben avraham and havlin , 2000 ) .
it refers to the probability of existence of a path between every pair of nodes in a graph , or equivalently , the graph being connected
whilst many previous studies have examined cortical network connectivity in schizophrenia and other disorders , none to our knowledge have employed the concept of percolation , an issue that we presently redress .
the percolation function is the cumulative density function ( cdf ) of percolation as a function of connectivity .
it is possible , in principle , to measure the percolation function in living neural tissue , by using progressive lesioning , for instance through the administration of inhibitory neurotransmitters ( breskin et al . ,
observations on human brain functional connectivity may be compared to the theoretical percolation function for random networks ( erds and rnyi , 1959 ) .
the percolation function cp(n ) of a random graph of n vertices and e edges is given by cp(n ) = e the presence of a critical threshold motivates us to revisit the notion of cortical dysconnectivity as a sudden breakdown of percolation .
there are , however , reasons to assume that the percolation threshold is neither the first , nor the most predominant , critical transition in the development of schizophrenia : brains are not random networks . in both the structural ( sporns and zwi , 2004 ) and functional ( salvador et al .
2006 ) domains , the hallmarks of brain organization include local clustering as expressed in high values for the clustering coefficient ( cc ) , high global connectedness as specified by a short characteristic path length ( cpl ) ( watts and strogatz , 1998 ) , and modularity ( murre and sturdy , 1995)a combination characteristic of modular small - world networks ( he et al . , 2009 ) .
graph - theoretical studies ( murre and sturdy , 1995 ; watts and strogatz , 1998 ) showed that small - world and modular networks can secure global connectivity with a small number of connections . for brains configured as modular small - worlds , a few connections will suffice to ensure percolation .
most likely , therefore , percolation is not the crucial bottleneck for brain pathologies such as schizophrenia .
we will propose as an alternative theoretical possibility that , instead , brain pathologies are associated with a breakdown in the local organization . in schizophrenia patients ,
functional connectivity in scalp eeg channels appears to reflect a loss of clustering after correcting for differences in the density of functional connections ( micheloyannis et al .
the question , therefore , arises , whether fragmentation can be understood as a critical breakdown in the ability of the brain to establish and maintain a modular small - world functional architecture .
here we show by numerical simulations that in neural activity networks , with loss of connectivity a self - organizing small - world neural network can not sustain its local clustering , well before global connectivity breaks down .
the iterated logistic map f(x)= 1 ax is unimodal on [ 1;1 ] [ 1;1 ] and capable of both periodic and chaotic behavior depending on its control parameter a. in this study , we construct networks of coupled logistic maps , all with parameter a = 1.7 such that the dynamics of a single unit are chaotic under iteration of a randomly chosen initial activation value . a unit x is coupled with coupling strength = 0.4 to any number mi of other units in the network such that its activation value xn + 1 at iteration n + 1 depends on the activation value of itself and all adjacent units at iteration n :
in this equation , b(i ) is the set of units adjacent to unit i , mi is the number of units adjacent to unit i. the coupling strength is divided by mi , and has a compensation term ( 1 ) to make sure that logistic map of an individual unit retains its mapping [ 1:1 ] [ 1:1 ] , and thus functions properly for any numbers of adjacent units . a network of this type can be used to study the buildup and breakdown of modularity resulting from hebbian adaptive structural self - organization .
it implements a simple rewiring rule based on synchronization of chaotic activity and rewires at most one connection per iteration , carefully keeping the network 's total number of connections constant throughout the process .
a single iteration of the network consists of four steps , to be repeated several times after an initial random inception :
initialize the network .
randomly establish e connections between v units to create a ( v , e ) random network , and initialize every unit with a random activation value [ 1:1 ] .
though values of v and e are chosen such that a network has a high probability of being connected , this is not required.update units .
synchronously update every unit 's activation value from its own and all its adjacent units ' activation values according to equation ( 1).select pivot and candidate .
randomly select one unit from the network ( the pivot ) . from all other units , select the one whose activation value is closest to the pivot 's .
then , from the units already adjacent to the pivot , select the one whose activation value is farthest from the pivot 's , and cut its connection to keep the number of connections constant .
if there is already a connection between the pivot and the candidate , or if the pivot has zero connections , nothing happens and this step is skipped.iteration completed .
randomly establish e connections between v units to create a ( v , e ) random network , and initialize every unit with a random activation value [ 1:1 ] .
though values of v and e are chosen such that a network has a high probability of being connected , this is not required .
synchronously update every unit 's activation value from its own and all its adjacent units ' activation values according to equation ( 1 ) .
randomly select one unit from the network ( the pivot ) . from all other units , select the one whose activation value is closest to the pivot 's .
then , from the units already adjacent to the pivot , select the one whose activation value is farthest from the pivot 's , and cut its connection to keep the number of connections constant .
if there is already a connection between the pivot and the candidate , or if the pivot has zero connections , nothing happens and this step is skipped .
networks implementing these iterative steps exhibit development from an initial random configuration to modular small - world configurations ( gong and van leeuwen , 2003 ; van den berg and van leeuwen , 2004 ; rubinov et al . , 2009b )
but as it turns out , both the consistent build - up of connective modularity on one hand , or the loss of structural coherence due to functional fragmentation on the other , are a result of changing dynamic activity depending critically on the number of connections in the network .
the influence of these numbers shows a close relationship to the percolation function of random graphs .
a common principle for neural network evolution is preferential attachment ( barabsi and albert , 1999 ) .
this mechanism leads to networks that are scale - free , but not modular small - worlds . only by combining preferential attachment with adaptive , hebbian rewiring ,
does a network emerge that is scale - free and also has modular small - world network structure ( gong and van leeuwen , 2003 ) . an adaptive rewiring scenario for evolving networks allows networks with initially random or regular structures to develop into modular small - world structures ( gong and van leeuwen , 2004 ; rubinov et al . , 2009b ) .
the scenario requires network units ( edges ) that produce ongoing , non - random , non - periodic oscillatory activity .
these could , for instance , be represented by spiking model neurons ( kwok et al . , 2007 ) or by nonlinear maps as an extremely simplified model of neural mass activity ( breakspear et al .
, 2003a , b ) . with these simple units as edges , the vertices of the network represents the couplings of a coupled nonlinear map ( kaneko , 1989 ) .
adaptive rewiring operates on this activity according to the general hebbian principle of what fires together wires together ( paulsen and sejnowski , 2000 ) . at successive points during the systems ongoing spontaneous activity
, connections are added between pairs of synchronously active but hitherto unconnected units , while connections between desynchronized units are removed ( see methods ) . over time
the network gradually assumes a modular , small - world structure ( figure 1 ) .
adaptive rewiring leads from an initial random network ( left ) , to modular small - world structure ( right ) in small iterative steps . coupled chaotic oscillators intermittently synchronize and desynchronize their activity spontaneously in patterns of great variability . after some time a momentarily synchronized pair of units that are not connected
receive a connection , which is removed from a pair that are connected but not synchronized . as this process continues , a modular , small - world structure emerges from an initially random configuration . to obtain a more detailed view of this phase transition , we use the adaptive rewiring scenario with coupled nonlinear maps ( kaneko , 1989 ) with initially randomly structured graphs , for a range of different numbers of vertices v : v = 300 , 400 , 500, ,1000 vertices and numbers of edges e that differ by small steps of 20 .
for each combination of v , e , across four million iterations we measured the cc and the cpl every one thousand iterations , resulting in a 4000 point record for each of five runs .
the maximum , minimum , and mean values of the last 2000 points in each run were averaged over the five runs as illustrated in figure 3 .
meanwhile a mixture of regular and irregular behavior is established in the network activity that is itself optimal for sustaining the small - world structure .
crucially , whilst low dimensional , ordered , and synchronized activity dominates within modular communities , high dimensional unsynchronized activity in connector hubs ensures that the system does not fragment ( rubinov et al .
attractors in the space of possible systems ( gong and van leeuwen , 2004 ) , which offers a potential explanation for their ubiquity in biological neural networks at different scales , including the entire brain ( barabsi and albert , 1999 ) . in this scenario , connectivity constitutes a critical limit for the evolution to small - world structure ( figure 2 ) .
when the number of edges is large enough , adaptive rewiring guarantees a robust evolution from random to small - world connectivity . below this limit , this evolution is frustrated , and fails to reach a stable asymptotic state . with reduced connectivity levels , we first encounter critical fluctuation : intermittently during some episodes , clusterings are formed intermittently , which are annihilated in other episodes
. this may reflect the intermittent occurrence of certain symptoms ( e.g. , delusions ) as the brain disease first becomes manifest .
for still lower connectivity levels , adaptive rewiring becomes completely ineffective ; this may reflect the advanced state of the disease .
self - organization from random to small - world critically in a network of 700 vertices .
the self - organization occurs through adaptive rewiring . whether a small - world emerges depends on the number of edges .
we compared the critical limit on the evolution to small - world structures to percolation thresholds of random networks with the same numbers of edges and vertices .
figure 4 shows that the observed minimum cc can be modeled as a linear function of cp(n ) , with k3 for offset and k4 for amplitude : ccpred = k3 + k4
cp(n ) .
parameter k3 was in the range [ 0.107:0.196 ] , parameter k4 in [ 0.392:0.459 ] and parameter k1 in [ 0.001:0.006 ] .
the behavior of these parameters across network sizes was not monotonic ( figure 4 ) .
parameter k2 however , the horizontal position of the anchor point , showed a universal scaling law to the anchor point in the percolation function of random graphs , namely ( table 1 ) : aswn(n ) = arand(n ) .
note : anchor point arand(n ) = n ln(n ) for classic random graphs of n vertices ; this anchor point indicates the percolation threshold , where the percolation function cp(n ) shows the greatest inflection .
aswn(n ) : anchor point for the small - world networks fitted according to figure 4 .
marquardt algorithm implemented in fityk . scaling power : the value of h in the equation aswn(n ) = arand(n ) .
a common principle for neural network evolution is preferential attachment ( barabsi and albert , 1999 ) .
this mechanism leads to networks that are scale - free , but not modular small - worlds . only by combining preferential attachment with adaptive , hebbian rewiring ,
does a network emerge that is scale - free and also has modular small - world network structure ( gong and van leeuwen , 2003 ) . an adaptive rewiring scenario for evolving networks allows networks with initially random or regular structures to develop into modular small - world structures ( gong and van leeuwen , 2004 ; rubinov et al . , 2009b ) .
the scenario requires network units ( edges ) that produce ongoing , non - random , non - periodic oscillatory activity .
these could , for instance , be represented by spiking model neurons ( kwok et al . , 2007 ) or by nonlinear maps as an extremely simplified model of neural mass activity ( breakspear et al .
, 2003a , b ) . with these simple units as edges , the vertices of the network represents the couplings of a coupled nonlinear map ( kaneko , 1989 ) .
adaptive rewiring operates on this activity according to the general hebbian principle of what fires together wires together ( paulsen and sejnowski , 2000 ) . at successive points during the systems ongoing spontaneous activity
, connections are added between pairs of synchronously active but hitherto unconnected units , while connections between desynchronized units are removed ( see methods ) . over time
the network gradually assumes a modular , small - world structure ( figure 1 ) .
adaptive rewiring leads from an initial random network ( left ) , to modular small - world structure ( right ) in small iterative steps . coupled chaotic oscillators intermittently synchronize and desynchronize their activity spontaneously in patterns of great variability . after some time a momentarily synchronized pair of units that are not connected
receive a connection , which is removed from a pair that are connected but not synchronized . as this process continues , a modular , small - world structure emerges from an initially random configuration . to obtain a more detailed view of this phase transition , we use the adaptive rewiring scenario with coupled nonlinear maps ( kaneko , 1989 ) with initially randomly structured graphs , for a range of different numbers of vertices v : v = 300 , 400 , 500, ,1000 vertices and numbers of edges e that differ by small steps of 20 .
for each combination of v , e , across four million iterations we measured the cc and the cpl every one thousand iterations , resulting in a 4000 point record for each of five runs .
the maximum , minimum , and mean values of the last 2000 points in each run were averaged over the five runs as illustrated in figure 3 .
meanwhile a mixture of regular and irregular behavior is established in the network activity that is itself optimal for sustaining the small - world structure .
crucially , whilst low dimensional , ordered , and synchronized activity dominates within modular communities , high dimensional unsynchronized activity in connector hubs ensures that the system does not fragment ( rubinov et al .
attractors in the space of possible systems ( gong and van leeuwen , 2004 ) , which offers a potential explanation for their ubiquity in biological neural networks at different scales , including the entire brain ( barabsi and albert , 1999 ) . in this scenario , connectivity constitutes a critical limit for the evolution to small - world structure ( figure 2 ) .
when the number of edges is large enough , adaptive rewiring guarantees a robust evolution from random to small - world connectivity . below this limit , this evolution is frustrated , and fails to reach a stable asymptotic state . with reduced connectivity levels , we first encounter critical fluctuation : intermittently during some episodes , clusterings are formed intermittently , which are annihilated in other episodes
. this may reflect the intermittent occurrence of certain symptoms ( e.g. , delusions ) as the brain disease first becomes manifest .
for still lower connectivity levels , adaptive rewiring becomes completely ineffective ; this may reflect the advanced state of the disease .
self - organization from random to small - world critically in a network of 700 vertices .
the self - organization occurs through adaptive rewiring . whether a small - world emerges depends on the number of edges .
we compared the critical limit on the evolution to small - world structures to percolation thresholds of random networks with the same numbers of edges and vertices .
figure 4 shows that the observed minimum cc can be modeled as a linear function of cp(n ) , with k3 for offset and k4 for amplitude : ccpred = k3 + k4
cp(n ) .
parameter k3 was in the range [ 0.107:0.196 ] , parameter k4 in [ 0.392:0.459 ] and parameter k1 in [ 0.001:0.006 ] .
the behavior of these parameters across network sizes was not monotonic ( figure 4 ) .
parameter k2 however , the horizontal position of the anchor point , showed a universal scaling law to the anchor point in the percolation function of random graphs , namely ( table 1 ) : aswn(n ) = arand(n ) .
note : anchor point arand(n ) = n ln(n ) for classic random graphs of n vertices ; this anchor point indicates the percolation threshold , where the percolation function cp(n ) shows the greatest inflection .
aswn(n ) : anchor point for the small - world networks fitted according to figure 4 .
marquardt algorithm implemented in fityk . scaling power : the value of h in the equation aswn(n ) = arand(n ) .
we propose that important insights into cortical activity and architecture can be obtained by modeling the activity - dependent rewiring of neural connections during development ( gong and van leeuwen , 2003 ; rubinov et al . , 2009b ) .
in our model , network connections evolve in accordance with the principle that the structure rewires in adaptation to spontaneous , on - going activity .
this evolution , however , is only guaranteed if there are sufficiently many connections available .
if connectivity is reduced below this number , the structure shifts toward randomness ; in particular , local clustering is reduced .
andreasen ( 1999 ) and friston and frith ( 1995 ) considered schizophrenia as fragmentation , understood as the breakdown of integration between widely distributed brain areas ( stephan et al . , 2006 , 2009 ) .
this breakdown can be associated with the loss of connectivity ( zalesky et al . , 2011 ) , in particular of input to layer 3 pyramidal cells , an effect which is well - documented ( e.g. , garey et al .
( 2011 ) observed widespread impairment in structural connectivity in schizophrenic patients , involving medial frontal , parietal / occipital and left frontal cortex .
it should be observed that the loss of connectivity that may lead to the onset of schizophrenia can be relatively subtle . across the population ,
inputs to layer 3 pyramidal cells are substantially reduced during late adolescence , the typical period for the onset of schizophrenia ( bourgeois et al . , 1994 ) .
given that brain connectivity is costly , it may well be that in normals , its density hovers just above the critical level ( the anchor point in figure 4 ) , but in early schizophrenia it may fall just below this point . the graph - theoretical concept of percolation tells us that a small decline in connectivity can lead to a sudden breakdown of global network coherence .
based on our results , however , we argue that fragmentation in brain pathologies such as schizophrenia may be considered theoretically as a breakdown in the local connectivity structure , prior to the loss of global coherence . the number minimally needed to secure local modularity , and hence to prevent it from shifting toward randomness in structure , is systematically related to , and greater than , that needed to secure global connectivity , even if the system has fallen into entirely random connectivity .
this result is of potential importance for understanding the pathophysiological processes that give rise to this disorder .
the loss of local clustering in our model is in accordance with observations in schizophrenic patients by micheloyannis et al .
et al . ( 2006 ) , the clinical group also showed longer path lengths than the controls , whereas in rubinov et al .
nevertheless , we might attribute the discrepancy to the fact that in both studies comparisons were made , for statistical reasons , between networks that were thresholded to have identical connectivity .
whereas the above - mentioned effects of clustering remain relatively unaffected by threshold setting , the differences in path length rapidly disappear for lower thresholds ( figure 1 in micheloyannis et al . , 2006 ) .
( 2003b ) reported that although there were no significant increases in the occurrence of nonlinear interdependence between pairs of electrodes in schizophrenia , there was an increase in the co - occurrence in multiple ( widespread ) instances of nonlinear interdependence .
this means that a relatively large number of global connections will have survived thresholding in rubinov 's study , leading to their observation of path length shortening .
it can not be concluded from rubinov 's study , therefore , that global connectivity is stronger in schizophrenics than in normals ; it could , however , be concluded that the global connectivity becomes stronger in schizophrenia relatively to their local connectivity .
such a conclusion would entirely be in accordance with the modularity breakdown observed in our model .
along the lines set out here , a shift in the balance from local to global connectivity is perfectly consistent with an overall loss of connectivity in early schizophrenia .
( 2003 ) introduced the notion of overbindingthe formation of excessive connections that are effectively random and , as such , do not enable distinguishing external from internal sources , thus providing conditions favorable for phenomena such as hallucination . a possible objection to our findings is the specific choice of our rewiring algorithm .
note , however , that in the present paper we sought to establish the principled possibility using the simplest possible model , rather than to establish the empirical validity through the most realistic model possible
. note that , as a consequence , the model contains only generic dynamical and adaptive principles .
we have discussed elsewhere the robustness of this model ( gong and van leeuwen , 2003 , 2004 ; van den berg and van leeuwen , 2004 ; kwok et al .
an important limitation is that the model inevitably makes over - simplifying assumptions . in particular
inter - modular connections are physically of longer range than intra - modular ones and , therefore , have a higher metabolic cost and a greater vulnerability .
preliminary analysis of models with more realistic constraints does not appear , however , to affect our conclusions .
clearly , a more differentiated model is needed to address empirical datasets such as ( rubinov et al . , 2009a ) , an important goal of future work .
however , it should also be noted that uncovering universal principles such as those reported here has the advantage of being
detail invariantthat is , robust across a range of potential constraints , whereas findings arising in detailed models may not be robust to changes in those details .
we observed universal scaling behavior in adaptive self - organization of clustered small - world networks : the connectivity needed for these network properties to emerge under hebbian rewiring scales with a universal power = 1.17 to the percolation function in random networks .
note , first , that > 1 might have been expected , given that the requirement to observe clustering and small - world structure are constraints additional to percolation .
what is surprising is that these requirements are met with alpha very close to unity ; near - linear scaling implies that these additional constraints can be realized with great efficiency . in terms of kolmogorov - complexity , small - worlds are compressible , whereas almost every possible network of n nodes and e edges ( or equivalently a bit string of length l = n(n 1 ) with e ones and l - e zeros ) will be incompressible ( li and vitnyi , 1993 ) . in this perspective , the ubiquity of small - world structure in real - world networks is quite astonishing : within human brains ( sporns and zwi , 2004 ; stam , 2004 ; eguluz et al .
bartolomei et al . , 2006 ; micheloyannis et al . , 2006 ; ponten et al .
, 2007 ; rubinov et al . , 2009a ; bassett et al . , 2010 ) , as well as between them : networks of scientific co - authorship ( newman , 2001 ) , collaborating movie actors ( watts and strogatz , 1998 ; amaral et al . , 2000 ) , social networks in general ( wasserman and faust , 1994 ) . here
we showed how such a network could arise with minimal connectivity close to random network percolation what is the reason for the universality of the scaling exponent ?
we may wonder whether the same exponent found in other domains , could help us understand the principle .
a study of class graphs in open - source , object - oriented software systems ranging from simple paint programs , peer - to - peer downloaders , racing games , database management software to a complete operating system , showed that the number of links between classes scales to the number of classes with an exact power = 1.17 .
the authors found that class graphs are small - world networks at the critical threshold for the breakdown of modularity , which happens when developments to the system are widely dispersed and affect many unrelated classes in apparently distant modules ( valverde and sol , 2007 ) .
the similarity of this finding to ours supports the view that the scaling exponent reflects a general feature in the emergence and breakdown of modular network structure .
the study of self - organizing modular small - world networks casts a new perspective on psychiatric illnesses characterized by disorganized cognition , such as schizophrenia , of which the expression has been attributed to fragmentation a subtle but pernicious disconnection ( friston , 1996 , p. 644 ) . rather than a breakdown in global connectivity
, we propose that fragmentation is to be understood as a failure to organize the functional connectivity of the brain into a modular small - world structure .
this is in accordance with the observed random shift in schizophrenic ( micheloyannis et al . , 2006 ;
random networks are considered extremely uneconomical ; in terms of cable length , an optimal configuration combines local modules with a limited number of large - scale connections ( murre and sturdy , 1995 ) . even though our model does not consider distance , in terms of network topology it is still the case that information travels efficiently both within locally connected circuits of modular small - world graphs and between their circuits , which makes these networks efficient for transport or communication ( latora and marchiori , 2001 ; bassett et al . , 2010 ) .
the scaling observations tell us that fragmentation is a result of a breakdown in local , rather than global structure . with progressive loss of connectivity ,
ultimately , it may not matter which connections are lost first , the result may be a cascade of changes that lead to the network falling apart . for diagnosis , however , a proper understanding of the early stages of the disease is crucial ; loss of modularity might offer a new perspective on the origins of the disease .
one simulation consists of one network of v units and e connections , which is randomly initialized and then iterated exactly 4,000,000 times , simultaneously rearranging its connections and activity patterns , according to the adaptive rewiring scenario .
the smallest simulation we adopt has v = 300 units and e = 2400 connections . during iteration ,
its cpl and its cc are taken every 1000th iteration ( 1000 , 2000 , , 40,00,000 ) resulting in a 4000 point record , with a value for cc and a value for cpl at each point .
although the speed of convergence depends on the size of a network , 4 , 000 , 000 iterations prove to be enough to clearly discern asymptotic behavior for all simulations used in this investigation ( figure 3 ) .
evolution under adaptive rewiring of maximum , minimum , and average cluster coefficient and characteristic path length .
( a ) the values of minimum , maximum and average cc for networks of 700 vertices and edges ranging from [ 7000 , 7020 , 7040, ,10,000 ] after extensive adaptive rewiring . note that beyond 9000 edges , cc - values tighten to a narrow range , indicating strong and consistent clustering behavior .
( b ) the values of minimum , maximum , and average cpl for networks of 700 vertices and edges ranging from [ 7000 , 7020 , 7040, ,10,000 ] after extensive adaptive rewiring . beyond 9000 edges
thus , for 700 vertices , at least 9000 edges are needed for adaptive rewiring to converge to small - world structure . from the 4000 point record ,
the maximum , minimum , and average values for both cpl and cc are calculated from the last 2000 points . for statistical robustness
, we do any single simulation five times , and average the five values over this simulation - quintuple , resulting in a maximum , minimum , and average cc and cpl for the ( v = 300 , e = 2400 ) network .
we then start a new quintuple of simulations , increasing the number of connections e by 20 , generating five networks with v = 300 units and e = 2420 connections , and calculate the maximum , minimum , and average cc and cpl values from these five new simulations .
we keep starting new quintuples , repeatedly increasing e by 20 , until e = 3300 and the batch of 300-quintuples is complete . from the entire batch , the six cc and cpl values of every ( v ,
e ) are taken to graph the asymptotic clustering and path - length behavior of networks of 300 units as it depends on the numbers of connections ( figure 4 ) .
gray lines represent minimal , maximal and average observed values for clustering coefficient , the dotted line is the predicted clustering coefficient , ccpred , a linear function of the percolation function cp(n ) of a random graph of n vertices : ccpred = k3 + k4 cp(n ) fitted with parameters k3 and k4 to the minimum observed clustering ; the arrow indicates its anchor point aswn(n ) with the corresponding number of edges in parentheses .
this process is then repeated for a batch of quintuples of networks with 400 units and numbers of connections 3600 , 3620 , , 5000 ( see figure 4 , top - right box ) .
we continue doing this for batches of networks with 500 , 600 , 700 , 800 , 900 , and 1000 units , with connections increasing by 20 , showing asymptotic clustering and path - length behavior depending on connectivity for networks of different sizes ( figure 4 ) .
note that although for 300 units , connections ranged from e = 2400 to e = 3300 , these numbers are different for larger networks .
for each of the eight batches , a phase transition was witnessed for both the cc and the cpl .
to pin down the exact location of the steepest inclination in the phase transition of the cc ( its center , or anchor point ) , the percolation function for classic random graphs was function - matched to the minimum cc of every batch .
the minimum cluster coefficient was chosen over the average and the maximum cluster coefficient because it has the steepest inclination , which facilitates the fitting best .
macquardt an iterative curve - fitting algorithm which operates by minimizing the summed squares of the residuals , in this case the difference between minimal cc - values of n - edge simulation quintuples on the one hand , and the ( erds and rnyi , 1959 ) percolation function 's value for n edges on the other .
macquardt algorithm is sensitive to local minima which makes it inefficient when using completely random initial values .
initial parameters were hand - guessed separately for each of the eight subgraphs in figure 4 , after which the algorithm was ran until convergence beyond the program 's six - digit resolution , a procedure that was repeated three times with small differences in the hand - guessed initial parameters .
the final values did not differ within the program 's six - digit resolution over the three repetitions , and convergence was very fast ( typically well before 100 iterations ) .
the fits show significant deviations from the data curve , due to intrinsic fluctuations in the data .
we , therefore , considered reliable the estimates of the scaling power and other model parameters .
even more reliable estimates could , in principle , be obtained by scaling up the network size to 2000 , 5000 , and 10,000 vertices , resources permitting , as computation time and data grow nonlinearly with network size .
the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest .
the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest .
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psychiatric illnesses characterized by disorganized cognition , such as schizophrenia , have been described in terms of fragmentation and hence understood as reduction in functional brain connectivity , particularly in prefrontal and parietal areas . however , as graph theory shows , relatively small numbers of nonlocal connections are sufficient to ensure global coherence in the modular small - world network structure of the brain .
we reconsider fragmentation in this perspective .
computational studies have shown that for a given level of connectivity in a model of coupled nonlinear oscillators , modular small - world networks evolve from an initially random organization . here
we demonstrate that with decreasing connectivity , the probability of evolving into a modular small - world network breaks down at a critical point , which scales to the percolation function of random networks with a universal exponent of = 1.17 .
thus , according to the model , local modularity systematically breaks down before there is loss of global coherence in network connectivity .
we , therefore , propose that fragmentation may involve , at least in its initial stages , the inability of a dynamically evolving network to sustain a modular small - world structure .
the result is in a shift in the balance in schizophrenia from local to global functional connectivity .
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Introduction
Methods
Results
Evolving networks
Percolation and self-organization in small-worlds
Discussion
Data
Conflict of interest statement
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the prevalence of diabetes in the u.s . has increased ( 1 ) , and cardiovascular ( cv ) complications remain the major cause of morbidity and mortality in persons with type 2 diabetes , contributing substantially to increased health care costs ( 2 ) . as individuals attempt to manage their diabetes , there is increasing recognition that comorbid behavioral problems , including both depressive symptoms and stress , are associated with poor glycemic control , poor lifestyle behaviors , and increased health services utilization ( 310 ) .
we ( 11 ) and others ( 3,4 ) have shown that these behavioral challenges are associated with inadequate medication adherence , which may be associated with adverse outcomes .
similarly , poor meta - bolic control may worsen depressive symptoms ( 5 ) , and the relationship appears to be bidirectional ( 8) . the prevalence of these comorbidities and their potential to worsen disease management have led some to develop interventions designed to address comorbid depressive symptoms or stress in persons with diabetes ( 1214 ) .
while many studies have documented the cross - sectional presence of these comorbidities and the effect on glycemic control in subjects with diabetes , only a limited number of studies have examined the potential impact on cv outcomes .
most of these studies have been limited to patients with depression recruited from established health care settings , and the true population - level impact is unknown .
likewise , there has not been adequate comparison of individuals with and without diabetes who have concurrent depressive symptoms and/or elevated levels of perceived stress to examine the unique consequences of behavioral and medical comorbidity .
the objective of the present report was to examine the association between the presence of depressive symptoms and/or increased levels of perceived stress , determined at baseline , and risk of incident cv events over 5 years of follow - up in a major national cohort study that used population - based sampling methods , collecting data in the home both in subjects with and without diabetes .
the description of the population for this analysis has previously been reported ( 15 ) . in short ,
the reasons for geographic and racial differences in stroke ( regards ) study is a population - based , prospective , longitudinal cohort study of 30,239 subjects aged > 45 years , 45% of whom are male , 55% female , 41% black , 59% white , 55% from the stroke belt
it was designed to examine factors associated with the geographic and racial differences in stroke incidence as well as excess stroke mortality in the southeastern u.s .
( i.e. , stroke belt ) relative to the rest of the nation and among blacks relative to whites .
the regards study included relevant measures for perceived stress and depressive symptoms and provides a unique opportunity to examine the impact of these behavioral comorbidities in individuals with and without diabetes and the incidence of adverse cv outcomes in a national sample of black and white adults .
the regards cohort was recruited between january 2003 and october 2007 and is evaluated by computer - assisted telephone interview every 6 months .
the initial recruitment call was used to obtain verbal consent and collect cv history , risk factors , and demographic characteristics , including age , sex , and race ( each subject self - reported race , and by design , the study compared only non - hispanic black and white subjects , excluding other racial / ethnic groups ) .
the study also included an in - home visit by a trained health professional for data collection .
participants were asked to provide all prescription and nonprescription medications they had taken in the past 2 weeks , and medication names were recorded during the in - home visit and subsequently coded into drug classes . written informed consent was obtained during the in - person evaluation .
the present analysis included all members of the cohort who had complete follow - up data .
a brief physical exam including blood pressure ( assessed as the average of two measurements obtained with an aneroid sphygmomanometer after the subject was in the seated position for at least 3 min with both feet on the floor ) and blood and urine samples , and an electrocardiogram ( ecg ) was conducted during an in - home visit by a trained health professional 34 weeks after the telephone interview .
height and weight was measured via standard procedures , and bmi was computed as the body weight in kilograms divided by the square of height in meters ( 2 ) .
all ecgs were read by a trained cardiologist in a centralized ecg reading laboratory at the wake forest university / baptist medical center in winston - salem , nc , using a predefined interpretation protocol ( 16 ) .
atrial fibrillation was determined to be present if there was a self - reported history ( asked as has a doctor or other health professional ever told you that you had atrial fibrillation ? ) or if characteristic findings were present on the ecg as defined in the protocol .
left ventricular hypertrophy was defined to be present or absent based on ecg findings using the sokolow - lyon limb lead criteria ( 17 ) as specified in the protocol .
hs - crp was determined by particle - enhanced immunonephelometry using the bnii nephelometer ( n high sensitivity crp ; dade behring , deerfield , il ) , while total and hdl cholesterol and glucose were measured by colorimetric reflectance spectrophotometry using the ortho clinical vitros 950/irc chemistry system ( johnson & johnson clinical diagnostics , new brunswick , nj ) in accordance with the national cholesterol education program guidelines ( 18 ) .
diabetes was defined as self - reported diabetes obtained during the telephone interview or a fasting glucose 126 mg / dl , a nonfasting glucose 200 mg / dl , or the presence of oral hypoglycemic medication or insulin each obtained during the home visit .
the primary exposure of interest was the presence , at baseline , of depressive symptoms and/or elevated levels of perceived stress among individuals with diabetes at baseline compared with those without diabetes .
the presence of depressive symptoms was assessed using the previously validated four - item center for epidemiologic studies depression ( cesd ) questionnaire ( 19 ) .
the cesd-4 is derived from the original 20-item cesd ( 20 ) and has been found to be highly correlated at 0.87 ( 19 ) .
these questions included responses that ranged from 0 to 12 based on the number of days the subject reported experiencing those feelings in the prior week , with higher scores indicating more depressive symptoms .
subjects with a cesd score 4 were determined to have an elevated level of depressive symptoms .
perceived level of stress was measured using a four - item version of the cohen perceived stress scale ( 21 ) , a validated and previously used ( 22 ) instrument for measuring the perception of personal stress .
it measures the extent to which respondents perceive their lives as unpredictable , uncontrollable , and/or overloaded .
subjects with cohen perceived stress scale scores > 4 were determined to have high levels of psychological stress , corresponding to the highest quartile of scores .
the primary outcomes of interest were physician - adjudicated cv events including stroke , myocardial infarction ( mi)/acute coronary heart disease ( chd ) , and cv death .
living participants or their proxies were contacted every 6 months via telephone to assess new - onset stroke , chd events , and cv mortality .
a trained interviewer administered a standardized questionnaire that specifically asks whether , since the last follow - up , they had been hospitalized for stroke or heart disease .
medical records were retrieved for all potential stroke- and chd - related hospitalizations and deaths . for suspected stroke ,
each event was adjudicated via medical record review by a neurologist - led medical review team .
stroke events were defined following the world health organization definition ( 23 ) but also included events with symptoms lasting < 24 h with neuroimaging consistent with acute ischemia or hemorrhage and cases where adjudicators agreed that the event was likely a stroke or death related to stroke but information was incomplete for world health organization or clinical classification .
after a report of a hospitalization or death that potentially could be related to cvd , medical records were retrieved , and the event was adjudicated by a physician - led medical review team , following published guidelines ( 24,25 ) .
specifically , medical records were examined for the presence of signs or symptoms suggestive of ischemia ; a rising and/or falling pattern in cardiac troponin or creatine phosphokinase - mb over 6 or more hours with a peak value greater than or equal to twice the upper limit of normal ( diagnostic cardiac enzymes ) ; and ecg changes consistent with ischemia or mi , guided by the minnesota code and classified as evolving diagnostic , positive , nonspecific , or not consistent with ischemia ( 26,27 ) .
participant deaths were detected by report of next of kin , online sources ( e.g. , the social security death index ) , and the national death index .
for information surrounding the circumstances of participant death , proxies or next of kin were interviewed . additionally , following published guidelines , medical records in the last year of life , death certificates , and autopsy reports
were collected and reviewed by the physician - led adjudicators to determine whether the death was a cvd death ( 24,25 ) .
data on traditional cv risk factors and socioeconomic factors were also collected in order to adjust for potential confounding in our analyses of the relationship between the presence of comorbid behavioral problems , diabetes , and adverse cv outcomes .
additional data collected included the following : annual household income ( < $ 20,000/year , $ 20,000$35,000/year , $ 35,000$75,000/year , and > $ 75,000/year ) , education level ( less than high school education , high school graduate , some college , and college graduate or higher ) , and health insurance ( yes or no ) .
also collected was a health history including a history of stroke or heart disease ( self - reported mi , coronary artery bypass grafting , bypass , angioplasty , stenting , or evidence of myocardial infarction on the study ecg ) .
smoking behavior was collected and categorized as nonsmoker , past smoker , or current smoker .
the current study examined the relationship between comorbid depressive symptoms and/or elevated levels of perceived stress , both measured at baseline , in subjects with and without diabetes and the adjudicated incidence of stroke , acute chd , and cvd death for events occurring through 30 december 2010 .
follow - up time for each participant was calculated from the date of the in - home visit to date of first stroke , acute chd , death , or last telephone follow - up .
the analysis cohort for this report was 22,003 after exclusion of participants with a history of stroke or heart disease at baseline ( n = 7,164 ) , missing diabetes status at baseline ( n = 845 ) , or missing follow - up data ( n = 171 ) . the initial analysis compared the demographic , socioeconomic , and cv risk characteristics of subjects with and without diabetes in three groups : 1 ) those reporting no comorbid depressive symptoms and without elevated levels of perceived stress , 2 ) those reporting either depressive symptoms or elevated levels of perceived stress , and 3 ) those reporting both depressive symptoms and elevated levels of perceived stress .
age - adjusted incidence rates were then compared for each type of cv event in individuals with and without diabetes in the same three comparison groups . the longitudinal relationship between baseline behavioral morbidity ( i.e. , the three comparison groups ) , in both those with and without diabetes , and subsequent adjudicated cv events during follow - up was then examined in a series of cox proportional hazards models .
these models examined the relationships first in an unadjusted ( crude ) model and then in additional models that incrementally added the following groups of variables : 1 ) demographic characteristics ( race , age , sex , and region [ stroke belt vs. non stroke belt ] ) , 2 ) socioeconomic factors ( income , education , and health insurance ) , and , finally , 3 ) cv risk factors ( current smoking , history of heart disease , left ventricular hypertrophy , atrial fibrillation , bmi , systolic blood pressure , total cholesterol , hs - crp , and statin use ) .
the description of the population for this analysis has previously been reported ( 15 ) . in short ,
the reasons for geographic and racial differences in stroke ( regards ) study is a population - based , prospective , longitudinal cohort study of 30,239 subjects aged > 45 years , 45% of whom are male , 55% female , 41% black , 59% white , 55% from the stroke belt
it was designed to examine factors associated with the geographic and racial differences in stroke incidence as well as excess stroke mortality in the southeastern u.s .
( i.e. , stroke belt ) relative to the rest of the nation and among blacks relative to whites .
the regards study included relevant measures for perceived stress and depressive symptoms and provides a unique opportunity to examine the impact of these behavioral comorbidities in individuals with and without diabetes and the incidence of adverse cv outcomes in a national sample of black and white adults .
the regards cohort was recruited between january 2003 and october 2007 and is evaluated by computer - assisted telephone interview every 6 months .
the initial recruitment call was used to obtain verbal consent and collect cv history , risk factors , and demographic characteristics , including age , sex , and race ( each subject self - reported race , and by design , the study compared only non - hispanic black and white subjects , excluding other racial / ethnic groups ) .
the study also included an in - home visit by a trained health professional for data collection .
participants were asked to provide all prescription and nonprescription medications they had taken in the past 2 weeks , and medication names were recorded during the in - home visit and subsequently coded into drug classes . written informed consent was obtained during the in - person evaluation .
the present analysis included all members of the cohort who had complete follow - up data .
a brief physical exam including blood pressure ( assessed as the average of two measurements obtained with an aneroid sphygmomanometer after the subject was in the seated position for at least 3 min with both feet on the floor ) and blood and urine samples , and an electrocardiogram ( ecg ) was conducted during an in - home visit by a trained health professional 34 weeks after the telephone interview .
height and weight was measured via standard procedures , and bmi was computed as the body weight in kilograms divided by the square of height in meters ( 2 ) .
all ecgs were read by a trained cardiologist in a centralized ecg reading laboratory at the wake forest university / baptist medical center in winston - salem , nc , using a predefined interpretation protocol ( 16 ) .
atrial fibrillation was determined to be present if there was a self - reported history ( asked as has a doctor or other health professional ever told you that you had atrial fibrillation ? ) or if characteristic findings were present on the ecg as defined in the protocol .
left ventricular hypertrophy was defined to be present or absent based on ecg findings using the sokolow - lyon limb lead criteria ( 17 ) as specified in the protocol .
hs - crp was determined by particle - enhanced immunonephelometry using the bnii nephelometer ( n high sensitivity crp ; dade behring , deerfield , il ) , while total and hdl cholesterol and glucose were measured by colorimetric reflectance spectrophotometry using the ortho clinical vitros 950/irc chemistry system ( johnson & johnson clinical diagnostics , new brunswick , nj ) in accordance with the national cholesterol education program guidelines ( 18 ) .
diabetes was defined as self - reported diabetes obtained during the telephone interview or a fasting glucose 126 mg / dl , a nonfasting glucose 200 mg / dl , or the presence of oral hypoglycemic medication or insulin each obtained during the home visit .
the primary exposure of interest was the presence , at baseline , of depressive symptoms and/or elevated levels of perceived stress among individuals with diabetes at baseline compared with those without diabetes .
the presence of depressive symptoms was assessed using the previously validated four - item center for epidemiologic studies depression ( cesd ) questionnaire ( 19 ) .
the cesd-4 is derived from the original 20-item cesd ( 20 ) and has been found to be highly correlated at 0.87 ( 19 ) .
these questions included responses that ranged from 0 to 12 based on the number of days the subject reported experiencing those feelings in the prior week , with higher scores indicating more depressive symptoms .
subjects with a cesd score 4 were determined to have an elevated level of depressive symptoms .
perceived level of stress was measured using a four - item version of the cohen perceived stress scale ( 21 ) , a validated and previously used ( 22 ) instrument for measuring the perception of personal stress .
it measures the extent to which respondents perceive their lives as unpredictable , uncontrollable , and/or overloaded .
subjects with cohen perceived stress scale scores > 4 were determined to have high levels of psychological stress , corresponding to the highest quartile of scores .
the primary outcomes of interest were physician - adjudicated cv events including stroke , myocardial infarction ( mi)/acute coronary heart disease ( chd ) , and cv death .
living participants or their proxies were contacted every 6 months via telephone to assess new - onset stroke , chd events , and cv mortality .
a trained interviewer administered a standardized questionnaire that specifically asks whether , since the last follow - up , they had been hospitalized for stroke or heart disease . for each positive response , the date and time of each event were recorded .
medical records were retrieved for all potential stroke- and chd - related hospitalizations and deaths . for suspected stroke ,
each event was adjudicated via medical record review by a neurologist - led medical review team .
stroke events were defined following the world health organization definition ( 23 ) but also included events with symptoms lasting < 24 h with neuroimaging consistent with acute ischemia or hemorrhage and cases where adjudicators agreed that the event was likely a stroke or death related to stroke but information was incomplete for world health organization or clinical classification .
after a report of a hospitalization or death that potentially could be related to cvd , medical records were retrieved , and the event was adjudicated by a physician - led medical review team , following published guidelines ( 24,25 ) .
specifically , medical records were examined for the presence of signs or symptoms suggestive of ischemia ; a rising and/or falling pattern in cardiac troponin or creatine phosphokinase - mb over 6 or more hours with a peak value greater than or equal to twice the upper limit of normal ( diagnostic cardiac enzymes ) ; and ecg changes consistent with ischemia or mi , guided by the minnesota code and classified as evolving diagnostic , positive , nonspecific , or not consistent with ischemia ( 26,27 ) .
participant deaths were detected by report of next of kin , online sources ( e.g. , the social security death index ) , and the national death index . for information surrounding the circumstances of participant death , proxies or next of kin
additionally , following published guidelines , medical records in the last year of life , death certificates , and autopsy reports were collected and reviewed by the physician - led adjudicators to determine whether the death was a cvd death ( 24,25 ) .
data on traditional cv risk factors and socioeconomic factors were also collected in order to adjust for potential confounding in our analyses of the relationship between the presence of comorbid behavioral problems , diabetes , and adverse cv outcomes .
additional data collected included the following : annual household income ( < $ 20,000/year , $ 20,000$35,000/year , $ 35,000$75,000/year , and > $ 75,000/year ) , education level ( less than high school education , high school graduate , some college , and college graduate or higher ) , and health insurance ( yes or no ) .
also collected was a health history including a history of stroke or heart disease ( self - reported mi , coronary artery bypass grafting , bypass , angioplasty , stenting , or evidence of myocardial infarction on the study ecg ) .
smoking behavior was collected and categorized as nonsmoker , past smoker , or current smoker .
the current study examined the relationship between comorbid depressive symptoms and/or elevated levels of perceived stress , both measured at baseline , in subjects with and without diabetes and the adjudicated incidence of stroke , acute chd , and cvd death for events occurring through 30 december 2010 .
follow - up time for each participant was calculated from the date of the in - home visit to date of first stroke , acute chd , death , or last telephone follow - up .
the analysis cohort for this report was 22,003 after exclusion of participants with a history of stroke or heart disease at baseline ( n = 7,164 ) , missing diabetes status at baseline ( n = 845 ) , or missing follow - up data ( n = 171 ) .
the initial analysis compared the demographic , socioeconomic , and cv risk characteristics of subjects with and without diabetes in three groups : 1 ) those reporting no comorbid depressive symptoms and without elevated levels of perceived stress , 2 ) those reporting either depressive symptoms or elevated levels of perceived stress , and 3 ) those reporting both depressive symptoms and elevated levels of perceived stress .
age - adjusted incidence rates were then compared for each type of cv event in individuals with and without diabetes in the same three comparison groups . the longitudinal relationship between baseline behavioral morbidity ( i.e. , the three comparison groups ) , in both those with and without diabetes , and
subsequent adjudicated cv events during follow - up was then examined in a series of cox proportional hazards models .
these models examined the relationships first in an unadjusted ( crude ) model and then in additional models that incrementally added the following groups of variables : 1 ) demographic characteristics ( race , age , sex , and region [ stroke belt vs. non stroke belt ] ) , 2 ) socioeconomic factors ( income , education , and health insurance ) , and , finally , 3 ) cv risk factors ( current smoking , history of heart disease , left ventricular hypertrophy , atrial fibrillation , bmi , systolic blood pressure , total cholesterol , hs - crp , and statin use ) .
the current study included a total of 22,003 subjects , of whom 42% were black , 58% female , and 56% living in the southeastern u.s . stroke belt . among all subjects at baseline , 18.6% subjects ( n = 4,090 ) had diabetes , 10% ( n = 2,202 ) reported increased depressive symptoms , and 28% ( n = 6,132 ) reported elevated levels of stress .
subjects with diabetes were more likely to report either elevated depressive symptoms or stress or both than were subjects without diabetes ( 36.8% vs. 29.5% ; p < 0.001 ) .
detailed demographic characteristics for the study group are given in table 1 , separated into those with and without diabetes and stratified by the presence of behavioral comorbidities . among subjects with diabetes ,
those reporting either increased depressive symptoms or stress or the combination were more likely to be women , black , live in the stroke belt , and have limited income .
subjects reporting either elevated depressive symptoms or stress or both had a higher prevalence of elevated baseline hs - crp values . across each category of behavioral comorbidity ( i.e. , none , either elevated depressive symptoms or stress , both elevated ) , subjects with diabetes had a higher prevalence of elevated baseline hs - crp values , higher mean systolic bp , and greater prevalence of statin use than those without diabetes .
baseline characteristics of regards participants with or without diabetes , by depressive symptom / stress category * p value for comparison of subjects with diabetes vs. subjects with no diabetes .
table 2 provides age - adjusted incidence rates per 1,000 person - years of follow - up for each of the three independent cv outcomes , broken out by the presence of behavioral comorbidities in those with and without diabetes . with respect to individual cv outcomes ,
the pattern of age - adjusted incidence rates was approximately twofold higher in those with diabetes than in those without diabetes across the range of comorbid behavioral illness . among individuals with diabetes and one or both behavioral comorbidities , age - adjusted incidence rates were highest for acute chd , followed by cv death , and stroke . among those without diabetes but with one or more behavioral comorbidities , incidence rates were again highest for acute chd but were followed by stroke and cv death .
event numbers , age - adjusted incidence rates per 1,000 person - years , and crude and adjusted hrs for stroke , acute chd , and cv disease death for individuals with stress or depressive symptoms and with stress and depressive symptoms compared with those with neither , for individuals with and without diabetes crude model adjusted for depressive symptom and stress category .
model 1 adjusted for crude model plus demographic factors ( race , sex , age , region ) .
model 2 adjusted for crude model plus demographic factors plus social and economic factors ( income , education , health insurance ) .
full model adjusted for crude model plus demographic factors , social and economic factors , and risk factors ( bmi , total cholesterol , hs - crp , atrial fibrillation , left ventricular hypertrophy , systolic blood pressure , statin use , current cigarette smoking ) .
table 2 also provides the results of a series of crude and progressively adjusted hazard ratio ( hr ) models for each of the three independent cv outcomes , demonstrating the progressive impact of comorbidity on acute chd and cv death among subjects with diabetes , relative to those without diabetes , even in fully adjusted models that accounted for demographic , socioeconomic , and cv risk factors . unlike the pattern for acute chd and cv death ,
the magnitude of the hrs for stroke was very similar in subjects with one versus both behavioral comorbidities and diabetes .
figure 1 illustrates this significant and progressive pattern of worsening fully adjusted hrs for cv death among those with behavioral comorbidities , relative to those with no behavioral comorbidities , in subjects with diabetes and in comparison with those who do not have diabetes . among individuals with diabetes , the presence of a single behavioral comorbidity either increased depressive symptoms or stress increased the risk of cv death by 53% relative to individuals with diabetes but without either behavioral comorbidity . among individuals with diabetes , the presence of both behavioral comorbidities increased depressive symptoms and stress
increased the risk of cv death by 115% relative to individuals with neither behavioral comorbidity , even after adjustment for a wide range of demographic and cv risk factors .
while there was a pattern of increasing risk of cv death among those with one or both behavioral comorbidities ( vs. neither ) in subjects without diabetes , the differences were substantially smaller ( 12% and 27% increases , respectively ) and were not statistically significant .
consequences of comorbid diabetes and elevated depressive symptoms and/or stress on cv death during follow - up among participants in the regards study .
this article joins a growing body of literature that demonstrates a compelling pattern of augmented risk for cv events or cv death associated with comorbid behavioral illness among individuals with diabetes that far exceeds that observed in individuals with these same illnesses but without diabetes .
while several studies have shown that formally diagnosed depression and/or depressive symptoms in subjects who are patients in an existing health care system are associated with incident chd , this is among the first studies to compare the impact of these behavioral comorbidities on cv outcomes and mortality in a population - based sample of subjects with versus without diabetes in the same study .
it is the first study to compare the effects of both depressive symptoms and stress together and separately in subjects with versus without diabetes , obtained by population - based sampling rather than recruitment in established health care settings .
further , the regards study has a large sample size ; includes oversampling of african americans in the stroke belt region , allowing important racial comparisons ; and also includes rich data obtained in the subject s home environment .
the study is novel in that , as a community - sampled study , it provides more precise estimates of the population burden and racial disparities associated with these comorbidities and their cv consequences than other studies in which patients were recruited in health care settings .
the study provides a much larger sample of african americans and illustrates the greater prevalence of comorbidities among african american women in particular , suggesting the need for more careful screening .
further , cv outcomes and cv death were carefully adjudicated events in the current study ; by contrast , no adjudication process occurred in many other studies .
our findings suggest that the adverse consequences observed in individuals with behavioral comorbidities and diabetes are best understood in a larger context or spectrum of linkages between physical and behavioral illness and subsequent outcomes .
elevated levels of stress and depressive symptoms , alone and together , were associated with a pattern of increased hrs for acute coronary disease and cv death in adjusted models both in subjects with and in subjects without diabetes , achieving statistical significance in those with diabetes .
together this pattern suggests the potential for progressive impact of single and multiple behavioral comorbidities on adverse cv outcomes and mortality that is of greatest concern in subjects with comorbid diabetes .
there was a significant pattern of increasing hrs for acute chd and cv death in those with diabetes and with one or both behavioral comorbidities ( none , stress or depressive symptoms alone , or both together ) .
while diabetes has long been known to increase cv outcomes , our findings suggest that , relative to those individuals with diabetes who have no behavioral comorbidity , those with either depressive symptoms or elevated levels of stress as well as those with both together have progressively increased risks for acute chd events and cv mortality during 5 years of follow - up , even when other risk factors are controlled for .
in particular , behavioral comorbidities such as stress or depressive symptoms are not usually screened for in busy primary care practices , and many patients are reluctant to share these symptoms with busy primary care providers .
further , many individuals are reluctant to seek care from mental health providers . among the demographic group in this study shown to have the highest prevalence of comorbid behavioral disease and diabetes ( i.e. , black females with limited income in the southeastern u.s . ) , there may also be distrust of the traditional health care delivery system . as a result , these comorbidities may go unrecognized and unmanaged .
this study therefore provides strong evidence for adverse consequences associated with these comorbidities and suggests the need for more careful research to identify optimal screening and treatment strategies that work in busy primary care practices or other community settings .
interestingly , while the presence of any behavioral comorbidity resulted in important increases in the hr for stroke , the increased hrs for one versus both behavioral comorbidities in subjects with diabetes were the same in fully adjusted models .
this may suggest that a different mechanism is operative in the increased relationship of behavioral comorbidities and stroke .
these findings build on and expand an established literature that links the cross - sectional and longitudinal presence of stress , depressive symptoms , and/or established depression with cv outcomes and/or mortality including in individuals with comorbid diabetes ( 10,2832 ) .
because our study measured these symptoms at baseline and then followed patients longitudinally , it suggests the potential for the chronic influence of these behavioral comorbidities in subjects with diabetes to across time lead to adverse cv outcomes .
indeed , the chronic nature of depressive symptoms and stress in individuals with type 2 diabetes has been described by fisher et al .
( 10,33 ) , who called for a more careful understanding of the conjoint physical and emotional burden in these patients .
further , these authors join our findings in calling for integrating the screening and management of the emotional side of diabetes into regular diabetes care . in addition , hamer et al .
( 34 ) demonstrated an association between stress and cv events with hr of 1.5 and with the suggestion that behavioral processes explained the largest proportion of the variance in the hazards model .
this raises an important question regarding the mechanism(s ) through which behavioral comorbidities result in increased cv events and/or cv death .
an early review by plante ( 35 ) suggested that stress may lead to depressive symptoms that are subsequently associated with accelerated atherosclerosis , endothelial dysfunction , inflammatory reactions , and interstitial disturbances that may predispose the subject to premature cv disease or death . in the current study ,
hs - crp levels were elevated in subjects with diabetes relative to those without diabetes , but among subjects with diabetes , there was a clearly progressive pattern of higher hs - crp levels among those with either versus both behavioral comorbidities .
these elevated hs - crp levels may suggest a pattern of worsening atherosclerotic disease in subjects with diabetes with one or both behavioral comorbidities relative to those with no comorbidity . a recent article by pizzi et al .
( 36 ) supports this and shows that depressive symptoms are associated with progressive longitudinal increases in carotid intima - media thickness relative to those who did not report these symptoms .
this progressive pattern of atherosclerosis associated with these symptoms or potentially other behavioral problems might be expected to be more common in subjects with diabetes and therefore contribute to adverse cv outcomes .
behavioral comorbidities may also interfere with self - care behaviors . among individuals with diabetes , a study by bonnet et al .
( 37 ) showed that those with anxiety or depressive symptoms were less likely to engage in healthy behaviors including physical activity , proper eating behaviors , and avoidance of smoking than were those without anxiety or depressive symptoms .
similarly , a meta - analysis by gonzalez et al . ( 3 ) that included studies of individuals with depressive symptoms showed that those with depressive symptoms or depression were more likely to be nonadherent with medications as well as with the larger treatment regimen .
( 38 ) suggest a stronger relationship between diabetes - related distress and glycemic control than between depressive symptoms and glycemic control , and recent data by this group ( 39 ) show that specific reductions in regimen - related distress can result in improvements in medication adherence , physical activity , and glycemic control , all of which may influence cv risk .
the scope of the implications from these important associations with cv outcomes should be considered in the context of the recent second diabetes attitudes , wishes and needs ( dawn2 ) study , which shows that these behavioral comorbidities know no geographic or ethnic boundaries and are highly prevalent in subjects with diabetes in multiple countries around the world ( 6 ) .
this suggests the need for additional research to understand the mechanisms by which these behavioral comorbidities are associated with cv outcomes in subjects with diabetes as well as to understand innovative intervention strategies that will not only improve the behavioral comorbidities but will also decrease the risk for cv outcomes .
clearly , more research is needed to understand whether the effective treatment of depressive symptoms and stress can lower cvd events in individuals with diabetes . and ,
as noted above , more work needs to be done to establish appropriate screening strategies for behavioral comorbidities that can be effectively disseminated in busy primary care settings where most individuals with diabetes are managed .
this study is a prospective cohort study and not a randomized clinical trial , and the incident outcomes should be understood as associations with baseline characteristics without clear evidence of causality .
by design , included only non - hispanic white and black subjects ; extrapolation to other ethnic groups or to other nations should not be undertaken .
the current study did not include a measure of diabetes duration , which may have influenced the risk of cv events .
the study included data on a large number of cv risk factors that were used to help minimize the influence of confounders ; however , there may have been other unmeasured risk factors that were associated with adverse cv outcomes .
the presence of elevated stress or depressive symptoms in community - dwelling subjects with diabetes was associated with an increased risk for acute chd or cv death .
moreover , subjects with diabetes who reported both stress and depressive symptoms together had the greatest risk for acute chd and cv death .
the presence of either or both behavioral comorbidities in subjects with diabetes was also associated with increased hrs for stroke .
elevated stress and/or depressive symptoms in subjects without diabetes resulted in increased hrs in fully adjusted models that were much smaller in magnitude and not statistically significant .
these findings demonstrate the persistent disparities and negative cv impact of these comorbidities at the population level and suggest the need for more careful integration of behavioral screening and management in primary care settings , where most patients with type 2 diabetes are managed .
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objectiveto evaluate the impact of comorbid depressive symptoms and/or stress on adverse cardiovascular ( cv ) outcomes in individuals with diabetes compared with those without diabetes.research design and methodsinvestigators examined the relationship between baseline depressive symptoms and/or stress in adults with and without diabetes and physician - adjudicated incident cv outcomes including stroke , myocardial infarction / acute coronary heart disease , and cv death over a median follow - up of 5.95 years in the national regards cohort study.resultssubjects included 22,003 adults ( 4,090 with diabetes ) ( mean age 64 years , 58% female , 42% black , and 56% living in the southeastern stroke belt ) .
elevated stress and/or depressive symptoms were more common in subjects with diabetes ( 36.8% vs. 29.5% ; p < 0.001 )
. in fully adjusted models , reporting either elevated stress or depressive symptoms was associated with a significantly increased incidence of stroke ( hr 1.57 [ 95% ci 1.05 , 2.33 ] vs. 1.01 [ 0.79 , 1.30 ] ) and cv death ( 1.53 [ 1.08 , 2.17 ] vs. 1.12 [ 0.90 , 1.38 ] ) in subjects with diabetes but not in those without diabetes . the combination of both elevated stress and depressive symptoms in subjects with diabetes was associated with a higher incidence of cv death ( 2.15 [ 1.33 , 3.47 ] ) than either behavioral comorbidity alone ( 1.53 [ 1.08 , 2.17 ] ) and higher than in those with both elevated stress and depressive symptoms but without diabetes ( 1.27 [ 0.86 , 1.88]).conclusionscomorbid stress and/or depressive symptoms are common in individuals with diabetes and together are associated with progressively increased risks for adverse cv outcomes .
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Introduction
Research Design and Methods
Study Design, Cohort Description, and Medication Information
Physical Examination and Laboratory Measures
Diabetes, Depressive Symptoms, and Stress Measures
CV Outcome Measures and Death
Measurement of Potential Confounders
Analysis
Results
Conclusions
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in order to prevent and control microbial proliferation in industrial settings , cleaning and disinfection plans are applied on a regular basis [ 1 , 2 ] . in food processing plants ,
the control of microbial contamination generally involves clean - in - place ( cip ) procedures which consist of running alternated cycles of detergent and disinfectant solutions with water rinses in high turbulence regimes through the plant and pipeline circuits without dismantling or opening the equipment [ 25 ] .
biocides are currently used in industrial processes as the most significant countermeasure to control microbial growth and proliferation .
industry moved progressively towards the use of surfactants that are less toxic and more biodegradable .
surfactants are classified according to the ionic physiognomies of their hydrophilic group as anionic , cationic , nonionic , and zwitterionic [ 6 , 8 ] .
quaternary ammonium compounds ( qacs ) are cationic surfactants that are commonly used because of their hard - surface cleaning , odor removal and antimicrobial properties . besides killing bacteria , the chemical nature of qacs can cause modifications on the properties of abiotic surfaces , decreasing their tension and therefore preventing attachment of microorganisms .
the antimicrobial mode of action of cationic surfactants is proposed by some authors as a sequence of events : attraction by the negatively charged cell surface ; adsorption to the cell wall through the hydrophobic headgroup ; reaction with the lipids and proteins that compose the cytoplasmic membrane ; and cell penetration and interaction with intracellular constituents [ 10 , 11 ] .
thus , qacs damage the outer layers of bacteria , thereby promoting the release of intracellular constituents .
antimicrobial efficacy tests require planning of an adequate strategy and should include all the parameters found in real settings .
aspects such as the proper contact time under known water hardness and conditions of high or low soil content should be considered . for an effective cleaning and disinfection plan
, the choice of the disinfectant must follow specific criteria such as compatibility with the surfaces to be disinfected , economic constraints , safety in the workplace , toxicological safety , and biological degradability .
it should , most of all , target the type of bacteria and the type of soiling .
in fact , disinfectants can be seriously affected by the presence of organic matter .
interfering substances have been studied in the last years and included in cleaning and disinfection plans regulated by the authorities such as the european standard en-1276 .
however , most of these studies only address the effects of bovine serum albumin ( bsa ) and water hardness [ 9 , 14 , 15 , 1921 ] .
evaluated the bactericidal activity of disinfectants referred in the german veterinary society guidelines as references for testing disinfectants used in dairy and food industries . in order to simulate the conditions found in practice , they used low fat milk as an organic load and reported the significance in choosing an appropriate disinfectant since the inclusion of a challenging substance ( organic material ) is important to access the proper bactericidal activity .
bessems demonstrated that a qac tested on three microorganisms ( pseudomonas aeruginosa , staphylococcus aureus , and candida albicans ) had a similar killing rate in the absence of interfering substances and after the inclusion of 17 dh water hardness , a strong reduction of the killing activity was found for the gram - negative bacteria
assessed the effect of dried yeast and human serum on the activity of benzalkonium chloride and concluded that the bactericidal activity of the qac was inhibited by solutions of both interfering substances .
this work provides information on the influence of potential interfering substances ( bovine serum albumin
ha ) on the antimicrobial activity of two qacs ( benzalkonium chloride and cetyltrimethyl ammonium bromide ) against bacillus cereus and pseudomonas fluorescens , as they are two major contaminants in the food industry , particularly the dairy industry , and are a known cause of produce spoilage and foodborne illnesses [ 2 , 2226 ] .
some of the interfering substances used throughout the experiments are proposed in the european standard en-1276 as potential interfering agents in disinfection while the others are extracellular polymeric substances ( eps ) from the biofilm matrix that have an important role in antimicrobial resistance .
the bacteria used in this work were pseudomonas fluorescens atcc 13525 and a bacillus cereus strain , isolated from a disinfectant solution and identified by 16s rrna gene sequencing .
bacterial strains were grown at a temperature of 30 2c and ph 7 , with glucose as the main carbon source .
culture medium consisted of 5 gl glucose , 2.5 gl peptone , and 1.25 gl yeast extract in phosphate buffer ( pb ) ( ph 7 , 0.025 m ) . a bacterial suspension was prepared by inoculation of a single colony grown on solid medium into a 1 l flask containing 250 ml of sterile nutrient medium .
this bacterial suspension was incubated overnight at the given temperature with agitation ( 120 rpm ) .
the qacs used throughout the experiments were benzalkonium chloride ( bac ) and cetyltrimethyl ammonium bromide ( ctab ) ( sigma , portugal ) .
preliminary studies with a concentration range between 0 and 5000 mgl were initially made . in order to ascertain the behaviour of bacteria to the qac ,
the selected concentrations for further studies were 3 , 5 , 10 , 20 , and 35 mgl .
the qacs were used individually and in combination ( both chemicals were combined in equal volumes and concentrations ) .
ha ( acros organics , fisher chemical , portugal ) , and yeast extract ye ( merck , portugal ) .
after the growth period , the suspensions were centrifuged ( 3999 g , 5 minutes ) , washed two times , and resuspended in pb to a final cell density of approximately 1 10 cellsml . in the case of the consortium ,
both bacterial suspensions were washed two times resuspended in pb to a final cell density of approximately 1 10 cellsml , and combined in equal volumes to obtain the same cell concentrations of the single species tests .
afterwards , all bacterial suspensions were exposed to several concentrations of qac for a period of 30 minutes .
the effects of the chemicals were evaluated by the assessment of the oxygen uptake rate due to glucose oxidation , according to simes et al . .
to investigate the influence of interfering substances on the antimicrobial efficacy , the same procedure was followed with the addition of 300 mgl of bsa , alg , ye , or ha to the bacterial suspension , simulating low concentrations of interfering substances according to the european standard en-1276 . three independent experiments , each with duplicate samples , were performed for each condition tested .
the methodology was performed according to johnston et al . for a period of 10 minutes .
bac and ctab were chemically neutralized by a sterile solution of ( w / v ) 0.1% peptone , 0.5% tween 80 , 0.1% sodium thiosulphate , and 0.07% lecithin dissolved in pb .
control experiments were performed to ascertain the effects of the 10-minute exposure to the neutralization solution , and no effects were detected on the respiratory activity of b. cereus and p. fluorescens ( data not shown ) .
after the neutralization step , the bacterial suspensions were centrifuged ( 3999 g , 5 min ) and resuspended in the same volume of pb .
the respiratory activity was ascertained by measuring oxygen uptake rates in a biological oxygen monitor ( yellow springs instruments 5300a ) .
demonstrated that this procedure is more adequate and rapid than the assessment of colony forming units to characterize the antimicrobial activity of biocides against heterotrophic aerobic bacteria .
samples were placed in the temperature - controlled vessel of the biological oxygen monitor ( t = 25 1c ) each containing a dissolved oxygen probe connected to a dissolved oxygen meter . before measuring
, the samples were aerated for 10 minutes to ensure oxygen saturation ( [ o2 ] = 8.6 mgl ) .
the vessel was closed , and the decrease of oxygen concentration was monitored over time .
the initial linear decrease corresponds to the endogenous respiration rate . to determine the oxygen uptake due to substrate oxidation , 12.5 l of a 5 gl glucose solution was added to each vessel .
the slope of the initial linear decrease in dissolved oxygen , after glucose injection , corresponds to the total respiration rate .
the difference between these two rates is the oxygen uptake rate due to glucose oxidation .
the inactivation was calculated using metabolic activities according to the following equation :
( 1)% inactivation=(mcmt)mc100 ,
where mc is the metabolic activity of the control experiments ( without antimicrobial exposure ) and mt is the metabolic activity of the bacterial solutions exposed to the antimicrobial .
if % inactivation > 0 there was inactivation of the microorganisms whereas if % inactivation < 0 there was metabolic potentiation .
the mbc for each situation was determined as the lowest concentration of qac or qac combination where no respiratory activity was detected . for each parameter
the statistical significance of the results was evaluated using the wilcoxon test ( confidence level 95% ) to investigate whether the differences between the resulting experimental values could be considered significant .
the antibacterial activity of bac , ctab , and their combination was investigated in the absence and in the presence of four selected interfering substances . in the absence of interfering substances bac caused the inactivation of b. cereus at 10 mgl , p. fluorescens at 35 mgl , and the consortium at 20 mgl .
ctab at 20 mgl completely inactivated b. cereus and at 35 mgl inactivated the total population of p. fluorescens and the consortium .
the combination of both qacs was synergistic in the inactivation of b. cereus ( total inactivation with 3 mgl ) and indifferent for p. fluorescens ( 35 mgl ) and the bacterial consortium ( 35 mgl ) .
the inclusion of the selected interfering substances influenced the antimicrobial activity of the qacs to some extent ( figures 13 ) .
the inactivation of b. cereus ( figure 1 ) was not affected by the presence of any interfering substances ( p > 0.05 ) , except with ha .
the antimicrobial action of the qacs against p. fluorescens ( figure 2 ) was not significantly influenced by the presence of most potential interfering substances ( p > 0.05 ) , except for ha where interference was observed ( p < 0.05 ) .
the antimicrobial activity of the qacs against the bacterial consortium ( figure 3 ) was affected by the presence of interfering substances .
ha reduced significantly the activity of ctab at higher concentrations ( p < 0.05 ) .
bsa and ye resulted in a significant reduction of the activity of the combination of qacs ( p < 0.05 ) .
linear correlations were determined to assess the relationship between qac concentrations and the inactivation data .
the effect of increasing qac concentration on bacterial inactivation shows that there are strong linear correlations ( r > 0.850 ) for the control assays , with the exception of b. cereus ( this bacterium was inactivated with low qac concentrations ) .
the most extreme cases are the treatments with ctab to p. fluorescens with alg as an interfering substance ( r = 0.771 ) and the bacterial consortium in the presence of ye ( r = 0.738 ) .
likewise , this decrease of linear correlation factors was found for p. fluorescens and for the consortium exposed to ha where the lowest correlation factor was 0.153 , which was obtained for p. fluorescens treated with ctab .
this phenomenon only happened when the qacs were used on p. fluorescens and the bacterial consortium in the presence of ye and ha .
the most significant cases of oxygen uptake rate increase were verified for p. fluorescens exposed to bac ( 5 to 35 mgl ) and ctab ( 3 to 35 mgl ) in the presence of ha and combination of qacs ( 3 to 10 mgl ) in the presence of ye .
a similar metabolic behaviour was found for the bacterial consortium exposed to bac ( 3 to 35 mgl ) and ctab ( 5 and 10 mgl ) for ha and qac combination ( 3 to 20 mgl ) with ye .
the mbc values for the different conditions tested ( single and combined qacs , in the absence and presence of potential disinfection interfering substances ) are shown in table 1 .
the presence of bsa increased the mbc of the combination of qacs for b. cereus ( 3 to 5 mgl ) and the consortium .
alg increased the mbc of bac for the consortium ( 20 to over 35 mgl ) and qacs combination ( 3 to 5 mgl ) for b. cereus .
ye increased the mbc of bac for b. cereus ( 10 to 20 mgl ) and qac combination ( 3 to 5 mgl ) .
the mbc values for the consortium of cells increased in the presence of ye ( bac20 to 35 mgl , ctab35 to over 35 mgl , and qac combination35 to over 35 mgl ) .
ha increased the mbc for all the scenarios , except of ctab when applied to b. cereus ( in this situation the mbc was reduced ) .
the mbc was reduced in other situations such as , for b. cereus , in the presence of alg when using bac and ctab ( 10 to 5 mgl and 20 to 5 mgl , resp . ) and in the presence of ye when using ctab ( 20 to 3 mgl ) .
fluorescens inactivation by ctab was reduced by bsa ( 35 to 20 mgl ) .
alg also reduced the antimicrobial activity of the combination of qacs against the bacterial consortium ( 35 to 20 mgl ) .
it is assumed that the organic material can potentially interfere with the antimicrobial agents by chemical and/or ionic interactions [ 15 , 33 ] .
therefore , it is necessary to know the role of each potential interfering substance in the antimicrobial activity in order to develop effective disinfection strategies .
the interfering substances tested are commonly found as residuals in the food industry ( from food products and from microbial contaminants , biofilms ) [ 18 , 27 ] . in this study
, higher inactivation rates were verified for b. cereus in comparison to p. fluorescens at the same qac concentration .
in fact , when b. cereus and p. fluorescens are combined in a 1 : 1 bacterial suspension , it is expected that the first is more affected than the second .
b. cereus is more susceptible due to the fact that it is a gram - positive bacterium that lacks an outer membrane , which typically provides increased protection to gram - negative bacteria .
this fact is corroborated by previous reports which stated that gram - positive bacteria are more susceptible to cationic surfactants than gram - negative bacteria [ 34 , 35 ] .
bsa was already studied as an interfering substance in disinfection practices [ 9 , 14 , 1921 , 36 ] .
the negative effect of bsa on the action of biocides against p. fluorescens was demonstrated by simes et al .
p. fluorescens treatment with ctab with the addition of 3 gl of bsa resulted in a 10-fold increase on the mbc of this qac [ 9 , 21 ] . in the present study ,
the efficacy of the combination of qacs against b. cereus and the cell consortium was also reduced .
this effect of bsa as an antimicrobial quencher is apparently due to the strong ability of qacs to react with proteins .
proteins can precipitate in the form of their anions , in this way , the negative - charged protein ions will cling to the positively charged molecules of the cationic compounds .
ctab is a biocide that targets the membrane and has a strong affinity for proteins .
bac is composed of a positively charged hydrophobic headgroup which clings to opposite charged surfaces [ 8 , 37 ] .
studied the effect of the alkyl chain of bac binding to bsa and dried yeast .
their conclusions were that bac is often inactivated by organic matter , either by adsorption to the bacterial surface or by adsorption to the organic matter in general .
these authors also suggested that the reduction in the activity of bac was probably related to more than one physical property of the compounds like the chain length ( longer chains result in more adsorption to the bacterial surface ) .
alg is a common constituent of the extracellular polymeric substances of the biofilm matrix [ 3840 ] .
a function frequently attributed to eps is their general protective effect on biofilm microorganisms against adverse conditions .
the eps matrix delays or prevents antimicrobials from reaching target microorganisms within the biofilm by diffusion limitation and/or chemical interaction with the extracellular proteins and polysaccharides [ 32 , 41 ] . in this study , alg either potentiated or hindered the antimicrobial activity of the selected qacs .
the presence of this interfering substance was not obvious on the inactivation of p. fluorescens .
on the other hand , the inactivation of b. cereus by bac and ctab and the consortium by the combination of qacs was easier in the presence of this interfering substance .
the bacterial consortium treatments with bac and b. cereus with the combination of qacs were hampered by the presence of alg .
davies et al . found that the production of alg was triggered by membrane perturbation induced by ethanol stress , nitrogen limitation , attachment to surfaces , or even high oxygen tension [ 42 , 43 ] .
this substance is suggested as one of the main biofilm resistance vectors either by reacting with the antimicrobials or by hindering antimicrobials diffusion to the cells .
the antimicrobial interference caused by alg is apparently due to electrostatic interactions between the anionic alg and the cationic - selected qacs .
the presence of ye as interfering substance resulted in three different outcomes on the antimicrobial activity of the qacs : ( 1 ) no effect / indifference , ( 2 ) the respiratory activity reduced , and ( 3 ) the respiratory activity potentiated .
this interfering substance worked mainly as a hinderer of the antimicrobial activity by increasing the mbc of b. cereus in all cases except for ctab , of p. fluorescens with the combination of qacs , and of the consortium of cells with ctab and the combination of qacs .
ye is listed in the european standard en-1276 as an interfering substance native to the brewery industry .
the constituents of ye are very similar to the components of the bacterial cells , thus , it is expected that the antimicrobial agents that target the bacterial cells are also drawn to ye . in a similar study by jon et al .
it was shown that the presence of dried yeast decreased the biocidal effectiveness of bac .
humic substances are found ubiquitously in the environment and can be found in the biofilm matrix [ 2 , 46 ] .
ha reduced the antimicrobial activity of the qacs in most of the cases , although in some cases it promoted the respiratory activity ( potentiation ) .
the presence of these compounds had the strongest effect compared to the remaining interfering substances .
studied the sorption mechanisms of anionic and cationic surfactants to natural soils concluding that the dominant sorption mechanism of surfactants to clay is cation exchange .
koopal et al . also verified the formation of complexes ha - cationic surfactant .
respiratory activity potentiation was verified with the addition of ha to p. fluorescens and ye to the bacterial consortium .
it is known that ha participates in cellular metabolism processes such as growth , respiration , photosynthesis , and nitrogen fixation . on the other hand , ha were proposed to replace synthetic surfactants such as sds , tween 80 , and triton x-100 in industrial applications such as textile dying or washing .
it is therefore possible that the inclusion of humic substances in a solution of qacs may interfere with the chemical characteristics of the solution .
the resultant mixture , with an apparent reduced antimicrobial efficacy , seems to potentiate the respiratory activity of the bacteria , particularly of p. fluorescens .
as qacs are membrane active agents , their use at sublethal concentrations could improve membrane permeability and consequently the nutrient influx , without compromising the bacterial viability . also , there is the hypothesis that the potentially interfering agents could be used as nutrients .
in fact , it was found that the growth rates of anaerobic and aerobic microorganisms increased when humic substances were added , which stimulated enzyme activity [ 52 , 53 ] . in a similar way ,
ha are likely to be used for growth in the same way as ye ; these might be broken down to smaller molecules that can be used by cells as a carbon or nitrogen sources .
the antimicrobial activity of the tested qacs was enhanced in some cases , where the interfering substances were present .
this is an unexpected result due to the recognized potential of alg , bsa , ha , and ye to interfere with disinfection .
this effect is probably due to the low concentration of interfering substances tested that caused both respiratory activity reduction and potentiation .
ethylenediamine tetraacetate ( edta ) was reported as early as 1965 to increase the biocidal effects of bac and chlorhexidine diacetate on pseudomonas aeruginosa .
reported that chitosan ( a polysaccharide ) potentiated the antimicrobial action of sodium benzoate on spoilage yeasts . in dairy plants
, disinfection is potentiated by pre - washes with alkali or enzyme - based cleaning agents .
most of these cases were observed for b. cereus ( four occurrences ) , one was observed for p. fluorescens , and another one was observed for the consortium of cells .
the mbc was improved by more than 50% in the cases of b. cereus and less than 30% for p. fluorescens and the consortium of cells . to our knowledge
there are no reported cases of antimicrobial agents potentiation by bsa , ye , or alg .
although the exact chemical structure of ha has not yet been determined , ha could be chemically similar to the tested qacs , presenting a positive hydrophilic head and a hydrophobic tail . with this structure ha could act as detergents in conditions such as those observed in the treatment of b. cereus with ctab .
the present work shows that increasing qacs concentrations lead to an increase in antimicrobial effectiveness .
this is valid mainly when the qacs were applied in the absence of interfering substances .
this means that disinfection was concentration dependent , as found for most of the antimicrobial chemicals .
however , the linear dependency of inactivation versus concentration is not verified for most of the tests where interfering substances were added .
this result evidences that the mathematical modelling of disinfection strategies requires a case - to - case analysis when interfering substances are present .
the overall results demonstrate that a disinfection process in the presence of the selected interfering substances can reduce the effectiveness of bac , ctab , and their combination .
the bacteria were inactivated equally by all qacs , although in the absence of interfering substances ctab was the most efficient solution .
p. fluorescens was the bacterium with the highest resistance to inactivation , followed by the bacterial consortium .
the tested interfering substances , referred in the european standard 1276 ( bsa and ye ) , and known eps constituents related with biofilm resistance ( alg ) resulted in mild interferences on the activity of the qacs .
ha were the interfering substance that resulted in the most severe effect by reducing the activity of qacs , causing , in some circumstances , significant respiratory activity potentiation .
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standard cleaning processes may not remove all the soiling typically found in food industry , such as carbohydrates , fats , or proteins .
contaminants have a high impact in disinfection as their presence may reduce the activity of disinfectants .
the influence of alginic acid , bovine serum albumin , yeast extract , and humic acids was assessed on the antimicrobial activities of benzalkonium chloride and cetyltrimethyl ammonium bromide against bacillus cereus vegetative cells and pseudomonas fluorescens .
the bacteria ( single and consortium ) were exposed to surfactants ( single and combined ) in the absence and presence of potential disinfection interfering substances .
the antimicrobial effects of the surfactants were assessed based on the bacterial respiratory activity measured by oxygen uptake rate due to glucose oxidation .
the tested surfactants were efficient against both bacteria ( single and consortium ) with minimum bactericidal concentrations ranging from 3 to 35 mgl1 .
the strongest effect was caused by humic acids that severely quenched antimicrobial action , increasing the minimum bactericidal concentration of the surfactants on p. fluorescens and the consortium .
the inclusion of the other interfering substances resulted in mild interferences in the antibacterial activity .
this study clearly demonstrates that humic acids should be considered as an antimicrobial interfering substance in the development of disinfection strategies .
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1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusions
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social scientists who explore factors mediating and moderating the relationships between social stressors and mental health , including drinking outcomes , have highlighted modes of coping [ 1 , 2 ] . these studies have explored behaviors which protect people from being psychologically harmed and cognitive appraisals which influence behaviors such as problem - focused coping or using alcohol to self - medicate distress .
however , studies have not considered the characteristics of the stressful situation itself that may make certain coping strategies more or less effective . in particular ,
psychiatric epidemiologic studies have tended to emphasize microlevel stressors ( e.g. , stressors in individuals ' role domains ) and , until recently , have ignored the linkages between macrolevel social forces and the daily stressors in people 's lives [ 79 ] .
however , macrolevel social conditions can affect the magnitude of stressors experienced in people 's lives and the extent to which they experience cumulative adversity .
this paper focuses on coping with the fallout from one type of macrolevel social stressor : the recent great recession .
this economic downturn constituted the most severe economic crisis in the united states since the great depression and had persisting economic effects ( e.g. , job loss , less desirable working conditions , loss of home , loss of retirement savings , lack of health care access , and social isolation ) which have been linked with deleterious drinking outcomes .
a key issue involving the effectiveness of alternative modes of coping with stressors derived from macrolevel social forces and protecting against deleterious drinking outcomes is the question of whether individual modes of coping outside of the political realm emphasized in the overall coping literature are most efficacious . or , alternatively , do stressors engendered by macrolevel social forces require unique forms of coping , that encompass the political realm ?
with respect to coping strategies employed in the wake of the great recession , politically oriented coping strategies might be of particular relevance due to the impact of governmental decision - making on the state of the economy .
politically oriented coping strategies might include political activism oriented to altering economic policies or support for campaigns by politicians offering solutions to economically based hardships . scholarly work on the occupy wall street movement , for example , documents involvement by individuals sharing their own economic struggles and collectively gathering to protest their precarious economic situation and uncertain economic future .
earlier work also suggests the salience of activities oriented toward changing politically based social realities such as through the act of voting or by collectively challenging community - level decisions such as school closings .
by contrast , the traditional coping literature has emphasized individual , nonpolitical modes of coping , such as emotional acceptance of the stressful situation , blaming one 's self for the situation , or taking individual actions such as looking for a job if unemployed .
while sociologists have recently accorded greater attention to the prevalence and alcohol - related consequences of macrolevel stressors , they have yet to address the extent to which politically oriented modes of coping may be the most efficacious ways to address problems stemming from macrolevel social forces or events . prior to the more recent focus on macrolevel stressors , kaplan and liu embraced the idea of collective coping as one means for individuals who maintained stigmatized personal identities to challenge and transform conventional socionormative systems through participation in social movements .
subsequently , thoits more explicitly addressed collective coping within the context of the stress paradigm involving acute and chronic stressors not limited to the specific area of stigmatized statuses .
she argued that individuals who find themselves in problematic situations can deliberately work to transform the meaning of their experiences and they can additionally use these experiences as a basis for helping or effecting changes in the lives of others
she proposed the concept of transformatory coping to include engagement in collective activist activities with others who share similar problems .
for example , parents of autistic children have lobbied governments for social services perceived to aid in their children 's development . in sum , ( 1 ) collective coping tactics ( such as politically oriented coping ) represent an unmeasured dimension of coping behaviors beyond that represented in the coping literature to date and ( 2 ) collective coping tactics may demonstrate a stronger association between stressors , particularly those stemming from macrolevel social forces , and deleterious drinking outcomes compared to the use of modes of coping previously emphasized in the literature on coping .
the present study extends previous work by empirically addressing the extent to which politically oriented coping activities engaged in as a response to a macrolevel social stressor , the great recession , are protective against alcohol - related outcomes compared with coping strategies focused outside of the political realm .
we hypothesize that politically oriented coping will be more protective against economic stressors linked with the great recession than nonpolitical modes of coping and will uniquely account for some portion of the associations between economy - related stressors and drinking outcomes .
further , we also examine whether politically oriented coping and coping outside of the political realm are more protective for men versus women in the face of macrolevel engendered stressors such as those involving the economic fallout from the great recession .
there is consistent evidence that women are more likely to use support - based coping strategies ( e.g. , seeking support from others such as partner family and friends ) in response to stress in contrast to men and some indication that avoidant coping techniques are associated with greater alcohol consumption among men but not women [ 1 , 2022 ] .
( in contrast , parity by gender in the use of individual active coping strategies and their significance for mental health outcomes is generally reported [ 1 , 21 ] . ) however , whether there is a corresponding propensity for men and women to differ in the use of politically oriented coping to offset the alcohol - related effects of economy - related stressors is less certain .
earlier research tended to argue that women were less politically interested , informed , and efficacious compared to men .
more recent work has shown that women and men differ in particular modes of participation ; women are more likely to vote and engage in individual political actions such as signing petitions or donating money , whereas men are more likely to be engaged in collective forms of action such as group protest activities .
thus , we hypothesize that there will be no overall differences in the extent to which women and men manifest politically oriented coping or in the effect that politically oriented coping has on drinking outcomes . following a transactional model of stress
[ 4 , 25 ] , we model coping as a mediator of the relationship between the stressor ( i.e. , stressful consequences of the great recession ) and the stress response ( i.e. , drinking outcomes ) .
data were derived from a study conducted in the united states between june , 2010 , and january , 2011 , that was undertaken in order to understand life change consequences of the major downturn in the economy known as the great recession .
respondents were selected by a random digit dial ( rdd ) phone survey of the continental united states , and those who consented to participate in the study were mailed questionnaires .
eligible respondents were selected from the households using the troldahl - carter - bryant method of respondent selection which involves the means to randomly select a respondent from all eligible household members .
respondents were told during the phone screen that a $ 50 american express gift card would be sent to the eligible respondent if he or she completed the questionnaire .
respondents were mailed an initial survey , a postcard reminder to nonresponders , and a second questionnaire if they still had not responded .
figure 1 encompasses a flow chart characterizing each stage in the data collection process through the completion of the questionnaires .
65.9% ( n = 663 ) of the respondents completing the screening calls returned the questionnaire .
the telephone screening cooperation rate and the mail survey response rate were each calculated using the conservative aapor response rate formula 3 .
the overall survey response rate is the product of the phone screening cooperation rate ( 35.5% ) and the mail questionnaire return response rate ( 65.9% ) or 16.8% .
we acknowledge that this response rate is less than ideal and further address this issue in the discussion of study limitations and note other indicators of the representativeness of the final sample .
selection weights were calculated for each of the cases to weight for the different probability of selection for each case .
poststratification weights were calculated for the dataset to ensure that the distribution of sample cases on important demographic variables ( age , race / ethnicity , and gender ) conformed to the distribution of these variables in census bureau 's 2008 united states population estimates .
it should be noted that estimates of alcohol consumption for the present sample did appear to conform to national estimates preweighting .
for example , the average number of drinks consumed in the past month on days when one drank for the present sample is 2.16 , versus the estimated average of 2.10 reported by the centers for disease control and prevention 's behavioral risk factor surveillance system for 2010 ( by gender , the averages are 2.35 for men and 1.89 for women in the present sample versus 2.43 for men and 1.81 for women in the cdc estimates ; by race / ethnicity , the averages are 2.16 for non - hispanic whites , 2.08 for asians , 2.12 for african americans , and 2.72 for hispanics in the present sample versus 2.26 for non - hispanic whites , 2.41 for asians , 2.19 for african americans , and 2.68 for hispanics in the cdc data ) .
it should additionally be noted that the respondents included in this sample reported an overall higher level of education than the general population based on 2008 census estimates .
analysis of variance revealed no significant variation by education in either the outcomes or support coping , avoidant coping , or politically oriented coping .
respondents with less than a high school degree are found to be marginally less likely ( p < 0.10 ) to use active coping strategies compared to those with a college degree or postcollege training . given this discrepancy as well as the fact that education is generally protective against problem drinking
predictor variables are economy - related stressors , coping strategies enacted outside of the political realm ( i.e. , active coping , support coping , and avoidant coping ) , politically oriented coping , and gender . the sociodemographic characteristics of age , education , and race / ethnicity are controlled in all analyses . to assess past - month drinking patterns , we use the quantity - frequency - variability index ( qfv ) developed by cahalan et al . .
frequency of drinking is measured as a count of the days on which alcohol was consumed in the past 30 days , and quantity of drinking is measured as the average number of drinks consumed on those days .
variability is calculated by the greatest number of drinks consumed on any one day in the past 30 days .
scores are calculated by multiplying responses to the quantity , frequency , and variability questions ( = 0.87 ) . as with the current data
, this index tends to approximate a continuous scale , and ample evidence supports its use as such ( see fitzgerald and mulford for a review ) .
our measure of problematic drinking is the 10-item bmast ( = 0.74 ) , which is a count measure of difficulties related to alcohol use over the past year .
respondents were asked to indicate yes or no in response to 10 items such as having an accident , losing a close friend , spouse , or loved one , being hospitalized , having trouble at work , and soliciting professional help because of one 's drinking . the bmast is one of the most widely used tools for assessing alcohol dependence and problems .
it correlates strongly with the full - length mast and evidence of its reliability and validity is widely available [ 31 , 32 ] .
moreover , we are not using the bmast as a diagnostic tool for depicting problem versus
nonproblem drinking , but as it is intended , as suggestive of different degrees of problematic drinking .
the measure of economy - related stressors is the life change consequences of the great recession ( lccgr ) instrument .
this construct was developed on the basis of qualitative analyses of transcripts derived from focus groups involving both genders and diverse racial / ethnic groups .
the items fall into seven categories : home ownership problems , such as difficulties in mortgage payments , difficulties in paying property taxes , or a drop in credit rating ; undesirable living situation , including having to live in a less desired location to save money or having gas and electricity or heat shut off due to an inability to pay bills ; problematic employment situation , including a pay - cut , furlough days , and increased feelings of competition with fellow employees ; unemployment or underemployment ; inadequate health insurance , including lack of medical or dental coverage , decreased quality of coverage , and inability to obtain coverage ; social role constraints , such as dissolution of spouse / partner relationship , decreased social life , and increased social isolation due to finances ; and inadequate sick time , including inadequate sick days and having to work despite poor health .
consistent with common practice , each score for this measure is a straight count of the number of stressors reported .
three dimensions of nonpolitical modes of coping are assessed : active coping , support coping , and avoidant coping .
these measures of coping are derived from subscales of the brief cope instrument and have previously been validated as stand - alone indices in community samples [ 3436 ] .
participants in the present study were asked whether they have used these coping strategies in response to the economic recession .
confirmatory factor analysis of the present data supports the inclusion of these items as three separate coping indices , consistent with prior research .
active coping ( = 0.84 ) includes eight items measuring acceptance , positive reframing , and planning and taking action in response to the economic recession .
support coping ( = 0.81 ) includes four items measuring use of emotional and instrumental support ( i.e. , receiving emotional support and getting advice and help from other people ) .
avoidant coping ( = 0.75 ) includes 10 items measuring self - distraction , behavioral disengagement , self - denial , blame , and a tendency to vent about or make fun of the situation . all items were rated on a four - point likert - type scale ranging from 0 ( i did not do this at all ) to 3 ( i did this a lot ) . the measure of politically oriented coping
is assessed by a four - item instrument ( = 0.79 ) drawn from the summed responses ( i.e. , not at all , a little , some , quite a bit , and a lot ) to four statements asking how often respondents , in response to the economic recession , have been ( 1 ) engaging in political activities such as signing petitions , leading or participating in rallies or marches , or writing to political representatives ; ( 2 ) organizing with others to challenge politicians currently in office ; ( 3 ) voting in elections to support politicians who share your political beliefs ; and ( 4 ) participating in groups trying to influence the policies of the government at the local , state , or national level .
the questions used to construct this index were derived from analyses of focus group transcripts ( see for details about these focus groups ) .
confirmatory factor analysis reveals that these items load on a single factor , supporting their inclusion as one index .
education is a categorical variable based on the educational attainment categories of ( 1 ) less than high school ( n = 45 ) ; ( 2 ) high school graduate ( n = 350 ) ; ( 3 ) college graduate ( n = 110 ) ; and ( 4 ) postcollege training ( n = 150 ) . race / ethnicity is a dummy variable including non - hispanic whites ( n = 436 ) , african americans ( n = 80 ) , hispanics ( n = 91 ) , asians ( n = 29 ) , and individuals who identify as an other race / ethnicity ( n = 17 ) . in all analyses ,
non - hispanic whites serve as the reference category . after examining bivariate correlations in order to assess the basic patterns of association among key study variables , we performed structural equation modeling ( sem ) using mplus software to examine the predictive significance of economy - related stressors for coping outside of the political realm and politically oriented coping tactics and the two drinking outcomes considered ( i.e. , past - month drinking and problematic drinking ) , net of the sociodemographic control variables .
we considered the potential for nonpolitical coping and politically oriented coping tactics to mediate the associations between economy - related stressors and each of the outcomes assessed in two models .
the first model tested for associations between economic stressors and the drinking - related outcomes .
the second model adds nonpolitical and politically oriented coping tactics to test the full mediation model .
this latter model assesses all of the direct and indirect paths between economic stressors and the drinking outcomes considered through the nonpolitical and politically oriented coping tactics investigated .
we formally tested for mediation using the procedures described by muthen and muthen for mplus software , which apply the tests described by mackinnon et al . .
finally , because the associations between social stress and drinking are found to vary by gender [ 33 , 39 ] , we examined whether any observed mediating effects of the coping strategies investigated vary by gender . for these tests ,
separate equations include the interaction term for gender by each coping strategy in the path models linking coping with drinking outcomes .
it is noteworthy that stressors related to the economy are associated with each of the alcohol - related outcomes and all of the coping resources considered : economy - related stressors are associated with more alcohol consumption and problematic drinking , as well as higher levels of active coping , support coping , avoidant coping , and politically oriented coping .
it is also noteworthy that only two of the coping strategies assessed are associated with the drinking outcomes .
avoidant coping and politically oriented coping are associated with both alcohol consumption and problematic drinking , but in opposite directions .
that is , greater avoidant coping is associated with greater alcohol consumption and problematic drinking , whereas greater politically oriented coping is associated with less alcohol consumption and problematic drinking .
the lack of correlation between active coping and support coping and each of the drinking outcomes , respectively , provides some indication that not all of the coping strategies may be useful in understanding the associations between economic stressors and drinking - related outcomes . additionally , the possibility that coping resources may vary by gender is not strongly supported by the pattern of correlations reported , with the exception that women reported significantly more emotional support than men . however , and consistent with previous research , women are found to drink less and less problematically .
the hypothesized associations between economy - related stressors , coping strategies , and the alcohol - related outcomes are further elaborated upon in the structural equation model .
estimation of the first model ( figure 2 ) , including only economic stressors , the drinking - related outcomes , and sociodemographic controls , produces a just identified model and , as such , meaningful fit statistics are not provided .
the standardized path coefficients demonstrate that economic stressors and each of the drinking - related outcomes considered are significantly and positively related .
net of the sociodemographic controls , greater economic strain is associated with greater alcohol consumption over the past month ( = 0.094 , s.e .
= 0.004 , and p < 0.01 ) and a higher incidence of problematic drinking over the past year ( = 0.181 , s.e .
sem analysis testing the second model , of the hypothesized associations between economy - related stressors , coping strategies , and the alcohol - related outcomes considered , is presented as figure 3 . in figure 3 ,
solid lines indicate effects that are statistically significant , and dashed lines indicate effects that are not statistically significant .
the model fit criteria provided by hu and bentler ( cfi > 0.95 ; rmsea < 0.06 ; srmr < 0.08 ) are used to assess the measurement model . based on these criteria , there is consistent evidence of good fit for this model ( cfi = 0.982 ; rmsea = 0.046 ;
this model demonstrates that economic stress is associated with higher levels of each of the coping resources considered .
that is , greater economic strain appears to predict a greater propensity to use both maladaptive coping tactics ( i.e. , avoidant coping ) and adaptive coping tactics ( i.e. , active coping , support coping , and politically oriented coping ) .
avoidant coping is associated with higher levels of past - month drinking and problematic drinking , whereas politically oriented coping is associated with less alcohol consumption and problematic drinking .
formal mediation tests next reveal that the effects of economy - related stressors on the alcohol - related outcomes assessed are partly explained by variation in avoidant coping and politically oriented coping .
significant indirect effects are found for the pathways from economy - related stress to both past - month drinking and problematic drinking . of the total effect of economy - related stress on past - month drinking ( 0.071 ) ,
0.047 is accounted for by the indirect effect of economy - related stress through avoidant coping and 0.024 is explained by the indirect influence of politically oriented coping .
a similar pattern emerges with respect to the relationship between economy - related stressors and problematic drinking .
the indirect effects of avoidant coping account for 0.046 of the total effect of economic stressors on problematic drinking ( 0.098 ) and politically oriented coping explains 0.032 .
taken together , these findings indicate that both adaptive and maladaptive coping strategies come to bear on drinking patterns associated with economic strain . on the one hand , economic strain appears to be associated with drinking more and more problematically , in the extent to which it is associated with a tendency to engage in avoidant coping strategies .
but , then , again , economic strain is also associated with greater politically oriented coping , which is protective against alcohol use and misuse .
moderation tests next determine whether the mediating effects of the coping strategies investigated vary by gender .
however , the mediating effect of avoidant coping for the economy - related stressor problematic drinking association differs for men and women in this sample . this effect is displayed in figure 4 , which presents the predicted pattern of gender contrasts in the effects of avoidant coping on problematic drinking based on the mean , plus and minus two standard deviation values of avoidant coping ( as displayed in table 1 ) .
as shown in figure 4 , the mediating effects of avoidant coping differ for men and women because the relationship between avoidant coping and problematic drinking is significantly less strong for women compared to men ( = 0.319 , s.e .
thus , the observation that economic strain is associated with greater problematic drinking in the extent to which it is associated with avoidant coping strategies appears to be more pronounced among men than among women .
the findings from this study support a broadened conceptualization of modes of coping in stress paradigm - oriented research to encompass politically oriented coping , in addition to modes of coping outside of the political realm which have predominated in studies of the coping - related moderators or mediators of the associations between social stressors and drinking outcomes .
moreover , given our focus on economic stressors deriving , at least in part , from the macrolevel social forces producing the great recession , we suggest that politically oriented coping is particularly salient as a mode of behavioral adaptation in relation to societally engendered stressors as opposed to stressors that are less affected by macrolevel social forces .
while this study specifically addressed deleterious drinking consequences of the recent great recession , social scientists have also delineated broader social - structural and political forces occurring over the last three decades which have led to pervasive job insecurity across all sectors of the us workforce and the erosion in the standard of living for most of the population [ 4143 ] .
these phenomena include globalization and the outsourcing of work , the downsizing of corporate entities , the shift from secure semiskilled industrial jobs which paid a living wage to low wage service sector jobs , an increase in contingent workers with lower pay , and lack of job security and fringe benefits . thus , politically oriented coping encompassing the goal of changing governmental policies ( e.g. , rallying for changes in the tax structure influencing the distribution of wealth throughout society , fighting for government stimulus policies oriented toward job creation , or rallying support for raising the government - mandated minimum wage ) may prove to be a more efficacious mode of coping with economic stressors and more protective against deleterious drinking outcomes compared to nonpolitically oriented active coping activities such as looking for a job when unemployed , especially if adequate numbers of jobs relative to demand do not exist and a large proportion of jobs that do exist pay less than a living wage .
consistent with our gender - linked hypothesis , our data showed that males and females did not differ either in the use of politically oriented coping or in the extent to which politically oriented coping was protective in relation to drinking outcomes .
however , our data showed that male but not female avoidant coping significantly predicted problem drinking .
thus , with regard to nonpolitically oriented coping with economic stressors , males clearly utilized a coping mode that was maladaptive .
this finding might be seen as congruent with early male socialization patterns which have been viewed as fostering men 's sense of self - importance rather than connectedness in social relationships insofar as men 's avoidance in dealing with economic problems may affect both themselves and others close to them .
alternatively , avoidant coping may predict problem drinking in men but not women to the extent to which males are socialized to drink more heavily than women .
our findings should be viewed within the context of the methodological limitations of this study .
the politically oriented coping measure which we developed for this study represents an initial attempt to operationalize this concept .
however , it is limited to 4 items in contrast to the much longer nonpolitically oriented coping instrument and does not differentiate politically oriented coping tactics into discrete modes , similar to the measurement of nonpolitically oriented coping .
although factor analysis supported the inclusion of the indicators of politically oriented coping as a single measure , additional work on the concept of politically oriented coping would be useful , with a differentiation between alternative types of politically oriented coping .
first , the differentiation between individual modes of political action ( e.g. , voting , signing petitions , and donating money ) and collective political activities ( participation and leadership roles in different types of political action groups ) would be useful .
secondly , some types of collective coping could be viewed as relatively more adaptive versus maladaptive .
for example , the tea party social movement was motivated by both economic and cultural concerns [ 45 , 46 ] .
however , one might differentiate between the tea party articulation of problem - focused social policies such as its belief in the need to diminish the size of government and racially oriented collective venting , such as screams of kill him by audience members in response to sarah palin 's critique of barak obama at rallies during the 2008 presidential election .
second , this study utilized cross - sectional data and , thus , likely provides only a snapshot of the complex processes linking economy - related stressors , coping strategies , and drinking outcomes . while our theoretical framework postulated that exposure to economic stressors coupled with particular modes of coping leads to problematic drinking
, it is possible that problem drinking coupled with maladaptive coping might make one prone to experiencing economic stressors such as losing one 's job .
moreover , we lack data regarding alcohol consumption prior to exposure to the stressors focused on in this study and the extent to which alcohol may have been used as a coping mechanism . thus ,
further longitudinal studies are necessary to more clearly delineate the causal directions of the relationships between economic stressors , modes of coping , and drinking outcomes .
third , the study methodology encompassed random - digit - dialing for recruiting the sample , thus only reaching individuals with landline telephone numbers .
consequently , individuals relying on cell phones only , along with households without access to any telephone , were not included in this study .
this potential noncoverage error is a source of concern because comparisons of our data with the us population revealed that the sample underrepresented african americans , latinos , and younger ( < age 40 ) and less - educated ( high school or less ) persons .
however , our data were weighted to reflect the demographics of the overall population , and we compared weighted and unweighted estimates of each of our dependent variables to determine if nonresponse and/or noncoverage may have introduced serious bias into one or more of them . in each instance , we found that the weighted values of each measure fell well within 1 sd of the unweighted values , suggesting that the distributions of our key measures were not appreciably influenced by the underrepresentation of particular demographic groups .
despite the noted limitations , this study extends prior research on the moderators and mediators of the social stressor - drinking outcome relationships to broaden notions of coping to include politically oriented coping .
future studies incorporating this mode of coping may more clearly elucidate the political dynamics involved in both the macrolevel production of social stressors and the deleterious alcohol - related consequences of these stressors .
this line of research would also have implications for the treatment of alcohol - related problems .
in particular , considerations of more adaptive modes of coping might go beyond recommending individual behaviors such as job seeking by unemployed individuals to also suggest politically oriented coping to collectively try to influence the social conditions such as unemployment levels that may give rise to the propensity to self - medicate distress through the use of alcohol .
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research derived from the stress paradigm suggests that certain types of coping ( e.g. , problem - focused coping instead of behavioral disengagement ) are protective against problem - related drinking to deal with social stressors .
going beyond the typical focus in the coping literature , we hypothesize that stressors engendered by macrolevel social forces may require coping actions within the political realm in contrast to modes of coping focused outside of the political realm .
a united states sample of 663 respondents completed a mail survey in 2010 , including measures of stressful consequences of the great recession , drinking patterns and problems , modes of coping encompassed in the brief cope instrument , and politically oriented coping .
structural equation modeling examined whether modes of coping mediated the links between stressors and drinking outcomes .
a substantial portion of the associations between stressors and drinking was explained by modes of coping .
politically oriented coping was protective against problem drinking for both genders .
future studies should further explore politically oriented coping in addition to modes of coping outside of the political realm when studying the relationships between macrolevel social stressors and deleterious drinking outcomes .
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1. Introduction
2. Methods
3. Results
4. Discussion
5. Conclusions
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in a contemporary health - care environment characterized by rapidly changing developments and relentlessly increasing knowledge , professional nurses need to develop critical thinking ( ct ) skills that will provide them with expertise in flexible , individualized , situation - specific problem - solving .
ct has been defined as a nonlinear and cycled process that allows people to make decisions on what to believe and what to do within a given context .
according to kostovich et al . , the ability to think critically is an essential attribute for today 's nurses , and the development of this skill in nursing students requires multiple approaches and techniques .
use of the nursing process is an essential element to cultivate ct and judgment skills in nursing students .
the nursing process is a nonlinear , dynamic activity , which focuses on the multifaceted aspects of a patient , their family , and the environment .
nurses and nursing students must develop and write nursing care plans to provide and organize nursing interventions based upon identified patients needs .
however , some researchers have reported that the current linear nursing care plan format does not meet the educational needs of students to develop ct skills and visualize the interconnectedness of patient clinical data .
in addition , nurse educators feel that the nursing care plans developed by students are not case sensitive and need in - depth comprehension of each client 's physical , psychological , social , and spiritual health . in the authors program ,
the traditional linear format nursing care plan used in the first five semesters was found not to meet the needs of sixth - semester nursing students .
these students need to be able to quickly conceptualize the plan of care in a functional format as they graduate and enter the practice arena , where they will be expected to care for multiple patients simultaneously and to rapidly use the nursing process to develop a nursing plan of care based upon priority needs of these patients .
concept map development is an alternative to using care plans as a method to document a plan of care based on evidenced - based practices .
, learners assimilate new concepts into their existing cognitive structure . when new concepts are integrated by identifying relationships with concepts already possessed , learning becomes meaningful .
hence , concept maps provide a format to visualize physiological , pathological , and psychological relationships and interactions in a concrete fashion .
visualization of patient care priorities through a holistic view of the patient can be achieved through concept mapping .
in addition , the minimal use of text or words makes it easy to scan for a word , phrase , or the general idea that is being explored .
some studies have explored the short - term effects of concept map teaching on students ct and found positive effects in clinical or classroom teaching .
concept mapping has also been described in an online course on distance education to adults in nursing to assess thinking processes of the students where it was found that they helped the students to self - assess their own thinking processes , thus facilitating reflective practice .
it has also identified by gerdeman et al . to improve clinical judgment skills in nursing students . however , contrary to previous positive results , some other studies reported contradictory results , for example in a quasi - experimental study , wheeler and collins found that the experimental group scored significantly higher on the overall analysis and evaluation scores in the pre and posttest comparisons , but no significant differences between the groups were found .
yeh and chen also found no significant differences of ct scores between their experimental and control groups , and one longitudinal study on concept mapping , conducted in a united states medical school with 1 year medical students , found no significant differences between ct scores in the pre- and post - tests .
possible explanations for the findings of these studies may stem from several factors , such as measurement error , instrumentation , the curriculum and various definition of ct adopted by the studies . on the other hand , some studies indicate that ct skill of nursing students in different countries had been different .
in iran , it can be stated that although ct is important in clinical judgments and decisions but during the training period , have had no significant development therefore the traditional education system needs evolution and revision in order to realize training purposes in line with fostering creative and efficient students . with regard to the need for finding best way to promote nursing students ct and the limited evidence of comparing concept mapping with traditional nursing care plan in clinical setting
, this study was performed to compare the effect of concept mapping with traditional nursing care plan on nursing students ct in clinical pediatric course .
a before - after experimental design with the control group was used to compare the effect of concept mapping and traditional linear nursing care planning on baccalaureate nursing student 's ct skills at shahrekord university of medical sciences in shahrekord , iran .
all 60 students from the nursing faculty of the shahrekord university of medical sciences , shahrekord , iran , who had enrolled in pediatric nursing clinical course , and in sixth - semester of their study in the year 2011 , were invited to participate in this study .
students were randomly divided into six equal groups of 10 students ; three groups as the control group and the other three groups as an experimental group . to prevent contact between the control and experimental groups , they take the pediatric clinical course in sequential time order . during the course
, students cared for patients from an assigned pediatric setting . because students were not able to have prior contact with their patients , they began developing their nursing care plans on the 1 day of their clinical experience .
they were asked to create a nursing care plan for a child they were caring for , which required gathering information on complete patient histories , relevant medications , treatments and medical diagnoses .
on the 2 day of clinical experience , clinical instructor who has the experience of concept mapping development taught the students in the experimental group how to create concept maps .
the students were informed in advance that a concept map should display each nursing diagnosis and more importantly , its relationship to the patient 's data , pathophysiology of the disease and interventions in a holistic view .
students were required to analyze assessment data and identify nursing diagnoses , relevant pathophysiology and interventions related to those diagnoses .
they used diagrams , color , and shapes to code the parts of the nursing process .
they also had 5 days after the clinical experience to reflect on and evaluate the effectiveness of their nursing care before the concept maps were due .
each student in the experimental group created nine concept maps during the course . during clinical group discussions
this activity provided all students with the opportunity to think out aloud about the accuracy and completeness of the nursing diagnoses , goals , nursing interventions and relationship depicted on their concept maps .
a sample concept map demonstrated by student students in the control group received traditional clinical teaching to create linear nursing care plans on a blank sheet .
the students were asked to read and analyze the patient profile data , formulate initial problems , prioritize nursing diagnoses and identify the relationship between nursing diagnoses , then develop and implement interventions to solve the problems , and to evaluate the effectiveness of their nursing care .
linear care plans were in text format without using any shapes , propositions or arrow to illustrate the interconnectedness between the data , nursing diagnosis and interventions .
they also had the opportunity to discuss their care plans in group discussion with guidance provided by the teacher .
nobody in the control group asked about concept mapping showing that control groups did not contaminate with the intervention by experimental groups . a well - known , validated and reliable tool was selected for this study .
ct skills were measured using the california critical thinking skill test ( cctst ) with subscales of analysis ( 9 items ) , evaluation ( 14 items ) , inference ( 11 items ) , and two types of reasoning ( deductive reasoning and inductive reasoning included 16 and 14 items respectively ) .
we selected this scale because it was tested for reliability and validity in iranian nursing students .
khalili and hossein zadeh reported that this scale in comparison with the other measuring tools of ct is more comprehensive .
the reliability coefficient of subscales after factor analysis in their study was in the range of 62 - 67% .
( l4 items ) note that a kr-20 range of 0.650.75 for this type of instrument is acceptable .
this instrument contains 34 items in multiple - choice format , 19 four - option multiple - choice items and 15 five - option multiple - choice items .
the range of possible total scores was 034 , with each subscale having a possible score range of 012 .
an overall score of < 11 indicated a lack of ct skills , a score of 1125 indicated average ct skills , and a score above 25 indicated strong ct skills . for a given ct skill , a subscale score of 4 or less indicated weakness , whereas a subscale score of 7 or above indicated strength .
data were collected before and after the program in a clinical setting at the beginning and end of clinical education .
agreement to use and evaluate concept mapping as a teaching strategy in clinical courses was obtained from the nursing department of shahrekord university of medical sciences , shahrekord , iran .
information about the study was given to every participant to assure the protection of human rights by the clinical teacher .
participants in the experimental group were informed that they would be free to change to the traditional teaching strategy at any time without effects on their course evaluation . to ensure students in the control group were not disadvantaged , after completing the study , they were taught concept mapping during a 1-day workshop .
chicago , il , usa ) software . in all analyses , p - 0.05 was considered as statistically significant .
we used independent and paired t - tests to compare mean scores between the experimental and control groups at pre- and post - tests .
a before - after experimental design with the control group was used to compare the effect of concept mapping and traditional linear nursing care planning on baccalaureate nursing student 's ct skills at shahrekord university of medical sciences in shahrekord , iran .
all 60 students from the nursing faculty of the shahrekord university of medical sciences , shahrekord , iran , who had enrolled in pediatric nursing clinical course , and in sixth - semester of their study in the year 2011 , were invited to participate in this study .
students were randomly divided into six equal groups of 10 students ; three groups as the control group and the other three groups as an experimental group .
to prevent contact between the control and experimental groups , they take the pediatric clinical course in sequential time order . during the course , students cared for patients from an assigned pediatric setting . because students were not able to have prior contact with their patients , they began developing their nursing care plans on the 1 day of their clinical experience .
they were asked to create a nursing care plan for a child they were caring for , which required gathering information on complete patient histories , relevant medications , treatments and medical diagnoses .
on the 2 day of clinical experience , clinical instructor who has the experience of concept mapping development taught the students in the experimental group how to create concept maps .
the students were informed in advance that a concept map should display each nursing diagnosis and more importantly , its relationship to the patient 's data , pathophysiology of the disease and interventions in a holistic view .
students were required to analyze assessment data and identify nursing diagnoses , relevant pathophysiology and interventions related to those diagnoses .
they used diagrams , color , and shapes to code the parts of the nursing process .
they also had 5 days after the clinical experience to reflect on and evaluate the effectiveness of their nursing care before the concept maps were due .
each student in the experimental group created nine concept maps during the course . during clinical group discussions
this activity provided all students with the opportunity to think out aloud about the accuracy and completeness of the nursing diagnoses , goals , nursing interventions and relationship depicted on their concept maps .
a sample concept map demonstrated by student students in the control group received traditional clinical teaching to create linear nursing care plans on a blank sheet .
the students were asked to read and analyze the patient profile data , formulate initial problems , prioritize nursing diagnoses and identify the relationship between nursing diagnoses , then develop and implement interventions to solve the problems , and to evaluate the effectiveness of their nursing care .
linear care plans were in text format without using any shapes , propositions or arrow to illustrate the interconnectedness between the data , nursing diagnosis and interventions .
they also had the opportunity to discuss their care plans in group discussion with guidance provided by the teacher .
nobody in the control group asked about concept mapping showing that control groups did not contaminate with the intervention by experimental groups .
ct skills were measured using the california critical thinking skill test ( cctst ) with subscales of analysis ( 9 items ) , evaluation ( 14 items ) , inference ( 11 items ) , and two types of reasoning ( deductive reasoning and inductive reasoning included 16 and 14 items respectively ) .
we selected this scale because it was tested for reliability and validity in iranian nursing students .
khalili and hossein zadeh reported that this scale in comparison with the other measuring tools of ct is more comprehensive .
the reliability coefficient of subscales after factor analysis in their study was in the range of 62 - 67% .
( l4 items ) note that a kr-20 range of 0.650.75 for this type of instrument is acceptable .
this instrument contains 34 items in multiple - choice format , 19 four - option multiple - choice items and 15 five - option multiple - choice items .
the range of possible total scores was 034 , with each subscale having a possible score range of 012 .
an overall score of < 11 indicated a lack of ct skills , a score of 1125 indicated average ct skills , and a score above 25 indicated strong ct skills . for a given ct skill , a subscale score of 4 or less indicated weakness , whereas a subscale score of 7 or above indicated strength .
data were collected before and after the program in a clinical setting at the beginning and end of clinical education .
agreement to use and evaluate concept mapping as a teaching strategy in clinical courses was obtained from the nursing department of shahrekord university of medical sciences , shahrekord , iran .
information about the study was given to every participant to assure the protection of human rights by the clinical teacher .
participants in the experimental group were informed that they would be free to change to the traditional teaching strategy at any time without effects on their course evaluation . to ensure students in the control group were not disadvantaged , after completing the study , they were taught concept mapping during a 1-day workshop .
data analysis was performed using statistical package for the social sciences ( spssinc . , chicago , il , usa ) software . in all analyses , p - 0.05
we used independent and paired t - tests to compare mean scores between the experimental and control groups at pre- and post - tests .
almost all students were 21 years old except one who was 22 years old ( range : 2122 ) .
the statistical analysis showed that two groups did not appreciably differ in age or sex .
there was no significant difference between the groups overall , and in all subscales before the program [ table 1 ] .
paired t - test showed significant improvement in overall and all subscales of cctst from pretest to posttest in both groups ( p < 0.001 ) [ tables 2 and 3 ] but t - test demonstrates that improvement in students ct skills in the experimental group was significantly greater than that in the control group after the program ( p < 0.001 ) [ table 4 ] .
student 's ct skills on the pretest comparison of overall and subscales of cctst - a in the experimental group comparison of overall and subscales of cctst - a in the control group student s ct skills on the posttest
this study was developed to examine whether concept mapping helped students develop better ct skills than the traditional method in an undergraduate pediatric clinical course . in the current study
, it was found that either concept map or traditional linear nursing care plan could promote ct skills in nursing students from lacking to an average ct standard .
however , similar to findings of other prior studies which observed improvements to nurses ct abilities following a concept mapping teaching strategy , the present study provided evidence to indicate that the effects of concept mapping were greater than those of traditional linear nursing care plans , with greater improvements to students overall and all subscales of ct skills as indicated by cctst scores . considering that the test of ct is based on the problem - solving process and the nursing process in the defined problem - solving stages , the use of which could improve ct skills .
the ct skills overall describe overall strength in using reasoning to form reflective judgments about what to believe or what to do and includes core reasoning skills such as analysis , inference , evaluation , induction , and deduction .
analytical reasoning skills enable people to identify the elements of a situation and determine how these parts interact . in the nursing process , students identify bio - psycho - social aspects of patients health and determine how these parts interact . in our study , students tried to understand the relationship between patient data , nursing process , interactions and connections between sign and symptoms , diagnostic data and medications as they developed traditional care plans or concept maps .
although both care plans and concept maps could reinforce the students analytical reasoning skills , concept mapping had a greater effect . according to cook et al . , the linear format of the nursing care plan is based upon the nursing process that does not always allow for a holistic picture of patient needs and does not allow for visualization of the interrelatedness of patient data .
in contrast , concept maps provide a format to visualize physiological , pathophysiological and psychological relationships and interactions in a concrete fashion , which is more effective to support quality analysis .
schuster also suggested that visualization of patient care priorities through a holistic view of the patient can be achieved through concept mapping .
nirmala and shakuntala also supported that the cross - links in concept maps are useful for correlating patients diagnoses , symptoms , treatment and interventions and then to thinking critically in clinical decision - making . according to facione et al .
inductive reasoning helps in isolating the cause of an ailment or arriving at a theory to explain the relationship between symptoms .
evaluative reasoning skills enable students to assess the credibility of resources of information and the claims they make . in our study , after the 5 weeks program , the concept mapping participants demonstrated greater ct abilities to assess the credibility of , and relationships between statements and to provide reasoning according to evidence and using deduction , comprehend , express and clarify meaning and to perform inductive reasoning tasks .
similar to some previous studies ( yeh and chen ) , our study found that the experimental group had significantly higher scores of inference and deduction than those of the control group in the posttest . when constructing concept maps , students need to draw logical conclusions from factual knowledge or promises known through a process of inference such as identifying the major concepts , determining the relationship between concepts and making propositions using cross - links before coming to conclusions . in summary , converting to a concept map format allow for visualization of all aspects of patient clinical data , physical assessment , disease process and the relationship between this information , facilitating ct in nursing students in a clinical area .
previous researches on the ct of nursing students in iranian faculties showed that learning in the educational system takes place at the initial cognitive levels , and higher levels such as analysis or synthesis and evaluation are less addressed .
in fact , less attention is paid to the growth of the ct power according to kermansaravi et al .
there are main and serious obstacles in the development of ct , one of which is the predominant use of traditional teaching methods in the current education system which is preventing from the development of decision making and troubleshooting ( or problem - solving ) skills in the learners and as a result limits the opportunities for students ct . considering that the test of ct is based on the problem - solving process and the nursing process is the defined problem - solving stages , the use of which is one - way emphasized in nursing education programs to the growth of ct .
our study suggested that preparing nursing care using the concept map is more effective in promoting ct in nursing students than traditional linear nursing care plans .
as with all studies with small samples , generalization of findings must be made cautiously .
however , this study adds to the growing body of knowledge that suggests concept mapping improves students abilities to see patterns and relationships .
although this study identified some short - term effects of the concept mapping program , its long - term effects remain unknown .
the sample set came from only one university in iran limiting the feasibility of generalization of results to other universities and countries .
future investigations should include multisite evaluations in a range of geographic locales with larger sample sizes .
as with all studies with small samples , generalization of findings must be made cautiously . however , this study adds to the growing body of knowledge that suggests concept mapping improves students abilities to see patterns and relationships .
although this study identified some short - term effects of the concept mapping program , its long - term effects remain unknown .
the sample set came from only one university in iran limiting the feasibility of generalization of results to other universities and countries .
future investigations should include multisite evaluations in a range of geographic locales with larger sample sizes .
when presented with complex health - care situations and the need to process vast amounts of information , clinical nurses must be in ownership of ct skills in order to make appropriate professional judgments and clinical decision making .
results from the present study support the application of concept mapping as a clinical teaching strategy to promote the development of ct skills .
concept mapping , in comparison with the traditional linear nursing care plan , resulted in greater improvements in all ct skills .
however , the 5 weeks program demonstrated only short - term effects , therefore , further longitudinal studies are suggested .
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introduction : concept map is a useful cognitive tool for enhancing a student 's critical thinking ( ct ) by encouraging students to process information deeply for understanding . however , the evidence regarding its effectiveness on nursing students ct is contradictory .
this paper compares the effectiveness of concept mapping and traditional linear nursing care planning on students ct.methods:an experimental design was used to examine the ct of 60 baccalaureate students who participated in pediatric clinical nursing course in the shahrekord university of medical sciences , shahrekord , iran in 2013.results:participants were randomly divided into six equal groups of each 10 student , of which three groups were the control group , and the others were the experimental group .
the control group completed nine traditional linear nursing care plans , whereas experimental group completed nine concept maps during the course .
both groups showed significant improvement in overall and all subscales of the california ct skill test from pretest to posttest ( p < 0.001 ) , but t - test demonstrated that improvement in students ct skills in the experimental group was significantly greater than in the control group after the program ( p < 0.001).conclusions : our findings support that concept mapping can be used as a clinical teaching - learning activity to promote ct in nursing students .
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INTRODUCTION
METHODS
Design
Setting and sample
Intervention procedure
Critical thinking measurement
Ethical review
Data analysis
RESULTS
DISCUSSION
Limitations
CONCLUSIONS
Financial support and sponsorship
Conflicts of interest
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tumor - associated cell cycle defects , manifesting in unscheduled proliferation , and the associated genomic and chromosomal instabilities are mediated by misregulation of cyclin - dependent kinases ( cdks ) .
because of the main role in the division cycle , cdks have been recognized as targets for anticancer therapy .
many small - molecule organic compounds , which have been identified as cdk modulators , are currently in preclinical or clinical development . however , no cdk inhibitors have gained marketing approval , despite 20 years of scientific investigation .
several studies have shown synergism when cdk inhibitors were combined with organic ( e.g. , doxorubicin , paclitaxel ) and inorganic ( e.g. , cisplatin , carboplatin ) cytotoxic drugs .
the first publications have appeared recently and include fe , cu , and pt complexes with cdk inhibitors derived from 6-benzylaminopurine , metal - based indolo-[3,2-d]benzazepines ( paullones ; ga , cu , ru , and os ) , and indolo-[3,2-c]quinolines ( ru , os ) . another class of compounds potentially suitable for targeted metal - based chemotherapy is that of 3-(1h - benzimidazol-2-yl)-1h - pyrazolo[3,4-b]pyridines .
these have been documented recently as potent cdk1 inhibitors with antiproliferative activity in hela ( cervical carcinoma ) , hct116 ( colon carcinoma ) , and a375 ( melanoma ) human cancer cell lines .
comparison of cdk1 inhibitory activity with the inhibiting activity in four other protein kinases ( vegf - r2 , her2 , aurora - a , and ret ) revealed selectivity for cdk1 . structural modifications consisting of a replacement of both bicyclic rings in 3-(1h - benzimidazol-2-yl)-1h - pyrazolo[3,4-b]pyridines by monocycles while retaining the imidazolyl pyrazole core have been proposed in order to obtain inhibitors with improved pharmacokinetic and solubility properties .
the most promising were suggested to be 3-(1h - benzimidazol-2-yl)-1h - pyrazolo[3,4-b]pyridines ( see chart 1 ) .
the inspection of substitution patterns on the benzimidazole moiety and structure activity relationships revealed that a methoxymethyl group in position 7b ( 4b ) is favorable for cdk1 inhibiting potency .
the role of the pyrazole nh group is also of note , since its methylation led to a significant reduction of cdk1 activity .
the effect of various heteroaryl groups in position 5a was also remarkable for the development of more - effective cdk inhibitors and antiproliferative agents .
our previous experience with metal - based indolo-[3,2-d]benzazepines prompted the use of the half - sandwich metal - arene moiety as a suitable scaffold to which 3-(1h - benzimidazol-2-yl)-1h - pyrazolo[3,4-b]pyridines may be attached .
organometallic compounds [ m(-arene)(yz)x ] ( where m = ru , os ) exhibit promising anticancer activity and are the focus of attention for several groups .
these compounds have shown activity toward classic ( dna ) and nonclassic ( e.g. , cdks ) targets in anticancer chemotherapy .
herein , we report ( i ) the modified synthetic approach to 3-(1h - benzimidazol-2-yl)-1h - pyrazolo[3,4-b]pyridines ( recall chart 1 : l1 , x = h , y = h ; l2 , x = br ; y = h ; l3 , x = br ; y = ch2och3 ) ; ( ii ) the synthesis and characterization of a new family of organoruthenium(ii ) ( 11a , 12a , 13a ) and osmium(ii ) ( 11b , 12b , 13b ) complexes of the general formula [ mcl(-p - cymene)l]cl , where l = 3-(1h - benzimidazol-2-yl)-1h - pyrazolo[3,4-b]pyridines ( l1l3 ) ( chart 1 ) ; ( iii ) stabilization of the 7b tautomer of methoxymethyl - substituted l3 by metal coordination as well as ( iv ) nmr spectroscopic characterization of two tautomers 7b - l3 and 4b-l3 in a metal - free state ; and ( v ) cell cycle effects , as well as the antiproliferative and cdk inhibitory activities of both metal - free ligands and organometallic complexes .
1h - pyrazolo[3,4-b]pyridine-3-carboxylic acid ( 1 ) was obtained via the oxidation of 3-methyl-1h - pyrazolo[3,4-b]pyridine by kmno4 in the presence of a base , followed by acidification with 37% hcl .
solvents [ toluene , ethanol ( etoh ) , tetrahydrofuran ( thf ) , diethyl ether ( et2o ) ] were dried using standard procedures .
3-methyl-1h - pyrazolo[3,4-b]pyridine ( 10.9 g , 0.08 mol ) and anhydrous sodium acetate ( 10.21 g , 0.13 mol ) were suspended in glacial acetic acid ( 42 ml ) .
bromine ( 20.42 g , 0.13 mol ) was added , and the resulting mixture was stirred at room temperature for 22.5 h and then at 110115 c for 2.53 h. afterward , water ( 300350 ml ) was added and the mixture was stirred at room temperature .
the formed light yellow precipitate was filtered off and dried in vacuo at 4050 c .
yield : 17 g. the raw product was used without further purification in the next step .
purification by column chromatography afforded a white powder ( sio2 , etoac , rf = 0.79 ; 12.5 g , 72.6% yield ) .
esi - ms in meoh ( positive ) : m / z 213 [ m+h ] , 235 [ m+na ] , 253 [ m+k ] ; ( negative ) : m / z 211 [ m h ] .
h nmr ( 500.32 mhz , meoh - d4 ) : 8.55 ( d , 1h , j = 2.16 hz , ch ) , 8.43 ( d , 1h , j = 2.15 hz , ch ) , 2.56 ( s , 3h , ch3 ) ppm .
h nmr ( 500.32 mhz , dmso - d6 ) : 13.42 ( brs , 1h , nh ) , 8.54 ( d , 1h , j = 2.19 hz , ch ) , 8.53 ( d , 1h , j = 2.18 hz , ch ) , 2.49 ( s , 3h , ch3 ) ppm .
colorless crystals of 20.5h2o suitable for x - ray diffraction ( xrd ) study were grown in etoac ( see figure s1 in the supporting information ) . to sodium hydroxide ( 9.54 g , 0.24 mol ) in water ( 150 ml ) was added the raw product 2 ( 7.1 g , 0.03 mol ) . after a dropwise addition of kmno4 ( 16.98 g , 0.11 mol ) in water ( 300 ml ) at 100 c over 2 h , the reaction mixture was further heated for 1 h. mno2 was filtered off from the hot reaction mixture and washed with hot water .
400 ml , and the yellow solution was acidified to ph 2 , using concentrated hcl .
yield : 2.28 g. the crude hydrochloride of 3 was used without further purification in the next step .
esi - ms in meoh ( positive ) : m / z 243 [ m+h ] , 265 [ m+na ] , 287 [ m+2na h ] ; ( negative ) : m / z 241 [ m h ] .
h nmr ( 500.32 mhz , meoh - d4 ) : 8.69 ( d , 1h , j = 2.22 hz , ch ) , 8.68 ( d , 1h , j = 2.22 hz , ch ) ppm .
h nmr ( 500.32 mhz , dmso - d6 ) : 14.65 ( s , 1h , nh ) , 13.45 ( brs , 1h , nh ) , 8.72 ( d , 1h , j = 2.23 hz , ch ) , 8.58 ( d , 1h , j = 2.25 hz , ch ) ppm .
nah ( 1.75 g , 0.07 mol ) was suspended in dry thf ( 200 ml ) . a solution of 2-amino-3-nitrobenzyl alcohol ( 5.23 g , 0.03 mol ) in dry thf ( 100 ml )
was added dropwise at 0 c and the mixture was stirred at the same temperature for 15 min . after a dropwise addition of mei ( 11.5 g , 0.08 mol ) ,
stirring was continued at room temperature for 3 h. a saturated aqueous solution of nahco3 ( 300 ml ) and meoh ( 300 ml ) then were added .
the organic phase was dried over na2so4 , filtered , and evaporated to yield a red oil ( 4.85 g ) .
the raw product was purified by column chromatography ( sio2 , etoac , or etoac / hexane 1/1 , first fraction , a red - orange oil crystallized to form a red solid at 4 c ; yield : 3.68 g , 65% ) .
h nmr ( 500.32 mhz , dmso - d6 ) : 8.00 ( d , 1h , j = 8.71 hz ,
c6h3 ) , 7.09 ( br , 2h , nh2 ) , 6.67 ( t , 1h , j = 8.45 hz , c6h3 ) , 4.48 ( s , 2h , ch2 ) , 3.31 ( s , 3h , ch3 ) ppm .
a mixture of 4 ( 1.4 g , 0.008 mol ) and 10% pd / c ( 0.18 g ) in dry etoh ( 55 ml ) was stirred under hydrogen atmosphere at room temperature for 1824 h. the catalyst was removed by filtration through gf-3-filter under argon and washed with dry etoh ( 5070 ml ) .
the filtrate was evaporated in vacuo to give a light - orange solid ( 1.17 g , 100% yield ) , which was used immediately in the next step .
h nmr ( 500.32 mhz , dmso - d6 ) : 6.52 ( dd , 1h , j = 1.87 hz , j = 7.25 , c6h3 ) , 6.426.36 ( m , 2h , c6h3 ) , 4.48 ( br , 2h , nh2 ) , 4.31 ( br , 4h , nh2+ch2 ) , 3.24 ( s , 3h , ch3 ) ppm .
n , n-carbonyldiimidazole ( cdi , 4.16 g , 0.026 mol ) was added in small portions to 1 ( 2.34 g ) in dry dmf ( 12 ml ) .
the mixture was stirred at room temperature for 2024 h. water ( 5 ml ) then was added and the suspension was stirred until all co2 was ceased .
the white precipitate was filtered off , washed with water ( 35 ml ) , and dried in a sublimator in vacuo at 60 c , to remove the imidazole as a contaminant .
esi - ms in methanol ( positive ) : m / z 214 [ m+h ] , 237 [ m+na ] ; ( negative ) : m / z 212 [ m h ] .
h nmr ( 500.32 mhz , dmso - d6 ) : 14.95 ( brs , 1h , nh ) , 8.91 ( s , 1h , chim ) , 8.74 ( dd , 1h , j = 1.61 hz , j = 4.51 hz , chpy ) , 8.63 ( dd , 1h , j = 1.59 hz , j = 8.12 hz , chpy ) , 8.14 ( t , 1h , j = 1.23 hz , chim ) , 7.51 ( dd , 1h , j = 4.46 hz , j = 8.06 hz , chpy ) , 7.20 ( s , 1h , chim ) ppm .
n , n-carbonyldiimidazole ( cdi , 16.2 g , 0.1 mol ) was added in small portions to crude 3 ( 11.58 g ) in dry dmf ( 70 ml ) .
the mixture was stirred at room temperature for 2024 h. water ( 10 ml ) then was added and the suspension was stirred until all co2 was ceased .
the white precipitate was filtered off , washed with water ( 1015 ml ) , and dried in a sublimator in vacuo at 60 c , to remove the imidazole as a contaminant .
esi - ms in methanol ( positive ) : m / z 315 [ m+na ] ; ( negative ) : m / z 291 [ m h ] .
h nmr ( 500.32 mhz , dmso - d6 ) : 14.95 ( brs , 1h , nh ) , 8.89 ( s , 1h , chim ) , 8.83 ( d , 1h , j = 2.12 hz , chpy ) , 8.75 ( d , 1h , j = 2.07 hz , chpy ) , 8.13 ( s , 1h , chim ) , 7.20 ( s , 1h , chim ) ppm .
the solution of 1,2-diaminobenzene ( 0.5 g , 4.67 mmol ) in dry dmf ( 2 ml ) was added to the suspension of 6 ( 0.94 g , 4.41 mmol ) in dry dmf ( 13 ml ) , and this reaction mixture was heated under argon at 85 c for 5 h. dmf then was evaporated in vacuo at 50 c and water ( 1012 ml ) was added .
the light - yellow precipitate was filtered off and dried in vacuo at 50 c .
yield : 0.86 g. the raw product was used without purification in the next step .
esi - ms in methanol ( positive ) : m / z 255 [ m+h ] , 277 [ m+na ] ; ( negative ) : m / z 253 [ m h ] .
h nmr ( 500.32 mhz , dmso - d6 ) : 14.30 ( brs , 1h , nhpz ) , 9.73 ( brs , 1h , conh ) , 8.64 ( dd , 1h , j = 1.59 hz , j = 4.44 hz , chpy ) , 8.56 ( dd , 1h , j = 1.53 hz , j = 8.02 hz , chpy ) , 7.397.36 ( m , 2h , chpy+chbz ) , 6.97 ( td , 1h , j = 1.31 hz , j = 7.73 hz , chbz ) , 6.82 ( dd , 1h , j = 1.2 hz , j = 7.89 hz , chbz ) , 6.64 ( td , 1h , j = 1.2 hz , j = 7.67 hz , chbz ) , 4.93 ( s , 2h , nh2 ) ppm .
( 10 ml ) was added to the suspension of 7 ( 2.28 g , 7.8 mmol ) in dry dmf ( 10 ml ) , and this mixture was heated under argon at 85 c for 7 h. dmf then was evaporated in vacuo at 50 c and water ( 20 ml ) was added .
yield : 2.2 g. the raw product was used without purification in the next step .
esi - ms in methanol ( positive ) : m / z 333 [ m+h ] , 355 [ m+na ] ; ( negative ) : m / z 331 [ m h ] .
h nmr ( 500.32 mhz , dmso - d6 ) : 14.54 ( brs , 1h , nhpz ) , 9.80 ( brs , 1h , conh ) , 8.73 ( d , 1h , j = 2.26 hz , chpy ) , 8.69 ( d , 1h , j = 2.22 hz , chpy ) , 7.34 ( dd , 1h , j = 0.98 hz , j = 7.88 hz , chbz ) , 6.99 ( td , 1h , j = 1.43 hz , j = 8.03 hz , chbz ) , 6.82 ( dd , 1h , j = 1.22 hz , j = 7.96 hz , chbz ) , 6.64 ( td , 1h , j = 1.19 hz , j = 7.73 hz , chbz ) , 4.94 ( s , 2h , nh2 ) ppm .
a mixture of 5 ( 1.17 g , 7.69 mmol ) and 7 ( 2.1 g , 7.2 mmol ) in dry dmf ( 45 ml ) was heated under argon at 85 c for 20 h. dmf then was evaporated in vacuo at 50 c and water ( 20 ml ) was added .
yield : 2.3 g. the product was used without further purification in the next step .
esi - ms in methanol ( positive ) : m / z 399 [ m+na ] ; ( negative ) : m / z 375 [ m h ] .
h nmr ( 500.32 mhz , dmso - d6 ) : 14.54 ( brs , 1h , nhpz ) , 9.85 ( brs , 1h , conh ) , 8.73 ( d , 1h , j = 2.13 hz , chpy ) , 8.68 ( d , 1h , j = 2.05 hz , chpy ) , 7.29 ( d , 1h , j = 7.56 hz , chbz ) , 7.03 ( d , 1h , j = 7.29 hz , chbz ) , 6.65 ( t , 1h , j = 7.79 hz , chbz ) , 4.78 ( brs , 2h , nh2 ) , 4.42 ( s , 2h , ch2 ) , 3.29 ( s , 3h , ch3 ) ppm . the raw product 8 ( 0.86 g ) was heated in a glacial acetic acid ( 10 ml ) at 125 c for 2.5 h. the solvent was evaporated under reduced pressure and the residue was dried in vacuo at 50 c , then resuspended in ch2cl2/meoh ( 4/1 , 25 ml ) , filtered off , and dried in vacuo to give l1 as a white powder ( 0.4 g ) .
the filtrate was evaporated and the residue was purified by column chromatography ( sio2 , etoac , rf = 0.58 ) to give an additional amount of l1 ( 0.22 g ) .
calcd for 110.15h2o0.1etoac ( mr = 246.76 g / mol ) : c , 65.22 ; h , 4.13 ; n , 28.38 . found : c , 65.56 ; h , 3.90 ; n , 28.39 .
esi - ms in methanol ( positive ) : m / z 237 [ m+h ] , 259 [ m+na ] ; ( negative ) : m / z 235 [ m h ] .
vis ( methanol ) , max , nm ( , m cm ) : 232 ( 25618 ) , 275 ( 15198 ) , 324 ( 22333 ) .
h nmr ( 500.32 mhz , dmso - d6 ) : 14.19 ( brs , 1h , h1a ) , 13.11 ( brs , 1h , h1b ) , 8.85 ( dd , 1h , j = 1.5 hz ,
j = 8.1 hz , h4a ) , 8.66 ( dd , 1h , j = 1.5 hz , j = 4.5 hz , h6a ) , 7.75 ( d , 1h , j
= 7.3 hz , h4b or h7b ) , 7.54 ( d , 1h , j = 7.7 hz , h4b or h7b ) , 7.39 ( dd , 1h , j = 4.5 hz , j = 8.0 hz , h5a ) , 7.24 ( m , 2h , h5b+h6b ) ppm .
c nmr ( 125.81 mhz , dmso - d6 ) : 153.13 ( c8a ) , 150.22 ( c6a ) , 146.99 ( c2b ) , 144.34 ( c8b or c9b ) , 136.06 ( c3a ) , 134.68 ( c8b or c9b ) , 131.82 ( c4a ) , 123.35 ( c5b or c6b ) , 122.11 ( c5b or c6b ) , 119.44 ( c4b or c7b ) , 118.65 ( c5a ) , 113.33 ( c9a ) , 112.01 ( c4b or c7b ) ppm . n nmr ( 50.70 mhz , dmso - d6 ) : 166.2 ( n1a ) , 121.3 ( n1b ) ppm . the raw product 9 ( 2.2 g ) was heated in a glacial acetic acid ( 30 ml ) at 125 c for 2 h. the solvent was evaporated under reduced pressure , and the residue was dried in vacuo at 50 c , then resuspended in ch2cl2/meoh ( 7/1 , 50 ml ) , filtered off and dried in vacuo to give l2 as a white powder ( 1.15 g ) .
the filtrate was evaporated and the residue was purified by column chromatography ( sio2 , etoac / hexane 2/1 , rf = 0.6 ) to give an additional amount of the product ( 0.27 g ) . yield : 1.42 g , 58% based on 7 .
calcd for 120.25h2o0.04etoac ( mr = 322.17 g / mol ) : c , 49.06 ; h , 2.76 ; n , 21.74 .
esi - ms in methanol ( positive ) : m / z 315 [ m+h ] , 337 [ m+na ] ; ( negative ) : m / z 313 [ m h ] .
vis ( methanol ) , max , nm ( , m cm ) : 234 ( 28154 ) , 282 ( 17628 ) , 335 ( 17198 ) .
h nmr ( 500.32 mhz , dmso - d6 ) : 14.43 ( brs , 1h , h1a ) , 13.19 ( brs , 1h , h1b ) , 8.99 ( d , 1h , j = 2.2 hz , h4a ) , 8.75 ( d , 1h , j = 2.3 hz , h6a ) , 7.77 ( d , 1h , j = 7.9 hz , h4b or h7b ) , 7.54 ( d , 1h , j = 7.9 hz , h4b or h7b ) , 7.26 ( m , 2h , h5b+h6b ) ppm .
c nmr ( 125.81 mhz , dmso - d6 ) : 151.49 ( c8a ) , 150.66 ( c6a ) , 146.42 ( c2b ) , 144.24 ( c8b or c9b ) , 135.61 ( c3a ) , 134.67 ( c8b or c9b ) , 133.35 ( c4a ) , 123.54 ( c5b or c6b ) , 122.27 ( c5b or c6b ) , 119.55 ( c4b or c7b ) , 114.87 ( c5a or c9a ) , 113.49 ( c5a or c9a ) , 112.09 ( c4b or c7b ) ppm . n nmr ( 50.70 mhz , dmso - d6 ) : 167.5 ( n1a ) , 121.3 ( n1b ) ppm .
the raw product 10 ( 2.3 g ) was heated in a glacial acetic acid ( 40 ml ) at 125 c for 2 h. the solvent was evaporated under reduced pressure , and the residue dried in vacuo at 50 c . after washing with ch2cl2 ( 30 ml ) , ch2cl2/meoh ( 2/1 , 57 ml ) the gray product was purified by column chromatography ( sio2 , etoac , rf = 0.68 ) to give a white powder ( 0.9 g ) . the filtrates were evaporated and the remaining solid was purified by column chromatography to give an additional amount of the product ( 0.3 g ) .
calcd for 13 : c , 50.29 ; h , 3.38 ; n , 19.55 . found : c , 50.03 ; h , 3.19 ; n , 19.19 .
esi - ms in methanol ( positive ) : m / z 381 [ m+na ] ; ( negative ) : m / z 357 [ m h ] .
vis ( methanol ) , max , nm ( , m cm ) : 235 ( 29776 ) , 282 ( 17544 ) , 337 ( 16958 ) .
nmr characterization of 7b - l3 and 4b-l3 tautomers ( 1/1.3 ) in dmso - d6 : h nmr ( 500.32 mhz , dmso - d6 ) , 7b - l3 : 14.47 ( brs , 1h , h1a ) , 13.25 ( brs , 1h , h1b ) , 8.99 or 8.98 ( d+d , ( 1 + 1.3)h , j = 2.3 hz , h4a+h4a ) , 8.75 ( d , ( 1 + 1.3)h , j = 2.3 hz , h6a+h6a ) , 7.72 ( dd , 1h , j = 1.8 hz ,
j = 6.8 hz , h4b ) , 7.287.21 ( m , ( 2 + 2.6)h , h5a , h6a+h5a , h6a ) , 4.80 ( s , 2h , h10b ) , 3.37 ( s , 3h , h11b ) ppm .
h nmr ( 500.32 mhz , dmso - d6 ) , 4b-l3 : 14.43 ( brs , 1.3h , h1a ) , 13.22 ( brs , 1.3h , h1b ) , 8.99 or 8.98 ( d+d , ( 1 + 1.3)h , j = 2.3 hz , h4a+h4a ) , 8.75 ( d , ( 1 + 1.3)h , j = 2.3 hz , h6a+h6a ) , 7.47 ( dd , 1.3h , j = 1.2 hz ,
j = 7.7 hz , h7b ) , 7.287.21 ( m , ( 2 + 2.6)h , h5a , h6a+h5a , h6a ) , 4.96 ( s , 2.6h , h10b ) , 3.44 ( s , 3.9h , h11b ) ppm .
c nmr ( 125.81 mhz , dmso - d6 ) , 7b - l3 : 151.51 ( c8a+c8a ) , 150.69 ( c6a or c6a ) , 150.65 ( c6a or c6a ) , 146.70 ( c2b ) , 144.46 ( c9b ) , 135.62 ( c3a+c3a ) , 133.45 ( c4a or c4a ) , 133.42 ( c4a or c4a ) , 133.29 ( c8b ) , 123.38 ( c6b ; c5b or c6b ) , 123.36 ( c6b ; c5b or c6b ) , 122.95 ( c7b ) , 122.16 ( c5b or c6b ) , 118.97 ( c4b ) , 115.04 ( c5a or c9a or c5a or c9a ) , 114.92 ( c5a or c9a or c5a or c9a ) , 113.49 ( c5a or c9a or c5a or c9a ) , 70.49 ( c10b ) , 57.95 ( c11b ) ppm .
c nmr ( 125.81 mhz , dmso - d6 ) , 4b-l3 : 151.51 ( c8a+c8a ) , 150.69 ( c6a or c6a ) , 150.65 ( c6a or c6a ) , 146.13 ( c2b ) , 142.50 ( c9b ) , 135.62 ( c3a+c3a ) , 134.42 ( c8b ) , 133.45 ( c4a or c4a ) , 133.42 ( c4a or c4a ) , 129.38 ( c4b ) , 123.38 ( c6b ; c5b or c6b ) , 123.36 ( c6b ; c5b or c6b ) , 122.16 ( c5b or c6b ) , 120.81 ( c5b ) , 115.04 ( c5a or c9a or c5a or c9a ) , 114.92 ( c5a or c9a or c5a or c9a ) , 113.49 ( c5a or c9a or c5a or c9a ) , 111.17 ( c7b ) , 69.90 ( c10b ) , 58.35 ( c11b ) ppm .
n nmr ( 50.70 mhz , dmso - d6 ) : 167.6 ( n1a+n1a ) , 121.4 ( n1b+n1b ) ppm .
a mixture of l1 ( 54.7 mg , 0.23 mmol ) and [ rucl2(-p - cymene)]2 ( 70 mg , 0.11 mmol ) in dry ethanol ( 25 ml ) was stirred at room temperature for 1 h. ethanol then was evaporated up to ca .
calcd for 11ah2o ( mr = 559.45 g / mol ) : c , 49.38 ; h , 4.50 ; n , 12.52 ; cl , 12.67 .
found : c , 49.68 ; h , 4.25 ; n , 12.11 ; cl , 12.26 .
esi - ms in methanol ( positive ) : m / z 470 [ m hcl cl ] , 507 [ m cl ] , 528 [ m hcl+na ] ; ( negative ) : m / z 468 [ m2hcl h ] , 505 [ m hcl h ] .
vis ( methanol ) , max , nm ( , m cm ) : 251 ( 16335 ) , 299 ( 26663 ) , sh 347 ( 16012 ) .
vis ( h2o ) , max , nm ( , m cm ) : sh 245 ( 10728 ) , 294 ( 18597 ) , 360 ( 9666 ) .
h nmr ( 500.32 mhz , dmso - d6 ) : 14.91 ( brs , 1h , h1b ) , 9.17 ( d , 1h , j = 7.7 hz , h4a ) , 8.82 ( d , 1h , j = 3.8 hz , h6a ) , 8.11 ( d , 1h , j = 7.1 hz , h4b ) , 7.84 ( d , 1h , j = 8.5 hz , h7b ) , 7.58 ( m , 3h , h5a+h5b+h6b ) , 6.43 ( m , 2h , h2c+h6c ) , 6.31 ( d , 1h , j = 5.3 hz , h3c or h5c ) , 6.12 ( d , 1h , j = 5.5 hz , h3c or h5c ) , 2.54 ( sep , 1h , h7c , under dmso - d6 peak ) , 2.21 ( s , 3h , h10c ) , 0.94 ( d , 3h , j = 6.6 hz , h8c or h9c ) , 0.91 ( d , 3h , j = 6.6 hz , h8c or h9c ) ppm .
c nmr ( 125.81 mhz , dmso - d6 ) : 153.97 ( c8a ) , 150.45 ( c6a ) , 146.52 ( c2b ) , 141.37 ( c9b ) , 134.70 ( c8b ) , 134.64 ( c3a ) , 131.48 ( c4a ) , 125.39 ( c5b or c6b ) , 124.92 ( c5b or c6b ) , 119.56 ( c5a ) , 117.85 ( c4b ) , 113.96 ( c7b ) , 111.54 ( c9a ) , 103.96 ( c4c ) , 103.74 ( c1c ) , 84.44 ( c2c or c6c ) , 83.73 ( c2c or c6c ) , 82.15 ( c3c or c5c ) , 80.84 ( c3c or c5c ) , 31.12 ( c7c ) , 22.19 ( c8c+c9c ) , 19.17 ( c10c ) ppm .
n nmr ( 50.70 mhz , dmso - d6 ) : 128.7 ( n1b ) ppm .
a mixture of l1 ( 42.5 mg , 0.18 mmol ) and [ oscl2(-p - cymene)]2 ( 70 mg , 0.09 mmol ) in dry ethanol ( 25 ml ) was stirred at room temperature for 2 h. ethanol then was removed under reduced pressure up to ca .
calcd for 11bh2o ( mr = 648.61 g / mol ) : c , 42.59 ; h , 3.88 ; n , 10.79 . found : c , 42.56 ; h , 3.57 ; n , 10.97 .
esi - ms in methanol ( positive ) : m / z [ m hcl cl ] , 596 [ m cl ] , 618 [ m hcl+na ] ; ( negative ) : m / z 595 [ m hcl h ] .
vis ( methanol ) , max , nm ( , m cm ) : sh 252 ( 17048 ) , 299 ( 20228 ) , 343 ( 19879 ) .
h nmr ( 500.32 mhz , meoh - d4 ) : 8.99 ( dd , 1h , j = 1.3 hz ,
j = 8.1 hz , h4a ) , 8.77 ( dd , 1h , j = 1.4 hz ,
j = 4.8 hz , h6a ) , 7.96 ( dd , 1h , j = 2.0 hz , j = 6.4 hz , h4b ) , 7.80 ( dd , 1h , j
= 1.9 hz , j = 6.1 hz , h7b ) , 7.647.59 ( m , 3h , h5a+h5b+h6b ) , 6.66 ( d , 1h , j = 5.6 hz , h2c or h6c ) , 6.59 ( d , 1h , j = 5.7 hz , h2c or h6c ) , 6.43 ( d , 1h , j = 5.6 hz , h3c or h5c ) , 6.21 ( d , 1h , j = 5.7 hz , h3c or h5c ) , 2.43 ( sep , 1h , j = 6.9 hz , h7c ) , 2.38 ( s , 3h , h10c ) , 0.96 ( d , 3h , j = 6.9 hz , h8c or h9c ) , 0.92 ( d , 3h , j = 6.9 hz , h8c or h9c ) ppm .
c nmr ( 125.81 mhz , meoh - d4 ) : 153.25 ( c8a ) , 148.91 ( c2b ) , 147.42 ( c6a ) , 140.49 ( c9b ) , 135.15 ( c3a ) , 134.25 ( c8b ) , 132.07 ( c4a ) , 125.47 ( c5b or c6b ) , 124.81 ( c5b or c6b ) , 118.48 ( c5a ) , 116.89 ( c4b ) , 112.98 ( c7b ) , 112.92 ( c9a ) , 97.71 ( c4c ) , 94.88 ( c1c ) , 76.44 ( c2c or c6c ) , 74.99 ( c2c or c6c ) , 71.93 ( c3c or c5c ) , 70.44 ( c2c or c6c ) , 31.37 ( c7c ) , 21.35 ( c8c or c9c ) , 21.09 ( c8c or c9c ) , 17.84 ( c10c ) ppm .
crystals of 11b4h2o suitable for xrd study have been obtained from a solution of 11b in ethanol .
a mixture of l2 ( 73.2 mg , 0.23 mmol ) and [ rucl2(-p - cymene)]2 ( 70 mg , 0.11 mmol ) in dry ethanol ( 25 ml ) was stirred at room temperature for 1 h. ethanol then was removed under reduced pressure up to ca .
calcd for 12ah2o ( mr = 638.35 g / mol ) : c , 43.28 ; h , 3.79 ; n , 10.97 .
esi - ms in methanol ( positive ) : m / z 548 [ m hcl cl ] , 608 [ m hcl+na ] ; ( negative ) : m / z 549 [ m2hcl h ] , 582 [ m hcl h ] .
vis ( methanol ) , max , nm ( , m cm ) : sh 254 ( 17778 ) , 303 ( 29953 ) , 351 ( 17387 ) .
h nmr ( 500.32 mhz , dmso - d6 ) : 14.34 ( brs , 1h , h1b ) , 9.21 ( s , 1h , h4a ) , 8.73 ( s , 1h , h6a ) , 8.06 ( d , 1h , j = 7.6 hz , h4b ) , 7.79 ( d , 1h , j = 8.5 hz , h7b ) , 7.52 ( m , 2h , h5b+h6b ) , 6.32 ( d , 2h , j = 5.8 hz , h2c+h6c ) , 6.22 ( d , 1h , j = 6.2 hz , h3c or h5c ) , 6.03 ( d , 1h , j = 6.1 hz , h3c or h5c ) , 2.52 ( sep , 1h , h7c , under dmso - d6 peak ) , 2.18 ( s , 3h , h10c ) , 0.94 ( d , 3h , j = 6.8 hz , h8c or h9c ) , 0.89 ( d , 3h , j = 6.9 hz , h8c or h9c ) ppm .
c nmr ( 125.81 mhz , dmso - d6 ) : 154.04 ( c8a ) , 151.24 ( c6a ) , 146.57 ( c2b ) , 141.37 ( c9b ) , 134.63 ( c8b ) , 133.43 ( c3a ) , 131.82 ( c4a ) , 125.33 ( c5b or c6b ) , 124.88 ( c5b or c6b ) , 117.78 ( c4b ) , 114.72 ( c5a or c9a ) , 113.85 ( c7b ) , 112.48 ( c5a or c9a ) , 103.96 ( c4c ) , 103.74 ( c1c ) , 84.44 ( c2c or c6c ) , 83.69 ( c2c or c6c ) , 82.16 ( c3c or c5c ) , 80.76 ( c3c or c5c ) , 31.09 ( c7c ) , 22.20 ( c8c or c9c ) , 22.17 ( c8c or c9c ) , 19.16 ( c10c ) ppm .
n nmr ( 50.70 mhz , dmso - d6 ) : 127.3 ( n1b ) ppm .
a mixture of l2 ( 56.4 mg , 0.18 mmol ) and [ oscl2(-p - cymene)]2 ( 70 mg , 0.09 mmol ) in dry ethanol ( 25 ml ) was stirred at room temperature for 2 h. ethanol then was removed under reduced pressure up to ca .
calcd for 12bh2o ( mr = 727.51 g / mol ) : c , 37.97 ; h , 3.33 ; n , 9.63 . found : c , 37.82 ; h , 3.02 ; n , 9.33 .
esi - ms in methanol ( positive ) : m / z 638 [ m hcl cl ] , 696 [ m hcl+na ] ; ( negative ) : m / z 672 [ m hcl h ] .
vis ( methanol ) , max , nm ( , m cm ) : sh 253 ( 21638 ) , 302 ( 30505 ) , 357 ( 21813 ) .
h nmr ( 500.32 mhz , meoh - d4 ) : 9.08 ( d , 1h , j = 2.1 hz , h4a ) , 8.84 ( d , 1h , j = 2.1 hz , h6a ) , 7.96 ( dd , 1h , j = 2.1 hz ,
j = 6 hz , h4b ) , 7.80 ( dd , 1h , j = 2.1 hz ,
j = 6.1 hz , h7b ) , 7.63 ( m , 2h , h5b+h6b ) , 6.69 ( d , 1h , j = 5.7 hz , h2c or h6c ) , 6.63 ( d , 1h , j = 5.6 hz , h2c or h6c ) , 6.48 ( d , 1h , j = 5.6 hz , h3c or h5c ) , 6.22 ( d , 1h , j = 5.7 hz , h3c or h5c ) , 2.42 ( sep , 1h , j = 6.9 hz , h7c ) , 2.39 ( s , 3h , h10c ) , 0.95 ( d , 3h , j = 6.9 hz , h8c or h9c ) , 0.91 ( d , 3h , j = 6.9 hz , h8c or h9c ) ppm .
c nmr ( 125.81 mhz , meoh - d4 ) : 153.04 ( c8a ) , 152.49 ( c6a ) , 148.15 ( c2b ) , 140.47 ( c9b ) , 134.58 ( c3a ) , 134.19 ( c8b ) , 131.13 ( c4a ) , 125.79 ( c5b or c6b ) , 125.05 ( c5b or c6b ) , 116.99 ( c4b ) , 115.39 ( c5a or c9a ) , 113.09 ( c7b ) , 112.11 ( c5a or c9a ) , 98.54 ( c4c ) , 95.52 ( c1c ) , 75.73 ( c2c or c6c ) , 75.25 ( c2c or c6c ) , 71.87 ( c3c or c5c ) , 69.99 ( c3c or c5c ) , 31.41 ( c7c ) , 21.34 ( c8c or c9c ) , 21.16 ( c8c or c9c ) , 17.84 ( c10c ) ppm .
crystals of 12b and 12b2ch3oh2h2o suitable for xrd study have been obtained from a solution of 12b in methanol .
a mixture of l3 ( 64 mg , 0.18 mmol ) and [ rucl2(-p - cymene)]2 ( 50 mg , 0.08 mmol ) in dry ethanol ( 25 ml ) was stirred at room temperature for 1 h. ethanol then was removed under reduced pressure up to ca .
calcd for 13a1.5h2o ( mr = 691.41 g / mol ) : c , 43.43 ; h , 4.23 ; n , 10.13 ; cl , 10.26 .
found : c , 43.81 ; h , 4.24 ; n , 10.11 ; cl , 10.57 .
esi - ms in methanol ( positive ) : m / z 592 [ m hcl cl ] , 650 [ m hcl+na ] ; ( negative ) : m / z 591 [ m2hcl h ] , 628 [ m hcl h ] .
vis ( methanol ) , max , nm ( , mcm ) : sh 252 ( 18744 ) , 306 ( 28529 ) , 354 ( 16889 ) .
h nmr ( 500.32 mhz , dmso - d6 ) : 13.88 ( brs , 1h , h1b ) , 9.41 ( s , 1h , h4a ) , 8.68 ( s , 1h , h6a ) , 7.99 ( d , 1h , j = 7.7 hz , h4b ) , 7.49 ( m , 2h , h5b+h6b ) , 6.29 ( m , 2h , h2c+h6c ) , 6.20 ( d , 1h , j = 5.5 hz , h3c or h5c ) , 6.00 ( d , 1h , j = 5.9 hz , h3c or h5c ) , 4.88 ( s , 2h , h10b ) , 3.39 ( s , 3h , h11b ) , 2.48 ( sep , 1h , h7c , under dmso - d6 peak ) , 2.17 ( s , 3h , h10c ) , 0.93 ( d , 3h , j = 6.9 hz , h8c or h9c ) , 0.87 ( d , 3h , j = 6.8 hz , h8c or h9c ) ppm .
c nmr ( 125.81 mhz , dmso - d6 ) : 155.35 ( c8a ) , 150.27 ( c6a ) , 147.43 ( c2b ) , 141.64 ( c9b ) , 133.07 ( c8b ) , 132.68 ( c3a ) , 131.68 ( c4a ) , 124.87 ( c5b or c6b ) , 124.59 ( c5b or c6b ) , 124.53 ( c7b ) , 117.18 ( c4b ) , 114.23 ( c5a or c9a ) , 112.78 ( c5a or c9a ) , 103.74 ( c4c ) , 103.09 ( c1c ) , 85.38 ( c2c or c6c ) , 83.68 ( c2c or c6c ) , 82.13 ( c3c or c5c ) , 81.05 ( c3c or c5c ) , 70.19 ( c10b ) , 58.03 ( c11b ) , 31.04 ( c7c ) , 22.22 ( c8c or c9c ) , 22.09 ( c8c or c9c ) , 19.13 ( c10c ) ppm .
n nmr ( 50.70 mhz , dmso - d6 ) : 122.9 ( n1b ) ppm .
the red xrd - quality crystals of [ rucl(-p - cymene)(l3h ) ] ( 13c ) were obtained from etoh / et2o and an etoh solution of 13a ( long crystallization ) .
the yellow crystals of [ rucl(-p - cymene)(l3)]cl[rucl(-p - cymene)(l3h)]ch3oh ( 13dch3oh ) suitable for xrd study have been obtained from methanolic solution of 13a ( short crystallization ) .
h nmr ( 13c , 500.32 mhz , dmso - d6 ) : 13.43 ( s , 1h ) , 9.18 ( d , 1h , j = 2.1 hz ) , 8.48 ( d , 1h , j = 2.2 hz ) , 7.94 ( d , 1h , j = 8.2 hz ) , 7.45 ( t , 1h , j = 7.8 hz ) , 7.39 ( d , 1h , j = 7.2 hz ) , 6.16 ( d , 1h , j = 6.0 hz ) , 6.13 ( d , 1h , j = 6.1 hz ) , 6.07 ( d , 1h , j = 5.9 hz ) , 5.89 ( d , 1h , j = 6.4 hz ) , 4.85 ( s , 2h ) , 4.04 ( s , 3h ) , 2.14 ( s , 3h ) , 0.93 ( d , 3h , j = 6.8 hz ) , 0.86 ( d , 3h , j = 6.9
a mixture of l3 ( 59 mg , 0.17 mmol ) and [ oscl2(-p - cymene)]2 ( 60 mg , 0.08 mmol ) in dry ethanol ( 25 ml ) was stirred at room temperature for 3 h. ethanol was evaporated up to ca .
calcd for 13b : c , 39.85 ; h , 3.48 ; n , 9.29 . found : c , 39.60 ; h , 3.32 ; n , 9.20 .
esi - ms in methanol ( positive ) : m / z 682 [ m hcl cl ] , 704 [ m2hcl+na ] ; ( negative ) : m / z 680 [ m2hcl h ] , 716 [ m hcl h ] .
vis ( methanol ) , max , nm ( , m cm ) : sh 256 ( 19825 ) , 303 ( 24634 ) , 357 ( 18850 ) .
h nmr ( 500.32 mhz , meoh - d4 ) : 9.33 ( d , 1h , j = 2.1 hz , h4a ) , 8.84 ( d , 1h , j = 2.2 hz , h6a ) , 7.91 ( dd , 1h , j = 1.2 hz ,
j = 7.8 hz , h4b ) , 7.647.58 ( m , 2h , h5b+h6b ) , 6.69 ( d , 1h , j = 5.6 hz , h2c or h6c ) , 6.62 ( d , 1h , j = 5.7 hz , h2c or h6c ) , 6.47 ( d , 1h , j = 5.5 hz , h3c or h5c ) , 6.21 ( d , 1h , j = 5.6 hz , h3c or h5c ) , 4.94 ( q , 2h , j = 12.4 hz , h10b ) , 3.49 ( s , 3h , h11b ) , 2.41 ( sep , 1h , j = 6.9 hz , h7c ) , 2.39 ( s , 3h , h10c ) , 0.94 ( d , 3h , j = 6.9 hz , h8c or h9c ) , 0.89 ( d , 3h , j = 6.9 hz , h8c or h9c ) ppm . c nmr ( 125.81 mhz , meoh - d4 ) : 153.18 ( c8a ) , 152.16 ( c6a ) , 148.36 ( c2b ) , 140.82 ( c9b ) , 134.35 ( c3a ) , 132.68 ( c8b ) , 131.68 ( c4a ) , 125.63 ( c5b or c6b ) , 124.85 ( c5b or c6b ) , 124.51 ( c7b ) , 116.72 ( c4b ) , 115.24 ( c5a or c9a ) , 112.26 ( c5a or c9a ) , 98.75 ( c4c ) , 95.50 ( c1c ) , 75.98 ( c2c or c6c ) , 75.34 ( c2c or c6c ) , 71.82 ( c3c or c5c ) , 70.29 ( c10b ) , 70.04 ( c3c or c5c ) , 57.17 ( c11b ) , 31.39 ( c7c ) , 21.34 ( c8c or c9c ) , 21.16 ( c8c or c9c ) , 17.85 ( c10c ) ppm .
the yellow crystals of [ oscl(-p - cymene)(l3)]cl[oscl(-p - cymene)(l3h)]0.75 ch3oh0.25h2o ( 13e0.75ch3oh0.25h2o ) suitable for xrd study have been obtained from a methanolic solution of 13b .
elemental analyses ( c , h , n , cl ) were performed by the microanalytical service of the institute of physical chemistry , university of vienna .
electrospray ionization mass spectrometry ( esi - ms ) was carried out with an esquire 3000 instrument ( bruker daltonics , bremen , germany ) , using solutions of compounds in methanol .
vis spectra were recorded on a lambda 20 uv vis spectrophotometer ( perkin elmer ) , using samples dissolved in methanol ( l1l3 , 11a , 12a , 13a , 11b , 12b , 13b ) and water ( 11a ) over 24 or 48 h , correspondingly . the one - dimensional ( h , c , n ) and two - dimensional spectra ( n , h hsqc , c , h hsqc , c , h hmbc , h , h cosy , h , h tocsy , h , h noesy ( l3 , 12a ) , h , h roesy ( l3 , 11a , 12a , 13a , 13b ) ) were recorded with a bruker model dpx500 ( ultrashield magnet ) system in dmso - d6 ( l1l3 , 11a , 12a , 13a ) , meoh - d4 ( 11b , 12b , 13b ) , d2o ( h nmr , 11a ) , and d2o / dmso - d6 ( h nmr , 12a ) , using standard pulse programs at 500.32 ( h ) , 125.81 ( c ) and 50.70 ( n ) mhz .
h signals are referenced relative to the solvent signals ( dmso - d6 at 2.51 , meoh - d4 at 3.33 ppm ) .
single crystals were positioned at distances of 35 , 40 , 40 , 40 , 40 , 40 , and 35 mm from the detector , and 1556 , 1131 , 518 , 1911 , 1176 , 1978 , and 1039 frames were measured , each for 30 , 60 , 30 , 20 , 70 , 60 , and 30 s over 1 scan width for 20.5h2o , 11b4h2o , 12b , 12b2ch3oh2h2o , 13c , 13dch3oh , and 13e0.75ch3oh0.25h2o , correspondingly .
crystal data , data collection parameters , and structure refinement details are given in table 1 .
the structures were solved by direct methods and refined by full - matrix least - squares techniques .
refinement of the structure of 12b revealed that the chloride counteranion occupies two statistically disordered positions with site occupation factor ( sof ) values of 0.57:0.43 , whereas , in 13c , the methoxymethylene group was found to be disordered over two positions with sof values of 0.80:0.20 .
similarly , the methoxymethylene group of one crystallographically independent complex in 13dch3oh or in both crystallographically independent complexes in 13e0.75ch3oh0.25h2o is disordered with sof values of 0.35:0.65 and 0.60:0.40 , 0.80:0.20 , correspondingly . the disorder was resolved with constrained anisotropic displacement parameters and restrained bond distances using eadp and sadi instructions of shelx97 , respectively .
structure solution was achieved with shelxs-97 and refinement with shelxl-97 , and graphics were produced with ortep-3 .
wr2 = { [w(fo fc)]/[w(fo)]}. gof = { [w(fo fc)]/(n p ) } , where n is the number of reflections and p is the total number of parameters refined .
a549 ( non - small cell lung carcinoma , human ) and sw480 ( colon carcinoma , human ) cells were kindly provided by brigitte marian ( institute of cancer research , department of medicine i , medical university of vienna , austria ) .
ch1 cells ( ovarian cancer , human ) were a gift from lloyd r. kelland ( crc centre for cancer therapeutics , institute of cancer research , sutton , u.k . ) .
cells were grown in 75-cm culture flasks ( iwaki ) in a complete medium ( i.e. , minimum essential medium supplemented with 10% heat - inactivated fetal bovine serum , 1 mm sodium pyruvate , 4 mm l - glutamine , and 1% nonessential amino acids from 100 stock ) as adherent monolayer cultures .
cultures were grown at 37 c under a humidified atmosphere containing 5% co2 and 95% air .
antiproliferative activity in vitro was determined by the colorimetric mtt assay ( mtt = 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2h - tetrazolium bromide , fluka ) . for this purpose
, cells were harvested from culture flasks by the use of trypsin and seeded in complete medium ( 100 l / well ) into 96-well plates ( iwaki ) in densities of 4 10 ( a549 ) , 1.5 10 ( ch1 ) , and 2.5 10 ( sw480 ) viable cells per well .
test compounds were dissolved in dmso first , appropriately diluted in complete medium , and instantly added to the plates ( 100 l / well ) , where the dmso content did not exceed 0.4% and 1% for the ligands and complexes , respectively . after exposure for 96 h , the medium was replaced with 100 l / well rpmi 1640 medium ( supplemented with 10% heat - inactivated fetal bovine serum and 4 mm l - glutamine ) plus 20 l / well mtt solution in phosphate - buffered saline ( 5 mg / ml ) , followed by incubation for 4 h. subsequently , the medium / mtt mixture was removed , and the formazan product formed by viable cells was dissolved in dmso ( 150 l / well ) .
optical densities were measured with a microplate reader ( tecan spectra classic ) at 550 nm ( and a reference wavelength of 690 nm ) to yield relative quantities of viable cells as percentages of untreated controls , and 50% inhibitory concentrations ( ic50 ) were interpolated from concentration effect curves .
calculations are based on at least three independent experiments with triplicates for each concentration level . to study the effects on the cell cycle of exponentially growing ch1 cells by flow cytometric analysis of their relative dna content
, cells were harvested from culture flasks , seeded in complete medium into 90-mm petri dishes ( 1 10 cells / dish ) and , after recovery for 24 h , exposed to various concentrations of the test compounds for 24 h. for this purpose , test compounds were diluted from dmso stocks with complete medium ( see above ) such that the effective dmso content did not exceed 0.5% . after exposure , treated and control cells were collected by scratching , washed with pbs , and stained with 5 g / ml propidium iodide overnight .
their fluorescence was measured with a facs calibur instrument ( becton dickinson ) , and the obtained histograms were analyzed with cell quest pro software ( becton dickinson ) .
at least two independent experiments were performed for each setting , and 2.5 or 3.0 10 cells were measured per sample .
the cdk - inhibitory capacities of test compounds were studied by a radiochemical assay using recombinant cdk1/cyclin b and cdk2/cyclin e isolated from sf21 insect cells and histone h1 as the substrate for phosphorylation , as described by marko et al .
briefly , mops - buffered assay mixtures containing the test compound ( with a maximum of 1% dmso ) , the respective kinase / cyclin complex , histone h1 , and 0.4 ci ( -p)atp per sample were incubated for 10 min at 30 c .
aliquots of the solution were spotted onto phosphocellulose squares , which had been washed three times with 0.75% phosphoric acid , followed by acetone .
the dried squares were measured in scintillation vials by -counting ( perkin elmer tri - carb 2800tr ; quanta smart software ) .
results were obtained in duplicate in at least two independent experiments , and ic50 values were calculated by interpolation .
several routes to 3-(1h - benzimidazol-2-yl)-1h - pyrazolo[3,4-b]pyridines have been proposed by johnson & johnson pharmaceutical research & development l.l.c .
the first one was developed for 3-(1h - benzimidazol-2-yl)-1h - pyrazolo[3,4-b]pyridines with an unsubstituted benzimidazole moiety and involved sulfur - induced benzimidazole ring formation via the treatment of 5-bromo-1h - pyrazolo[3,4-b]pyridine-3-carboxaldehyde with 1,2-diaminobenzene .
the poor reproducibility of this synthesis prompted the exploration of an alternative way , via 5-bromo-1h - pyrazolo[3,4-b]pyridine-3-carboxylic acid , which was used for the preparation of 3-(1h - benzimidazol-2-yl)-1h - pyrazolo[3,4-b]pyridines with a substituted benzimidazole moiety .
the patented route to 5-bromo-1h - pyrazolo[3,4-b]pyridine-3-carboxylic acid ( 3 ) consists of four steps ( see scheme 1 , steps i iv ) : oxidation of 3-methyl-1h - pyrazolo[3,4-b]pyridine by kmno4 in the presence of a base with subsequent acidification with h2so4 , esterification of 1h - pyrazolo[3,4-b]pyridine-3-carboxylic acid ( 1 ) in the presence of h2so4 in methanol , bromination of 1h - pyrazolo[3,4-b]pyridine-3-carboxylic acid methyl ester in an acoh / acona mixture , and hydrolysis of 5-bromo-1h - pyrazolo[3,4-b]pyridine-3-carboxylic acid methyl ester in the presence of naoh , followed by acidification with hcl .
reagents and conditions : ( i ) kmno4 , naoh , 3 h , 100 c , h2so4 ; ( ii ) methanol , h2so4 , reflux , 68 h , nahco3 , 42% ( i + ii ) ; ( iii ) br2 , acoh , acona , 115 c , overnight , chromatographic purification , 43% ; ( iv ) naoh , meoh , reflux , 4 h , hcl , 100% ; ( v ) br2 , acoh , acona , 115 c , 2.53 h ; and ( vi ) kmno4 , naoh , 3 h , 100 c , hcl , 2435% ( v + vi ) .
we performed the synthesis of 3 in two steps ( see scheme 1 , steps v and vi ) : bromination of 3-methyl-1h - pyrazolo[3,4-b]pyridine in acoh / acona mixture and oxidation of crude 5-bromo-3-methyl-1h - pyrazolo[3,4-b]pyridine ( 2 ) by kmno4 in a basic medium , followed by acidification with 37% hcl , with an overall yield of 2435% .
5-bromo-3-methyl-1h - pyrazolo[3,4-b]pyridine ( 2 ) is a known compound , the synthesis of which is well - documented . for the benzimidazole ring formation , 1,2-diaminobenzene and 1-methoxymethyl-2,3-diaminobenzene
the reported synthesis of 1-methoxymethyl-2,3-diaminobenzene ( 5 ) from 2,3-diaminobenzyl alcohol afforded the desired product in 34% yield ( see scheme 2 , step i ) .
however , the instability and low yield of diamines , as well as the necessity to purify the desired ether using column chromatography , stimulated the search for a more - convenient procedure that was subsequently proposed : etherification of 2-amino-3-nitrobenzyl alcohol , followed by the reduction of 1-methoxymethyl-2-amino-3-nitrobenzene ( 4 ) with 10% pd / c in ethanol under a hydrogen atmosphere afforded 5 in 6575% yield ( see scheme 2 , steps ii and iii ) .
reagents and conditions : ( i ) nah , mei , dry thf , 0 c , 30 min , room temperature , overnight , chromatographic purification , 34% yield ; ( ii ) nah , mei , dry thf , 0 c , 15 min , room temperature , 3 h , chromatographic purification , 6575% yield ; ( iii ) 10% pd / c , ethanol , h2 , room temperature , 1824 h , 100% yield .
patented benzimidazole ring formation by cyclization of the 3-carboxyl group of 5-bromo-1h - pyrazolo[3,4-b]pyridine-3-carboxylic acid ( 3 ) with substituted 1,2-diaminobenzenes was realized via amide formation using coupling reagents , followed by treatment with glacial acetic acid .
hatu ( n , n , n,n-tetramethyl - o-(7-azabenzotriazol-1-yl)uronium hexafluorophosphate ) was used as a coupling reagent .
three 3-(1h - benzimidazol-2-yl)-1h - pyrazolo[3,4-b]pyridines ( l1l3 ) ( where x = h , br ; and y = h , ch2och3 ) have been synthesized in this work ( see chart 1 ) : 3-(1h - benzimidazol-2-yl)-1h - pyrazolo[3,4-b]pyridine ( l1 ) is a new compound that we have prepared as a model ligand for coordination to metals ; 3-(1h - benzimidazol-2-yl)-5-bromo-1h - pyrazolo[3,4-b]pyridine ( l2 ) was previously known as boc - protected compound prepared via 5-bromo-1h - pyrazolo[3,4-b]pyridine-3-carboxaldehyde;5-bromo-3-(4-methoxymethyl-1h - benzimidazol-2-yl)-1h - pyrazolo[3,4-b]pyridine ( l3 ) was synthesized via 5-bromo-1h - pyrazolo[3,4-b]pyridine-3-carboxylic acid ( 3 ) and patented as a potential cdk inhibitor and antiproliferative agent . for the synthesis of l1l3 , we used n , n-carbonyldiimidazole ( cdi ) as an amide - coupling reagent , because it is relatively inexpensive and the side products , carbon dioxide and imidazole , could be easily removed from the reaction mixture .
cdi - mediated amidation of acids 1 and 3 was performed as shown in scheme 3 ( steps i and ii ) . in the first step ,
the acyl - imidazolides 6 and 7 were obtained in dry dmf at room temperature and isolated as white solids .
the yields based on 3-methyl-1h - pyrazolo[3,4-b]pyridine are : 2529% ( 6 ) and 1316% ( 7 ) . in the second step , monoacylation of phenylenediamines ( 1,2-diaminobenzene and 1-methoxymethyl-2,3-diaminobenzene ( 5 ) ) using acyl - imidazolides was performed in dmf at 8085 c .
reagents and conditions : ( i ) cdi , dry dmf , room temperature , 2024 h ; ( ii ) 1,2-diaminobenzene or 5 , dry dmf , 8085 c ; 520 h ; and ( iii ) glacial acoh , 120125 c , 2.54.5 h , chromatographic purification .
amides 810 were used without further purification in ring closure reactions in a glacial acetic acid at 120125 c and afforded the desired 3-(1h - benzimidazol-2-yl)-1h - pyrazolo[3,4-b]pyridines : l1 ( 5560% ) , l2 ( 5861% ) , and l3 ( 4451% ) , based on 6 and 7 ( see scheme 3 , step iii ) .
the reported synthesis of l3 is a one - pot approach for amide formation using hatu with 57% yield , followed by cyclization under acidic conditions ( acetic acid ) with 87% yield .
thus , the ligands l1 , l2 , and l3 have been prepared in 7 , 8 , and 11 steps , correspondingly .
finally , the ligands l1l3 were reacted with [ mcl2(-p - cymene)]2 ( where m = ru , os ) in a 2:1 molar ratio in dry ethanol at room temperature to give [ mcl(-arene)(l)]cl complexes ( 11a , 11b , 12a , 12b , 13a , 13b ) in quantitative yields .
crystallization of [ rucl(-p - cymene)(l3)]cl ( 13a ) in etoh or etoh / et2o resulted in xrd - quality crystals of [ rucl(-p - cymene)(l3h ) ] ( 13c ) , while the crystallization of 13a in methanol afforded crystals of composition [ rucl(-p - cymene)(l3)]cl[rucl(-p - cymene)(l3h)]ch3oh ( 13dch3oh ) .
the osmium(ii ) analogue 13e0.75ch3oh0.25h2o was obtained via the crystallization of 13b in methanol . the full assignment of proton , carbon , and nitrogen resonances for l1l3 , 11a , 11b , 12a
, 12b , 13a , and 13b is quoted in tables s1s3 in the supporting information .
3-(1h - benzimidazol-2-yl)-1h - pyrazolo[3,4-b]pyridines with an unsubstituted benzimidazole moiety ( l1 , l2 ) display one set of signals .
l1 is characterized by three doublets of doublets for h4a , h5a , h6a ( 8.85 , 7.39 , 8.66 ppm , correspondingly ) of pyrazolopyridine moiety , two doublets for h4b , h7b ( 7.54 and 7.75 ppm ) , the overlapped h5b , h6b proton signals in one multiplet ( 7.24 ppm ) for a benzimidazole moiety and two singlet resonances for h1a and h1b ( for atom numbering , see chart 1 ) . the substitution of h5a by bromine ( l2 ) results in reduced multiplicity for the h4a and h6a signals ( two doublets at 8.99 and 8.75 ppm ) , whereas the benzimidazole moiety spectrum remains almost unchanged .
two singlets were attributed to nh protons and related to pyrazolopyridine ( h1a , at 14.19 ( l1 ) , 14.43 ( l2 ) ppm ) and benzimidazole ( h1b , at 13.11 ( l1 ) , 13.19 ( l2 ) ppm ) moieties .
nmr spectra for 11a , 11b , 12a , and 12b , where l1 and l2 coordinate as bidentate ligands via n2a and n3b with the formation of a stable five - membered chelate cycle , show one set of signals .
there was no evidence for monodentate or tridentate coordination of ligands with the formation of dinuclear or polynuclear complexes .
coordination of l1 and l2 to ruthenium(ii ) made it possible to assign the two doublets to proton resonances of h4b and h7b of the benzimidazole moiety . in the h ,
h roesy plots , one of them has cross - peaks with a ch cymene ring and the nearest to them is h4b ( e.g. , at 8.11 ( 11a ) , 8.06 ( 12a ) ppm ) .
a singlet at 14.91 ( 11a ) , 14.34 ( 12a ) ppm was assigned to nh proton and showed no couplings with other atoms .
the nitrogen resonance shift at 128.7 ( 11a ) , 127.3 ( 12a ) ppm is closer to the benzimidazole nh chemical shift in metal - free ligands ( 121.3 ( l1 , l2 ) ppm ) and , therefore , was assigned as n1b ( see table s3 in the supporting information ) .
the h1b resonance shows a significant shift by 1.8 and 1.15 ppm for 11a and 12a , respectively , upon ligand coordination ( l1 and l2 ) to the metal(ii)-arene moiety .
the nearest to the metal binding site pyrazolopyridine proton h1a was not detected for 11a , 11b , 12a , and 12b , and the proton resonance of h1b was also not seen for 11b or 12b .
l3 displays two sets of signals originated from 7b - l3 and 4b-l3 tautomers ( see chart 1 ) . the signals of pyrazolopyridine moieties are partially overlapped ( e.g. , h1a ( h1a ) at 14.47 and 14.43 ppm , h4a ( h4a ) at 8.99 and 8.98 ppm , h6a ( h6a ) at 8.75 ppm ) , whereas the signals of the benzimidazole moieties are better resolved ( see table s1 in the supporting information ) .
according to the h , h roesy plot , one of the ch2 groups at 4.80 ppm couples with the nh proton at 13.25 ppm , indicating their attribution to the 7b - l3 tautomer , whereas another nh proton at 13.22 ppm gives a cross - peak with h7b at 7.47 ppm and belongs to 4b-l3 tautomer ( figure 1 ) .
tautomers 7b - l3 and 4b-l3 are present in solution in 1:1.3 molar ratio and give , for the substituted benzimidazole moiety , two singlets ( h1b at 13.22 ppm , h1b at 13.25 ppm ) , two doublets of doublets ( h7b at 7.47 ppm , h4b at 7.72 ppm ) , one multiplet ( h5b , h6b , h5b , h6b at 7.287.21 ppm ) , two ch2 ( 4.80 ( h10b ) , 4.96 ( h10b ) ppm ) and two ch3 singlets at 3.37 ( h11b ) and 3.44 ( h11b ) ppm .
the absence of cross - peaks between h7b and h4b in the h , h cosy plot supports their relation to the two different molecules . the binding site in 4b-l3 tautomer is sterically shielded by a methoxymethyl group .
consistent with this nmr spectra display , only one set of signals is shown for 13a and 13b with the coordination of the 7b - l3 tautomer to ruthenium(ii ) and osmium(ii ) via n2a and n3b ( see scheme 4 ) .
the preference for coordination of the 7b - l3 tautomer is also confirmed by h , h roesy plots : h4b ( 7.99 ( 13a ) , 7.91 ( 13b ) ppm ) has couplings with ch protons of cymene ring .
the cross - peak between h10b at 4.88 ppm ( 13a ) and nh at 13.88 ppm ( 13a ) enabled the assignment of this singlet to a benzimidazole moiety ( h1b ) .
in addition , the chemical shifts for the c atoms of the benzimidazole moiety ( c4b , c7b , c5b , c6b ) of the coordinated ligand and metal - free 7b - l3 tautomer are very similar ( see table s2 in the supporting information ) .
the nitrogen resonance at 122.9 ppm ( 13a ) compares well to the benzimidazole nh chemical shift in metal - free l3 at 121.4 ppm . as for 11a , 11b , 12a , and
12b , the nearest to the coordination place pyrazolopyridine proton h1a resonance was not detected .
according to the h , h roesy plots of 11a , 12a , 13a , and 13b only ch cymene ring protons have couplings with the nearest h4b benzene ring proton , suggesting that the isopropyl or methyl groups are further away from the h4b proton .
the results of the xrd studies of [ oscl(-p - cymene)(l1)]cl4h2o ( 11b4h2o ) , [ oscl(-p - cymene)(l2)]cl ( 12b ) , [ oscl(-p - cymene)(l2)]cl2ch3oh2h2o ( 12b2ch3oh2h2o ) , [ rucl(-p - cymene)(l3h ) ] ( 13c ) , [ rucl(-p - cymene)(l3)]cl[rucl(-p - cymene)(l3h)]ch3oh ( 13dch3oh ) and [ oscl(-p - cymene)(l3)]cl[oscl(-p - cymene)(l3h)]0.75ch3oh0.25h2o ( 13e0.75ch3oh0.25h2o ) are shown in figures 2 , figure s2 in the supporting information , and figures 36 , respectively .
all complexes have a typical three - legged piano - stool geometry of ruthenium(ii ) and osmium(ii ) arene complexes , with an -bound p - cymene ring forming the seat and three other donor atoms ( two nitrogens n1 and n5 of the corresponding 3-(1h - benzimidazol-2-yl)-1h - pyrazolo[3,4-b]pyridine and one chlorido ligand ) as the legs of the stool . selected bond distances and angles are quoted in the figure captions .
all complexes crystallize as racemates , because of the presence of the stereogenic metal center .
fragment of the crystal structure of 11b4h2o showing nitrogen atoms n3 and n4 , which act as proton donors in intermolecular hydrogen bonding interactions n3hcl2 [ n3cl2 3.067(7 ) , n3hcl2 177.3 ] and n4ho3 [ n4o3 2.671(9 ) , n4ho3 170.9 ] ; thermal ellipsoids have been drawn at 50% probability level .
selected bond lengths and angles : os cl1 , 2.421(2 ) ; os
c(arene)av , 2.198(32 ) ; c1n2 , 1.349(11 ) ; n2n1 , 1.366(10 ) ; n1c6 , 1.357(11 ) ; c6c7 , 1.428(12 ) ; c7n5 , 1.345(11 ) ; n5c13 , 1.391(11 ) ; n1os n5 , 75.6(3) ; n1os cl1 , 85.5(2) ; n5os cl1 , 83.4(2). the bidentate 3-(1h - benzimidazol-2-yl)-1h - pyrazolo[3,4-b]pyridines , which can have different substituents in positions 5a and 7b , reveal different acid base properties .
it can act as a neutral organic ligand , with nitrogen atoms n2 and n4 as proton donors involved in intermolecular hydrogen bonding interactions n4ho1(x + 1 , y + 1 , z + 2 ) [ n4o1 , 2.730(4 ) ; n4ho1 , 176.1 ] and n2ho3(x + 2 , y + 1 , z + 1 [ n2o3 , 2.697(4 ) ; n4ho1 , 172.3 ] with one methanol and one water molecule , respectively , as is the case for 12b2ch3oh2h2o ( figure 3 ) or n2hcl2(cl2x ) and n4hcl1(x , y , z + 2 ) [ n4cl1 , 3.213(9 ) ; n4ho1 , 162.91 ] in 12b ( see figure s2 in the supporting information ) , correspondingly .
the ligand can be protonated at n3 ( n3hcl2 ) and deprotonated at n2 [ n2h o4 ( x + 1 , y + 1 , z + 1 ) with n2o4 2.923(9 ) , n2h
o4 138.7 ] ( overall charge zero ) with atom n4 as a proton donor to one of the four co - crystallized water molecules n4ho3 , as occurs in 11b4h2o ( see figure 2 ) .
ortep plot of the structure of the cation [ oscl(-p - cymene)(l2 ) ] in 12b2ch3oh2h2o .
thermal ellipsoids have been drawn at the 50% probability level . selected bond lengths and angles : os cl1 , 2.4043(1 ) ; os
c(arene)av , 2.197(26 ) ; c1n2 , 1.361(5 ) ; n2n1 , 1.344(4 ) ; n1c6 , 1.336(5 ) ; c6c7 , 1.447(6 ) ; c7n5 , 1.335(5 ) ; n5c13 , 1.380(5 ) ; n1os n5 , 74.73(13) , n1os cl1 , 84.29(10) ; and n5os cl1 , 83.25(10). in 13c , the ligand was found to be deprotonated at n2 , acting as a proton acceptor in the intermolecular hydrogen bonding interaction n2h n4 ( i denotes symmetry transformations x , y + 1.5 , z + 0.5 ) used to generate equivalent atoms ) [ n2n4 , 2.896(7 ) ; n2h n4 , 155.6 ] ( see figure 4 ) . ortep plot of the complex [ rucl(-p - cymene)(l3h ) ] ( 13c ) .
thermal ellipsoids have been drawn at 30% probability level . selected bond lengths and angles : ru cl , 2.399(2 ) ; ru n1 ,
c(arene)av , 2.186(31 ) ; c1n2 , 1.366(8 ) ; n2n1 , 1.353(7 ) ; n1c6 , 1.362(7 ) ; c6c7 , 1.426(9 ) ; c7n5 , 1.335(8 ) ; n5c13 , 1.398(8 ) ; n1ru n5 , 75.9(2) ; n1ru cl , 87.35(17) ; and n5ru cl , 85.73(17). complexes 13dch3oh and 13e0.75ch3oh0.25h2o crystallized both in the centrosymmetric triclinic space group p1. the asymmetric unit in both consists of a neutral complex [ mcl(-p - cymene)(l3h ) ] and a complex cation [ mcl(-p - cymene)(l3 ) ] ( m = ru or os ) , a chloride counteranion and co - crystallized solvent ( methanol or methanol / water )
. deprotonation of the organic ligand in [ mcl(-p - cymene)(l3h ) ] is corroborated by the presence of hydrogen bonding of the type n2ah n2b ( x + 1 , y + 1 , z + 1 ) in the crystal structure of the ruthenium complex 13dch3oh ( figure 5 ) and a similar interaction n2bh n2a ( x , y + 1 , z ) [ n2bn2a , 2.810(9 ) ; n2bh n2a , 170.1 ] in the crystal of the osmium analogue 13e0.75ch3oh0.25h2o . in figure 6 the structure of [ oscl(-p - cymene)(l3h ) ]
is shown . in both structures , the atoms n4a and n4b act as proton donors in strong hydrogen bonding interactions to the chloride counteranion , namely , n4ah
cl2 ( x + 1 , y + 2 , z + 1 ) [ n4cl2 , 3.275(6 ) ; n4ah cl2 , 176.9 ] , n4bh
cl2 [ n4bcl2 , 3.211(5 ) ; n4bh cl2 , 176.0 ] ( 13dch3oh ) and n4ah cl2 ( x + 1 , y + 1 , z + 1 ) [ n4acl2 , 3.273(8 ) ; n4ah cl2 , 177.0 ] , n4bh
cl2 [ n4bcl2 , 3.204(7 ) ; n4bh cl2 , 177.0 ] ( 13e0.75ch3oh0.25h2o ) .
fragment of the crystal structure of [ rucl(-p - cymene)(l3)]cl[rucl(-p - cymene)(l3h)]ch3oh ( 13dch3oh ) showing the intermolecular hydrogen bonding n2a hn2b [ n2an2b ( x + 1 , y + 1 , z + 1 ) , 2.814(7 ) ; n2a hn2b , 163.3 ] . selected bond lengths and angles : ru1a cl1a , 2.3952(17 ) ; ru1a
c(arene)av , 2.194(33 ) ; c1a n2a , 1.358(8 ) ; n2a n1a , 1.354(7 ) ; n1a c6a , 1.350(8 ) ; c6a c7a , 1.441(9 ) ; c7a n5a , 1.335(8 ) ; n5a c13a , 1.381(8 ) ; n1a ru1a n5a , 75.6(2) ; n1a
ru1a cl1a , 84.45(15) ; and n5a ru1a cl1a , 85.05(15). ortep plot of [ oscl(-p - cymene)(l3h ) ] in [ oscl(-p - cymene)(l3)]cl[oscl(-p - cymene)(l3h)]0.75ch3oh0.25h2o ( 13e0.75ch3oh0.25h2o ) .
selected bond lengths and angles : os1a cl1a , 2.402(2 ) ; os1a n1a , 2.068(7 ) ; os1a n5a , 2.086(7 ) ; os1a c(arene)av , 2.192(25 ) ; c1a n2a , 1.349(10 ) ; n2a n1a 1.369(9 ) ; n1a c6a , 1.361(10 ) ; c6a c7a , 1.431(11 ) ; c7a n5a , 1.339(10 ) ; n5a c13a , 1.398(11 ) ; n1a os1a n5a , 75.1(3) ; n1a
os1a cl1a , 83.56(18) ; and n5a os1a cl1a , 84.2(2). ligands l1l3 , as well as the corresponding ruthenium(ii ) ( 11a13a ) and osmium(ii ) ( 11b13b ) arene complexes , were studied with regard to their capacity of inhibiting cell growth in vitro in the human cancer cell lines ch1 ( ovarian carcinoma ) , sw480 ( colon carcinoma ) , and a549 ( non - small cell lung cancer ) , yielding the ic50 values listed in table 2 .
all compounds show the strongest effect in the generally chemosensitive ch1 cells , whereas the more chemoresistant a549 cells are also less affected by the compounds investigated here .
> l2 > l1 , indicating that bromination ( l2 , l3 ) and , even more so , the double substitution ( bromination and an additional replacement of h by a methoxymethyl group ( l3 ) ) are advantageous , with regard to activity .
these structure activity relationships are also reflected in the rank orders of the corresponding ruthenium and osmium complexes for the ruthenium complexes , 13a > 12a > 11a , and for the osmium complexes , 13b > 12b > 11b .
however , the ic50 values are shifted to higher concentrations ( see figure 7 ) .
the differences between the ruthenium and osmium analogues are mostly small ( ic50 values differ only up to a factor of 2.4 ) , with the osmium complexes being at least as active as their ruthenium counterparts .
ch1 denotes ovarian cancer , human ; sw480 denotes colon carcinoma , human ; and a549 denotes non - small - cell lung carcinoma , human .
50% inhibitory concentrations ( mean value standard deviation from at least three independent experiments ) obtained by the mtt assay ( 96-h exposure ) . concentration
effect curves of each ligand , compared to the corresponding ruthenium and osmium complexes , in ch1 ovarian cancer cells ( mtt assay , 96 h exposure ) : ( a ) l1 , 11a , 11b ; ( b ) l2 , 12a , 12b ; and ( c ) l3 , 13a , 13b . both the presence of substituents in the ligands and complexation result in a marked shift of antiproliferative activity . since 3-(1h - benzimidazol-2-yl)-1h - pyrazolo[3,4-b]pyridines have been reported to be potent cdk inhibitors , we expected the investigated ligands and complexes to induce cell cycle perturbations . to confirm this assumption in a sensitive cell line ,
exponentially growing ch1 cells were treated with the compounds for 24 h , stained with propidium iodide , and analyzed for their dna content by fluorescence - activated cell sorting ( facs ) .
surprisingly , the metal - free ligands l1l3 turned out to exert only modest effects on cell cycle distribution , with a slight increase of the g2/m fraction from 32% in the untreated control to 53% by 20 m l2 as the strongest effect observed in this setting ( higher concentrations of this compound led to disintegration of cells already after 24 h ) .
however , the less cytotoxic ruthenium complexes 12a and 13a , both bearing substituted ligands ( l2 and l3 correspondingly ) , cause a more pronounced g2/m phase arrest , as reflected by an increase of this cell fraction to 65% at 80 m and 59% at 40 m , respectively , and a concomitant decrease of the g0/g1 fraction to 21% and 24% ( compared to 42% in controls ) , whereas ruthenium complex 11a bearing an unsubstituted ligand l1 is devoid of activity on the cell cycle . on the other hand
, the osmium complexes do not generally show stronger effects on the cell cycle than the metal - free ligands , perhaps with the exception of 13b , which induces an increase of the g2/m fraction up to 53% at 80 m , accompanied by a decline of the g0/g1 fraction to 31% ( see figure 8) .
concentration - dependent effects of metal - free ligands ( top ) , and the corresponding ruthenium ( middle ) and osmium complexes ( bottom ) , on the cell cycle distribution of ch1 cells ( ( ) g0/g1 , ( ) s , and ( ) g2/m phase ) .
although the lack of generally pronounced cell cycle effects does not argue for a strong role of cdk inhibition in the mechanism of action of the investigated compounds , inhibitory potencies were studied in cell - free experiments with two recombinant cdk / cyclin complexes , by means of the histone h1 kinase assay .
results reveal that all compounds are capable of inhibiting kinase activities in a concentration - dependent manner , being more effective on cdk2/cyclin e than on cdk1/cyclin b ( see figure 9 ) .
in contrast to the observed cell cycle effects , but in accordance with antiproliferative activities , cdk - inhibitory potency of the metal - free ligands is consistently higher than that of the metal complexes . in particular , only l1l3 effectively inhibit cdk2/cyclin e in low concentrations ( 1 m or 10 m ) , whereas all complexes require higher concentrations to exert > 50% inhibitory effects . as in the mtt assay ,
differences between the effects of corresponding ruthenium and osmium complexes are minor , compared to the differences from those of the metal - free ligands .
concentration - dependent effects of metal - free ligands ( l1l3 ) as well as corresponding ruthenium ( 11a13a ) and osmium complexes ( 11b13b ) , on kinase activities of recombinant cdk1/cyclin b ( top ) and cdk2/cyclin e ( bottom ) .
the known multistep synthesis of 3-(1h - benzimidazol-2-yl)-1h - pyrazolo[3,4-b]pyridines , which we have modified , essentially afforded three organic compounds ( l1l3 ) that possess cdk - inhibiting properties .
these were used as bidentate ligands , and six novel organometallic complexes of the general formula [ mcl(-p - cymene)(l)]cl , where m = ru ( 11a , 12a , 13a ) or os ( 11b , 12b , 13b ) and l = l1l3 , have been synthesized and comprehensively characterized using spectroscopic and x - ray diffraction methods .
complexation of l1l3 with ruthenium or osmium yielded compounds with increased solubility in the biological medium , yet lowered antiproliferative activity in human cancer cell lines .
modulation of biological activities by substitution at the ligands can be accomplished in the metal - free molecules and their metal complexes in a similar way .
the known cdk - inhibitory activity of the ligands could be confirmed in cell - free experiments , in particular in cdk2/cyclin e , and their stronger effects on cdk activity parallel their higher capacity of inhibiting cancer cell growth in vitro , compared to their metal complexes .
nevertheless , the lack of pronounced effects on the cell cycle of chemosensitive ovarian cancer cells argues against a major role for inhibition of cell growth , at least in this setting .
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six organometallic complexes of the general formula [ miicl(6-p - cymene)(l)]cl , where m = ru ( 11a , 12a , 13a ) or os ( 11b , 12b , 13b ) and l = 3-(1h - benzimidazol-2-yl)-1h - pyrazolo[3,4-b]pyridines ( l1l3 ) have been synthesized .
the latter are known as potential cyclin - dependent kinase ( cdk ) inhibitors .
all compounds have been comprehensively characterized by elemental analysis , one- and two - dimensional nmr spectroscopy , uv vis spectroscopy , esi mass spectrometry , and x - ray crystallography ( 11b and 12b ) .
the multistep synthesis of 3-(1h - benzimidazol-2-yl)-1h - pyrazolo[3,4-b]pyridines ( l1l3 ) , which was reported by other researchers , has been modified by us essentially ( e.g. , the synthesis of 5-bromo-1h - pyrazolo[3,4-b]pyridine-3-carboxylic acid ( 3 ) via 5-bromo-3-methyl-1h - pyrazolo[3,4-b]pyridine ( 2 ) ; the synthesis of 1-methoxymethyl-2,3-diaminobenzene ( 5 ) by avoiding the use of unstable 2,3-diaminobenzyl alcohol ; and the activation of 1h - pyrazolo[3,4-b]pyridine-3-carboxylic acids ( 1 , 3 ) through the use of an inexpensive coupling reagent , n , n-carbonyldiimidazole ( cdi ) ) .
stabilization of the 7b tautomer of methoxymethyl - substituted l3 by coordination to a metal(ii ) center , as well as the nmr spectroscopic characterization of two tautomers 7b - l3 and 4b-l3 in a metal - free state are described .
structure
activity relationships with regard to cytotoxicity and cell cycle effects in human cancer cells , as well as cdk inhibitory activity , are also reported .
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Introduction
Experimental Section
Results and Discussion
Conclusion
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between april 1999 and march 2001 , 32 ( 26 men and 6 women ; age range , 7 - 62 years ; mean age , 43 years ) of 195 patients who had undergone ldlt underwent 42 sessions of conventional arteriography in search of bleeding foci of arterial origin within 6 months of the operation .
arterial bleeding was suspected clinically if the symptoms or signs were as follows : abnormally increased drainage of fresh blood through the jackson - pratt ( jp ) tube in 26 sessions , hematochezia or melena in 8 sessions , hemothorax or hemoptysis in 5 sessions , hemobilia in 2 sessions , or an acute decrease in the hemoglobin level in one session .
six patients underwent two or three sessions of arteriography or tae , because of recurrent arterial bleeding .
however , the bleeding foci were different in each of the sessions conducted for the same patient . in all patients ,
the arterial bleeding occurred within two months ( mean , 14 days ; range , 1 - 56 days ) after ldlt .
arteriography was performed within three days ( mean , 1 day ) after the detection of arterial bleeding , depending on the patient 's condition .
the underlying causes of hepatic failure and ldlt in these 32 patients were hepatitis b - related liver cirrhosis with or without hepatocellular carcinoma ( n=29 ) , secondary biliary cirrhosis due to intrahepatic stones ( n=1 ) , fatal fulminant hepatitis due to wilson 's disease ( n=1 ) , and biliary atresia ( n=1 ) .
the grafts involved in the ldlt included the right lobe ( n=21 ) , left lobe ( n=8 ) , dual left lobe ( n=2 ) , and left lateral segment ( n=1 ) . in all patients
, informed consent was obtained from the patient or the patient 's family prior to arteriography .
once the right or left femoral artery was punctured , a 5-f end - hole catheter was introduced over a 0.035-inch guide wire ( terumo ; radiofocus , tokyo , japan ) . following the superior mesenteric and common hepatic arteriographies , which were performed to evaluate the patency of the portal and hepatic arterial anastomoses and potential bleeding foci , the suspected arteries destined for selective arteriography were selected .
a provisional identification of the bleeding arteries was made based on the patient 's symptoms or signs , radiological findings , including dynamic ct and rbc scans , and the surgical information obtained during ldlt . in the patients with abnormally increased drainage of fresh blood through the jp tube , selective arteriographies were performed of several specific arteries , for which the arterial bleeding might be stagnant in the jp tube placed area . in those patients with hematochezia or melena , inferior mesenteric or left gastric arteriography was performed following superior mesenteric and common hepatic arteriographies , according to the available clinical information . in those patients with hemothorax or hemoptysis , selective arteriographies of the inferior phrenic artery ,
intercostal arteries adjacent to the pleural drain tubes , subclavian artery and/or bronchial artery were performed according to the available clinical information .
aortography for the purpose of gaining an understanding of the arterial anatomy prior to selective arteriography was not routinely performed .
however , aortography was performed to rule out the possible of there being any missed bleeding foci , if the selective arteriographies of the presumptive arteries did not demonstrate any active bleeding foci . if the bleeding foci were present on the arteriograms , then either tae or surgery was undertaken to control the bleeding , subject to the mutual agreement of the surgeons and interventional radiologists .
tae was intended to be the primary treatment , however , surgical management was considered as the primary treatment if the bleeding foci were technically too difficult or dangerous to be embolized , such as in the case of a hepatic arterial anastomotic site or hepatic resection margin . for tae
, the bleeding arteries were superselected using a 3f - microcatheter ( microferret ; cook , bjaerverskov , denmark ) and embolized with 0.018-inch - hilal microcoils ( cook , bloomington , u.s.a . ) or 0.018-inch - tornado microcoils ( cook , bloomington , u.s.a . ) .
gelatin sponge particles ( gelfoam ; upjohn , kalamazoo , mi ) were also used in the case of several specific arteries ( intercostal , inferior phrenic and epigastric arteries ) prior to embolization using microcoils , to minimize any rebleeding from collateral vessels .
if the bleeding foci were absent on the selective arteriograms and aortogram , explorative laparotomy or clinical observation was chosen by the surgeons , depending on the patient 's condition . in all patients , a complete retrospective review of the medical and surgical records and radiological imaging were performed .
the following items were documented retrospectively : the presumptive causes and locations of arterial bleeding , the technical and clinical success rates of tae , and the complications .
a bleeding focus was defined as an extravasation of the contrast media or a pseudoaneurysm that was a likely cause of bleeding .
the arterial bleeding was divided into four categories according to the possible related causes : surgical bleeding was defined as an arterial bleeding related to the ldlt itself and included bleeding from the hepatic resection margin , hepatic arterial anastomotic site , incision wounds , and the hepaticojejunostomy site .
iatrogenic bleeding was defined as bleeding associated with percutaneous invasive procedures , such as transhepatic biliary drainage , central venous access and chest tube insertion .
spontaneous bleeding was defined as bleeding that developed spontaneously , such as gastrointestinal bleeding remote from the hepaticojejunostomy site .
non - classifiable bleeding was defined as bleeding that was not able to be classified properly .
the technical success of tae was defined as the complete disappearance of arterial bleeding following tae . technically impossible or incomplete
clinical success was defined as an amelioration of the presenting signs or symptoms of arterial bleeding following tae .
major complications were defined as those necessitating an increased level of care , surgery , prolonged hospital stay , permanent adverse sequelae or death .
between april 1999 and march 2001 , 32 ( 26 men and 6 women ; age range , 7 - 62 years ; mean age , 43 years ) of 195 patients who had undergone ldlt underwent 42 sessions of conventional arteriography in search of bleeding foci of arterial origin within 6 months of the operation .
arterial bleeding was suspected clinically if the symptoms or signs were as follows : abnormally increased drainage of fresh blood through the jackson - pratt ( jp ) tube in 26 sessions , hematochezia or melena in 8 sessions , hemothorax or hemoptysis in 5 sessions , hemobilia in 2 sessions , or an acute decrease in the hemoglobin level in one session .
six patients underwent two or three sessions of arteriography or tae , because of recurrent arterial bleeding .
however , the bleeding foci were different in each of the sessions conducted for the same patient . in all patients ,
the arterial bleeding occurred within two months ( mean , 14 days ; range , 1 - 56 days ) after ldlt .
arteriography was performed within three days ( mean , 1 day ) after the detection of arterial bleeding , depending on the patient 's condition .
the underlying causes of hepatic failure and ldlt in these 32 patients were hepatitis b - related liver cirrhosis with or without hepatocellular carcinoma ( n=29 ) , secondary biliary cirrhosis due to intrahepatic stones ( n=1 ) , fatal fulminant hepatitis due to wilson 's disease ( n=1 ) , and biliary atresia ( n=1 ) .
the grafts involved in the ldlt included the right lobe ( n=21 ) , left lobe ( n=8 ) , dual left lobe ( n=2 ) , and left lateral segment ( n=1 ) .
in all patients , informed consent was obtained from the patient or the patient 's family prior to arteriography .
once the right or left femoral artery was punctured , a 5-f end - hole catheter was introduced over a 0.035-inch guide wire ( terumo ; radiofocus , tokyo , japan ) . following the superior mesenteric and common hepatic arteriographies , which were performed to evaluate the patency of the portal and hepatic arterial anastomoses and potential bleeding foci , the suspected arteries destined for selective arteriography were selected .
a provisional identification of the bleeding arteries was made based on the patient 's symptoms or signs , radiological findings , including dynamic ct and rbc scans , and the surgical information obtained during ldlt . in the patients with abnormally increased drainage of fresh blood through the jp tube , selective arteriographies were performed of several specific arteries , for which the arterial bleeding might be stagnant in the jp tube placed area . in those patients with hematochezia or melena , inferior mesenteric or left gastric arteriography was performed following superior mesenteric and common hepatic arteriographies , according to the available clinical information . in those patients with hemothorax or hemoptysis , selective arteriographies of the inferior phrenic artery ,
intercostal arteries adjacent to the pleural drain tubes , subclavian artery and/or bronchial artery were performed according to the available clinical information .
aortography for the purpose of gaining an understanding of the arterial anatomy prior to selective arteriography was not routinely performed .
however , aortography was performed to rule out the possible of there being any missed bleeding foci , if the selective arteriographies of the presumptive arteries did not demonstrate any active bleeding foci . if the bleeding foci were present on the arteriograms , then either tae or surgery was undertaken to control the bleeding , subject to the mutual agreement of the surgeons and interventional radiologists .
tae was intended to be the primary treatment , however , surgical management was considered as the primary treatment if the bleeding foci were technically too difficult or dangerous to be embolized , such as in the case of a hepatic arterial anastomotic site or hepatic resection margin . for tae
, the bleeding arteries were superselected using a 3f - microcatheter ( microferret ; cook , bjaerverskov , denmark ) and embolized with 0.018-inch - hilal microcoils ( cook , bloomington , u.s.a . ) or 0.018-inch - tornado microcoils ( cook , bloomington , u.s.a . ) .
gelatin sponge particles ( gelfoam ; upjohn , kalamazoo , mi ) were also used in the case of several specific arteries ( intercostal , inferior phrenic and epigastric arteries ) prior to embolization using microcoils , to minimize any rebleeding from collateral vessels . if the bleeding foci were absent on the selective arteriograms and aortogram , explorative laparotomy or clinical observation was chosen by the surgeons , depending on the patient 's condition .
in all patients , a complete retrospective review of the medical and surgical records and radiological imaging were performed .
the following items were documented retrospectively : the presumptive causes and locations of arterial bleeding , the technical and clinical success rates of tae , and the complications .
a bleeding focus was defined as an extravasation of the contrast media or a pseudoaneurysm that was a likely cause of bleeding .
the arterial bleeding was divided into four categories according to the possible related causes : surgical bleeding was defined as an arterial bleeding related to the ldlt itself and included bleeding from the hepatic resection margin , hepatic arterial anastomotic site , incision wounds , and the hepaticojejunostomy site .
iatrogenic bleeding was defined as bleeding associated with percutaneous invasive procedures , such as transhepatic biliary drainage , central venous access and chest tube insertion .
spontaneous bleeding was defined as bleeding that developed spontaneously , such as gastrointestinal bleeding remote from the hepaticojejunostomy site .
non - classifiable bleeding was defined as bleeding that was not able to be classified properly .
the technical success of tae was defined as the complete disappearance of arterial bleeding following tae . technically impossible or incomplete
clinical success was defined as an amelioration of the presenting signs or symptoms of arterial bleeding following tae .
major complications were defined as those necessitating an increased level of care , surgery , prolonged hospital stay , permanent adverse sequelae or death .
conventional arteriography demonstrated 42 bleeding foci of arterial origin in 30 sessions ( 23 patients ) .
the number of bleeding foci detected were one in 22 sessions ( 15 patients ) , two in six sessions ( 6 patients ) , and more than three in two sessions ( 2 patients ) .
the bleeding foci originated most frequently from the hepatic artery ( 14/42 , 33% ) and intercostal artery ( 11/42 , 26% ) .
based on a complete review of the clinical , interventional and surgical data , the bleeding foci were classified into 4 categories corresponding to the most likely causes of arterial bleeding ( table 2 ) . in this way
, it was demonstrated that , in addition to surgical bleeding ( 36% ) , iatrogenic bleeding ( 40% ) was also a major cause of arterial bleeding after ldlt .
tae was technically successful in 33 of the 42 foci of active arterial bleeding ( table 1 ) ( figs . 1 , 2 )
however , tae failed in the remaining 9 foci of active arterial bleeding , due to technically difficult superselection of the corresponding bleeding artery or the potential high risk associated with tae ( fig .
3 ) . these bleeding foci consisted of hepatic artery anastomotic sites ( n=2 ) , hepatic resection margins ( n=4 ) and hepaticojejunostomies ( n=3 ) , and they were treated by surgery .
however , in three sessions , additional immediate surgery had to be conducted following successful tae for the evacuation of hematoma .
however , there was no evidence of active arterial bleeding in the operation field in any of these cases .
in 20 of the 21 sessions , the patients showed a clinical improvement in the active arterial bleeding after successful tae .
one patient did not show any clinical improvement , because of recurrent arterial bleeding of the same focus .
this patient had been treated by anti - coagulation therapy due to cerebral infarction , resulting in a severe iatrogenic bleeding tendency , and died of multifocal hemorrhage including cerebral hemorrhage several days after tae .
the overall technical and clinical success rates for the 30 sessions of tae were 21 ( 70% ) and 20 ( 67% ) , respectively .
these relatively low success rates can be ascribed to the three bleeding foci ( hepatic resection margins , hepatic artery anastomotic sites and hepaticojejunostomy sites ) , in which embolization was either incomplete or impossible . in the case of the three bleeding foci ,
if these bleeding foci were excluded , the overall technical and clinical success rates were 19 ( 100% ) and 18 ( 95% ) out of 19 sessions .
six ( 19% ) patients underwent two or three sessions of arteriography , because of recurrent arterial bleeding .
however , different bleeding foci were treated in each session of arteriography conducted for the same patient . recurrent bleeding at the same foci was present in only one patient , as described above . on the other hand
these cases were managed either by surgery ( 6 patients ) or close clinical observation ( 5 patients ) with transfusion , depending on the patient 's condition . in three of the former six patients ,
surgery allowed the discovery of active arterial bleeding at the hepatic artery anastomotic site ( n=1 ) , hepatic resection margin ( n=1 ) , and venous oozing ( n=1 ) at the anastomosis of the hepatic vein and vena cava .
however , a retrospective review of the selective angiographic images of these patients did not demonstrate the presence of any bleeding focus . in the remaining three patients ,
surgery did not lead to the discovery of any bleeding focus , and they only underwent hematoma evacuation .
after either conservative or surgical management , the signs of active bleeding ameliorated in all 11 patients . during the 6 month follow - up period after ldlt , there were no major tae - related complications .
conventional arteriography demonstrated 42 bleeding foci of arterial origin in 30 sessions ( 23 patients ) .
the number of bleeding foci detected were one in 22 sessions ( 15 patients ) , two in six sessions ( 6 patients ) , and more than three in two sessions ( 2 patients ) .
the bleeding foci originated most frequently from the hepatic artery ( 14/42 , 33% ) and intercostal artery ( 11/42 , 26% ) .
based on a complete review of the clinical , interventional and surgical data , the bleeding foci were classified into 4 categories corresponding to the most likely causes of arterial bleeding ( table 2 ) . in this way
, it was demonstrated that , in addition to surgical bleeding ( 36% ) , iatrogenic bleeding ( 40% ) was also a major cause of arterial bleeding after ldlt .
tae was technically successful in 33 of the 42 foci of active arterial bleeding ( table 1 ) ( figs . 1 , 2 )
. however , tae failed in the remaining 9 foci of active arterial bleeding , due to technically difficult superselection of the corresponding bleeding artery or the potential high risk associated with tae ( fig .
3 ) . these bleeding foci consisted of hepatic artery anastomotic sites ( n=2 ) , hepatic resection margins ( n=4 ) and hepaticojejunostomies ( n=3 ) , and they were treated by surgery .
however , in three sessions , additional immediate surgery had to be conducted following successful tae for the evacuation of hematoma .
however , there was no evidence of active arterial bleeding in the operation field in any of these cases . in 20 of the 21 sessions , the patients showed a clinical improvement in the active arterial bleeding after successful tae .
one patient did not show any clinical improvement , because of recurrent arterial bleeding of the same focus .
this patient had been treated by anti - coagulation therapy due to cerebral infarction , resulting in a severe iatrogenic bleeding tendency , and died of multifocal hemorrhage including cerebral hemorrhage several days after tae .
the overall technical and clinical success rates for the 30 sessions of tae were 21 ( 70% ) and 20 ( 67% ) , respectively .
these relatively low success rates can be ascribed to the three bleeding foci ( hepatic resection margins , hepatic artery anastomotic sites and hepaticojejunostomy sites ) , in which embolization was either incomplete or impossible . in the case of the three bleeding foci ,
the technical success rate was only 2 ( 18% ) out of 11 sessions . if these bleeding foci were excluded , the overall technical and clinical success rates were 19 ( 100% ) and 18 ( 95% ) out of 19 sessions .
six ( 19% ) patients underwent two or three sessions of arteriography , because of recurrent arterial bleeding .
however , different bleeding foci were treated in each session of arteriography conducted for the same patient .
recurrent bleeding at the same foci was present in only one patient , as described above .
on the other hand , arteriography showed no active bleeding focus in 12 sessions ( 11 patients ) .
these cases were managed either by surgery ( 6 patients ) or close clinical observation ( 5 patients ) with transfusion , depending on the patient 's condition . in three of the former six patients ,
surgery allowed the discovery of active arterial bleeding at the hepatic artery anastomotic site ( n=1 ) , hepatic resection margin ( n=1 ) , and venous oozing ( n=1 ) at the anastomosis of the hepatic vein and vena cava .
however , a retrospective review of the selective angiographic images of these patients did not demonstrate the presence of any bleeding focus . in the remaining three patients ,
surgery did not lead to the discovery of any bleeding focus , and they only underwent hematoma evacuation .
after either conservative or surgical management , the signs of active bleeding ameliorated in all 11 patients . during the 6 month follow - up period after ldlt , there were no major tae - related complications .
according to our results , arterial bleeding after ldlt was not confined to surgical causes .
surgical bleeding was one of the most common causes of bleeding after ldlt , however , iatrogenic bleeding was even more common than surgical bleeding in our retrospective study .
coagulopathy after ldlt is inevitable for a certain period of time in most liver transplant recipients , until the graft 's function is normalized .
the risk of iatrogenic bleeding is also high in the early post - ldlt period .
thus , clinicians should make a particular effort to prevent iatrogenic bleeding during percutaneous procedures after ldlt . some amount of surgical bleeding from the hepatic artery at the hepatic arterial anastomotic site , hepatic resection margin or hepaticojejunostomy site is inevitable , because ldlt generates a liver graft with variable - sized cut surfaces and multiple anastomotic sites .
in fact , bleeding from the hepatic artery was the most common cause of bleeding in our study . however , performing tae in the case of bleeding from the hepatic artery following ldlt was virtually impossible , because of the difficulty involved in the superselection of the bleeding arteries , due to the existence of fine arterial feeders , hepatic arterial anastomotic stenosis , a tortuous arterial course or multifocal occurrences .
furthermore , performing tae of the hepatic artery runs the risk of hepatic infarction or failure following inadvertent diffuse proximal embolization ( 16 ) .
( 17 ) reported that three of nine patients with extrahepatic pseudoaneurysm following liver transplantation underwent effective coil embolization , however all patients subsequently underwent retransplantation due to graft ischemia .
accordingly , we achieved technical success in only four of the 14 foci involving bleeding from the hepatic arterial branches .
although tae can be performed in some patients with massive bleeding from the hepatic artery , we consider that surgical management is the treatment of choice for the management of surgically induced bleeding from the hepatic artery anastomotic site , hepatic resection margin or hepaticojejunostomy site .
the cause of bleeding from the intercostal artery was iatrogenic bleeding in most cases , resulting from either placement of a drain tube or central venous catheterization .
the cause of bleeding from the inferior phrenic artery was uncertain , however it might have been related to various causes , such as intraoperative retractor injury , chest tube insertion or spontaneous bleeding , and it must be routinely evaluated to rule out active bleeding . knowledge of the clinical symptoms or signs of arterial bleeding provided by the clinicians is very helpful prior to the performance of arteriography . a jp drain was the most common clinical clue to the presence of arterial bleeding after ldlt .
bleeding can occur from the hepatic , intercostal , inferior phrenic or pancreaticoduodenal arteries or from the jejunal branches of the superior mesenteric artery .
unusually , bleeding from the deep circumflex iliac and inferior epigastric arteries was manifested as jp bleeding in one case .
therefore , in our opinion , arteriograms of the inferior phrenic and intercostals arteries , as well as of the superior mesenteric and common hepatic arteries , should be surveyed in cases of jp drain bleeding .
in addition , those arteries supplying the lower thoracic and abdominal walls should also be considered as possible sources of arterial bleeding . as in the case of general surgical patients , ldlt recipients are also subject to bleeding from stress ulceration , peptic ulcer and angiodysplasia of the colon ( 4 , 6 , 18 ) .
not uncommonly , this bleeding tends to be resolved only by proper medical treatment ( 4 ) .
clinical observation may be sufficient whenever the patient 's condition tolerates it . in our study , four patients with hematochezia or melena showed negative outcomes on arteriography , but improved clinically without surgery or tae . in fact , tae was very effective in cases of positive arteriography , and all four patients with arteriographically proven gastrointestinal bleeding improved after only one session of tae . in the conventional arteriogram , a negative outcome does not always guarantee the absence of actual bleeding . in our study ,
surgery demonstrated active bleeding foci in three of six patients without active bleeding on selective arteriography .
this discrepancy seems to have been caused by minor or intermittent arterial bleeding or venous oozing .
in contrast , surgery did not lead to the detection of any bleeding focus in the three remaining patients . in conclusion ,
technical limitations were encountered when using tae for the management of hepatic arterial bleeding . however , in the other locations , tae seems to be effective and safe for the control of arterial bleeding after ldlt .
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objectiveto analyze the causes of arterial bleeding after living donor liver transplantation ( ldlt ) and to evaluate the efficacy of transcatheter arterial embolization ( tae).materials and methodsforty - two sessions of conventional arteriography were performed in 32 of the 195 patients who underwent ldlt during the past 2 years .
this was done in search of bleeding foci of arterial origin .
tae was performed with microcoils or gelatin sponge particles .
the causes of arterial bleeding , the technical and clinical success rates of tae and the complications were retrospectively evaluated.resultsforty-two bleeding foci of arterial origin were identified on 30 sessions of arteriography in 21 patients .
the most common cause of bleeding was percutaneous procedures in 40% of the patients ( 17 of the 42 bleeding foci ) followed by surgical procedures in 36% ( 15/42 ) .
the overall technical and clinical success rates of tae were 21 ( 70% ) and 20 ( 67% ) of the 30 sessions , respectively .
the overall technical success rate of tae for the treatment of bleeding from the hepatic resection margin , hepatic artery anastomotic site and hepaticojejunostomy was only 18% ( 2/11 ) , whereas for the treatment of bleeding in the other locations the technical and clinical success rates of tae were 100% and 95% , respectively .
no procedure - related major complications occurred.conclusionin the case of arterial bleeding after ldlt , percutaneous procedure - related hemorrhages were as common as surgery - related hemorrhages .
there were technical difficulties in using tae for the treatment of hepatic arterial bleeding .
however , in the other locations , tae seems to be safe and effective for the control of arterial bleeding in ldlt recipients .
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MATERIALS AND METHODS
Patient Population
Conventional Arteriography and Transcatheter Arterial Embolization
Analysis
RESULTS
Causes and Locations of Arterial Bleeding
Transcatheter Arterial Embolization
DISCUSSION
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delivery of exogenous nucleic acids such as dna into cells ( transfection ) holds great promise for disease prevention and therapy ( aka : gene therapy).(1 ) genetic material delivered into the cell modifies the function of such cells generally by altering the production of proteins .
the selection of appropriate dna carriers is a common problem for gene therapy , and better gene delivery agents are still being sought .
viral vectors ( such as retroviral , lentiviral , adenoviral ) and nonviral vectors ( such as liposomal and polymeric ) have both been used as gene delivery agents in the past .
although viral vectors are efficient carriers due to their natural ability to penetrate cells , they have also often been shown to provoke undesirable immune responses , thus limiting their usefulness.(2 ) nonviral vectors often prove less immunogenic , but improvement is needed to increase their overall transfection efficiency . to increase such efficiency ,
effort should be directed at increasing the vector s ability to promote efficient dna condensation , intracellular transport , and sustained gene expression .
additionally , the cytocompatibility of nonviral vectors must be well understood before they can be further optimized and become a viable option for gene therapy .
fullerene ( c60 ) derivatives have been extensively studied for a variety of medical applications(8 ) including their use as neuroprotective agents,(9 ) hiv-1 protease inhibitors,(10 ) bone - disorder therapy agents,(11 ) x - ray contrast agents,(12 ) and slow - release agents for drug delivery.(13 ) recently , c60-based reagents have also been examined as dna transfection vectors and tested for the ability to mediate gene transfer . these first generation c60-based transfection vectors have shown promise , but a few have also exhibited high cytotoxicity.(16 ) as many of the c60 derivatives previously described in the literature have only been slightly soluble in aqueous solutions , it has also been suggested that one possible reason for such observed cytotoxicity was due to the use of organic solvents that were necessary to dissolve those first generation c60-based transfection agents .
thus , the search for new materials and enhanced protocols are key objectives in the continuing development of c60-based transfection vectors , including the design , construction , and testing of new reagents that are water soluble and able to effect enhanced gene delivery without promoting significant cytotoxicity
. herein we report transfection efficiencies , cytotoxicity profiles , and biophysical structure / activity studies for a new class of water - soluble c60 vectors prepared using the hirschbingel reaction which is one of the simplest , highest - yielding , and most versatile c60 functionalization methods known . as shown in figure 1 ,
the structurally derivatized c60 vectors reported herein were synthesized to yield either positively charged , negatively charged , or neutral chemical structures when solvated under physiological conditions .
these vectors , which have been analyzed for their ability to promote dna transfection , offer the opportunity to elucidate the roles that hydrophobicity ( due to the fullerene core ) , hydrophilicity ( due to the water soluble substituents ) , and overall charge state displayed by the c60 derivatives have on dna transfection , gene expression , and cytotoxicity .
depiction of the structures of the derivatized c60 vectors ; the positively charged amino - c60 compounds ( iiv ) , the neutral serinolamide - c60 compound ( v ) , and the negatively charged c3-c60 compound ( vi ) ( chemical structures depicted at neutral ph in aqueous solution ; counterions in solution not shown ) .
materials for culturing cells , including dulbecco s modified eagle s medium ( dmem ) , trypsin , and phosphate buffer saline ( pbs ) , were obtained from gibco ( gibco life , grand island , ny ) .
complete osteogenic medium containing minimum essential medium ( mem ) , 10 mm -glycerophosphate , 50 g / ml ascorbic acid , and 10 vol % fetal bovine serum was obtained from gemini bio - products ( calabasas , ca ) .
a plasmid dna ( 7.2 kbp ) containing the gene for a red - shifted wavelength variant of the enhanced green fluorescent protein ( egfp ) under the transcriptional control of the cytomegalovirus ( cmv ) immediate - early gene promoter was used as the dna construct from which reporter gene expression was measured in the transfection assays .
plasmid dna was prepared in bulk from transformed bacterial sources and purified to homogeneity using commercial kits ( qiagen ; germantown , md ) employing anion - exchange chromatography .
the amino - c60 adducts ( iiv ) , the seri - c60 adduct ( v ) , and the c3-c60 adduct ( vi ) were synthesized and characterized according to previously published procedures .
stock solutions of individual compounds ivi were prepared by weight and filtered for sterilization using a cellulose acetate membrane filter ( 0.2 m pore diameter ) prior to use in forming transfection mixtures .
standard solutions were prepared at a concentration 10 g/l for each of these compounds in both tris edta ( te ) solution ( 10 mm tris , 1 mm edta , ph = 7.4 ) , and in serum - free dmem in separate reaction vials .
nih 3t3 mouse fibroblast cells ( atcc # crl-1658 ) and hek 293 cells ( human embryonic kidney ; atcc # crl-1573 ) were cultured in dmem containing 10% fbs in a 5% co2 atmosphere at 37 c prior to collection and plating in individual assay wells used for transfection studies . for subculture of cells , routinely on reaching 80% confluence ,
the cells were washed with pbs , detached using trypsin - edta , harvested by centrifugation , resuspended in culture medium and counted in a coulter counter prior to plating in multiwell dishes for subsequent transfection assays .
marrow stromal cells ( mscs ) were harvested from wistar rats as previously described.(24 ) mscs were cultured for 6 days in the presence of 10 m dexamethasone in complete osteogenic media before use in transfection studies and cultured under identical environmental conditions to 3t3 and hek 293 cells .
mixtures of individual derivatized c60 vector material and plasmid dna were allowed to form a complex in serum - free dmem , as described below , prior to adding to cells in the transfection assays .
each c60-vector solution was added dropwise to a solution of plasmid dna to obtain a final volume of 200 l .
each resulting solution was then vortexed briefly and allowed to stand for 30 min at room temperature to allow complete assembly of the vector / dna complex , which was then used immediately in the transfection experiment .
the final dna mass of each solution remained constant at 10 g while the concentration of the c60 derivative was varied to obtain a range of c60-reagent - to - dna - base - pair ( bp ) ratios ( defined here as
the value of dna mass was fixed at 10 g per well since it showed the most optimum transfection among different dna masses used ( 112 g ) for transfection at r = 16.8 .
transfections of nih 3t3 cells were carried out with c60/dna complexes in a range of r values ( 0.4242 ) .
for hek 293 cells and mscs , transfections were performed only at r = 4.2 and r = 16.8 .
cells were plated at 40,000 cells / cm in 96 well tissue culture plates and incubated overnight to permit cell attachment to the well surface .
the culture medium was then replaced by the serum - free transfection mixture for various time periods ( 2 h , 8 h , 24 or 48 h ) ( transfection time ) . after exposing the cells for the respective periods of time to the serum - free transfection mixture
, the cells were washed with pbs and the medium was replaced with complete medium ( containing fbs ) .
the cells were then further incubated for 8 h , 24 or 48 h ( incubation time ) before gfp fluorescence was measured using a flow cytometer . for comparative purposes
, control cell populations were also transfected with plasmid dna alone ( no c60 vector ) , or with plasmid dna complexed with an optimal level of one of two commercially available transfection reagents ; such that the dna was complexed with either 25 kda polyethylenimine ( pei ) , or with cytopure transfection reagent , which is reported by the manufacturer to exhibit very low levels of cytotoxicity .
the dna / pei complexes were assembled using a well - established protocol(25 ) and the cytopure / dna complexes were assembled as per the manufacturer s instructions and optimized to obtain an optimal level of dna transfection / gfp expression within the nih 3t3 cell type .
below is a brief description of the conditions which gave the optimal level of transfected nih 3t3 cells using 25 kda pei and cytopure . for pei ,
dna / polymer complexes were prepared in serum - free dmem to achieve a ratio of polymer to dna of 4 .
the complexes ( 100 l ) were then incubated at 25 c for 1015 min and then added to cell wells that contained 100 l of serum - free dmem .
for cytopure , 1.1 l of cytopure stock was diluted to 50 l with serum - free dmem .
the cytopure mixture was then added slowly to 1 g of dna diluted to 50 l with serum - free dmem .
the transfection mixture was vortexed , left standing for 15 min at room temperature and added to cell wells that contained 100 l of serum - free dmem .
after 24 h of transfection , the cells were washed and the medium was replaced with the serum - containing medium . for purposes of this study ,
transfection as described herein also relates to cell viability for sufficient time to ensure gfp gene expression , which was determined by cell fluorescence levels above a defined background threshold level ( determined using nontransfected cells to set lower detection limit parameter ) with flow cytometry .
cells were prepared for flow cytometry by trypsinization after being washed with sterile pbs to remove cell debris and any residual gene - delivery agents .
cells that were transfected successfully expressed gfp protein and were detected at 470/515 nm ( excitation / emission ) by the flow cytometer .
transfection efficiency has been determined as the percent of cells that express gfp per study sample relative to the total number of cells passing through the flow cytometer per study sample ( 10,000 events counted for each transfection sample).(25 ) the c60/dna complex solutions were prepared as described above .
c60 vector solutions without dna ( 010 g/l ) were prepared by diluting the c60 stock solution with serum - free dmem .
the concentration of cultured cells was determined prior to cell plating using a coulter counter and the cells were plated on 96 well clear - bottom plates at a density of 40,000 cells / cm . after incubating overnight to allow cell attachment , the medium in each well was replaced either with 200 l of the c60 vector alone or with c60 vector / dna solutions prepared as described above .
the cells were exposed to the solutions for either 2 , 8 or 24 h before the cytotoxicity was determined .
control cell samples were plated and grown under identical conditions as those described for treated cell samples , with the exception that neither c60 compounds nor transfection mixtures were added to the control cells .
the live / dead viability / cytotoxicity assay ( molecular probes , eugene , or ) was used to determine cytotoxicity levels in cells treated with either c60-vector or c60-vector / dna solutions .
this assay makes use of 2 different fluorescent dyes , calcein am and ethidium homodimer ( ethd-1 ) , to measure the number of live and dead cells respectively . the lipid permeable dye , calcein am , when taken up by living cells with intact plasma membranes , emits green fluorescence ( excitation / emission : 495/515 nm ) upon being cleaved by esterase enzymes in the cells .
conversely , the ethd-1 dye emits red fluorescence ( excitation / emission : 528/617 nm ) on binding to nucleic acids released from dead cells , and is excluded by the intact cell membranes of viable cells .
this assay was preferred over the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide ( mtt ) assay for evaluation of cell viability because c60-vectors compounds i and ii interacted and attached to the mtt - formazan making the formazan crystals insoluble for the next colorimetric assay .
similar effects have been reported for single - walled carbon nanotubes.(26 ) the reagents were prepared and added to the wells according to the manufacturer s guidelines . at the time points indicated , the media from the wells was aspirated and replaced with the live / dead reagent , after washing the cells with pbs .
the plates were then incubated in the dark at room temperature for additional 30 min before being measured for fluorescence .
fluorescence of each sample well was measured by using a fluorescent microplate reader ( tecan safire ) .
fluorescence of each sample well was normalized to percent of both live ( no c60 compounds or transfection mixtures treatment condition ) and dead ( 70% methanol treated ) control cell groups .
these normalized fractions are defined as the fraction of live and dead cells , respectively , in each of the c60-treated samples .
since gfp fluorescence has excitation / emission wavelengths of 470/515 nm , close to that of calcein am , there was a possibility that gfp fluorescence could interfere with the calcein am ( live ) measurement leading to potential errors for those wells containing c60 vector / dna mixtures .
thus , a baseline fluorescence intensity of each sample well at 495/515 nm was obtained before the addition of the live cell / dead cell dyes .
this intensity was then subtracted from the final calcein am detection intensity . for the dynamic light scattering ( dls ) experiments ,
a standard solution with concentration 10 g/l of each of the c60 vectors was prepared in each of these solvents : hplc grade water , 10 mm tris / edta solution , and serum - free dmem .
these solvents were filtered through a 0.22 m syringe filter ( millpore , millex- gp ) prior to use .
the size of standard 100 nm polystyrene beads in tris / edta solution and in serum - free dmem was measured to confirm that no discrepancies were detected with the data acquisition system .
dls measurements were conducted using a wyatt technologies dawn eos instrument equipped with a quasi - elastic light scattering interface module ( wyatt qels unit ) .
the scattering angle used for detection was 90 at a constant temperature of 25 c .
data analysis and size distribution computations were performed using the vendor supplied software ( astra v ) and a multi- digital recorder ( wyatt qels ) .
statistical analysis was performed between groups for the live / dead assay and for flow cytometry studies .
groups were analyzed by anova with significance defined by a p - value < 0.05 , and pairwise comparison was performed using the tukey test .
the cytotoxicity studies had four samples per group , and the transfection studies had three samples per group .
initial screening was performed on each of the six different derivatized c60 samples , compounds ivi ( figure 1 ) .
the different c60-vector / dna complexes were assembled at various r values ( 0.4242 ) and each analyzed for its ability to affect transfection in nih 3t3 cells under serum - free conditions .
serum - free conditions were used in these studies to ensure that derivatized nanomaterial / dna complexes were not compromised by serum lipids and/or proteins , and that the dna complexes were imported into the cells based on the individual physical properties of the complexes themselves , without the potential aid of serum proteins acting as carrier .
the best transfection efficiencies were routinely observed after allowing transfection mixtures to remain on cells for 24 h ( transfection time ) , then removing the transfection mixtures and incubating cells for an additional 24 h ( incubation time ) in the presence of serum for maximal gene expression to be detected . unless otherwise noted , all transfection efficiency data presented below were for cells transfected for 24 h under serum - free conditions and incubated for an additional 24 h in the presence of serum .
all c60-conjugated transfection mixtures showed the ability to transfect cells with exogenous dna with statistically significant differences relative to unconjugated dna alone ( naked dna ) ( table 1 ) .
transfection efficiency as reported in table 1 is defined as the ratio of gfp - positive cells as determined by fluorescence under flow cytometry per total number of cells counted in the flow cytometer .
no statistical difference was observed between the transfection efficiencies of the different groups at r = 0.42 and r = 4.2 . for r values above 4.2 , compounds iii and iv exhibited a statistical difference in transfection efficiencies compared to the other compounds . both of these compounds exhibited an increased transfection efficiency relative to the other c60 compounds studied , with a maximum transfection efficiency of 31% at r = 16.8 . at larger values of r
the efficiency dropped off . at r = 16.8 , their transfection efficiencies were comparable to that of 25 kda pei containing and cytopure transfection reagent containing complexes .
25 kda pei is a widely used and well - studied nonviral vector , while cytopure is a biodegradable polymeric transfection reagent that exhibits very low cytotoxicity and is reported to work on a broad spectrum of cell types , including hard - to - transfect primary cell isolates .
c60 vectors are listed in order of their measured efficiency at promoting transfection in nih 3t3 cells under serum - free conditions . to assess the transfection ability of c60 vectors in different cell types ,
studies were also performed on two other cell types : hek 293 cells and mscs .
hek 293 cells were chosen since they are another standard cell line used routinely for transfection assays , while mscs were chosen due to their eminence as primary cells for tissue engineering .
based on the initial screening results of each of the various derivatized c60 compounds for transfection - promoting ability in nih 3t3 cells as reported in tables 1 and 2 , transfection studies were also performed on hek 293 cells and mscs using compounds iii and iv at ratios of r = 4.2 and r = 16.8 .
however , hek 293 cells ' transfection efficiencies were lower than those observed for nih 3t3 cell at similar r values .
mscs exhibited low transfection efficiencies overall , and no increase in transfection efficiency was observed with increase in r values .
transfected cells were visualized by fluorescence microscopy for morphological analysis after transfection ( micrographs not shown ) .
positive transfection of cells with the plasmid coding for green fluorescent protein could be easily discerned by this technique .
however , variations in transfection efficiency among the different cell types tested here is not surprising , as cell type is a major factor in determining the level of transfection and gene expression from nonviral vectors as noted previously.(27 ) differences in cell characteristics , such as cell mitosis rates , can play an important role in transgene expression , and may explain the differences in the level of gene expression among the various cell lines studied here .
transfection efficiency values are presented as a quotient of gfp ( + ) cells relative to the total viable cells present in the transfected sample well as measured by flow cytometry .
figures 2 a , b and c display the fraction of viable nih 3t3 cells as a function of the c60 vector ( no dna present ) concentration after 2 , 8 , or 24 h post - treatment incubation times , respectively .
incubation with c60 compounds for 2 h resulted in a viable cell fraction greater than 50% for all c60 vector solutions .
lowest overall cell viability was observed with compound i ( 0.55 0.08 ) at 10 g/l , and no statistically significant increase in cell viability was observed with decreasing compound i concentration . for all other c60 compounds tested
, there was a general trend of increased cell viability with decreased c60 compound concentration .
fraction of live nih 3t3 cells after incubation with the different c60 vectors as a function of derivatized fullerene concentration after ( a ) 2 h , ( b ) 8 h , or ( c ) 24 h c60-incubation times respectively , under serum - free conditions .
error bars represent mean standard deviation for n = 4 . at the 8 h c60 incubation time , the fraction of live cells was higher than 50% for all c60 solutions tested except those exposed to compound i. for compound i , the cell viability was the lowest ( 0.51 0.04 ) at 10 g/l , but no statistically significant increase in cell viability was observed with decreasing compound i concentration . for all other c60 vectors ,
there was a general trend of increased cell viability with decreased compound concentration , but at each compound concentration tested , the cell viability was lower in the 8 h time point than in the corresponding sample concentration at the 2 h time point .
at the 24 h c60 incubation time point , only cells treated with compounds iii and iv exhibited greater than 50% cell viability at all concentrations tested , with an increase in cell viability with decreased c60 compound ( iii and iv ) concentration .
all other compounds showed a general trend of increased viability with a decrease in compound concentration , with cell viability values lower than 50% at 10 g/l .
figures 3 a , b and c display the cell viability of nih 3t3 cells cultured in the presence of the various c60-vector / dna mixtures as a function of both c60 compound concentration and r values after 2 , 8 or 24 h post - transfection time , respectively .
cell viability at the two hour post - transfection time point for compound i / dna and compound ii / dna mixtures increased from 0.19 0.0 at 10 g/l ( r = 42 ) to 0.70 0.15 at 0.1 g/l ( r = 0.42 ) .
the fraction of live cells was greater than 50% for all other c60 derivative / dna solutions at the 2 h post - transfection time point .
fraction of live nih 3t3 cells after incubation with the different c60/dna transfection mixtures as a function of c60 compound concentration and varying r value after ( a ) 2 h , ( b ) 8 h , or ( c ) 24 h post - transfection incubation time , under serum - free conditions respectively .
error bars represent the mean standard deviation for n = 4 . at the 8 h post - transfection time point ,
the fraction of live cells for compound i / dna and compound ii / dna was less than 50% at all concentrations and r values tested . for all other samples ,
the trend was similar to that of the 2 h post - transfection time point , with cell viability greater than 50% and an increase in viability observed with a decrease in transfection mixture concentration .
at the 24 h time point , cells treated with the compound v / dna mixture exhibited greater than 50% cell viability at all concentrations tested , while compound i / dna , compound ii / dna exhibited less than 50% cell viability .
cells treated with compounds iii / dna , iv / dna and vi / dna mixtures resulted in cell viabilities less than 50% at 4 g/l ( r = 16.8 ) and 10 g/l ( r = 42 ) , but greater than 50% viability when added at lower concentrations .
dls and optical microscopy studies were performed to help elucidate the biophysical structure / activity relationships that might exist between the various c60 complexes .
these studies were carried out to help generate correlations between the different c60 vectors , and the various c60 vector / dna structural attributes resulting in the different transfection efficiencies and cytotoxicity results observed under serum - free conditions .
table 3 presents the dls measurements on the various c60 vector compounds ivi when dissolved alone in either water ( 4 g/l ) , in a 10 mm tris / edta solution ( 4 g/l ) , in serum - free dmem ( 4 g/l ) , or when dissolved in complex with dna in 10 mm tris / edta ( 4 g/l , r = 16.8 ) or as a complex with dna in serum - free dmem ( 4 g/l , r = 16.8 ) .
the concentration of 4 g/l was chosen for dls experiments since compounds iii and iv exhibited maximum transfection efficiency at this concentration of c60 .
the rh values in table 3 provide qualitative information about the transfection mixture aggregate behavior which can readily be seen in figures 4 and 5 .
the rh parameter obtained from the dls measurements , and shown in table 3 , corresponds to the radius of a monodisperse hypothetical sphere having the same diffusion constant as that of the c60 compound aggregates or the c60/dna mixture aggregates under study .
pdi values between 0 and 0.05 indicate more monodisperse particles , and values above 0.05 indicate broadened size distributions.(30 ) clearly , the rh values shown in table 3 for the various c60 compounds and c60/dna complexes are average values for quite polydisperse populations of aggregates .
previous work by our group and others using atomic force microscopy ( afm ) and cryo transmission electron microscopy ( cryo - tem ) have shown that derivatized c60 compounds form aggregates that are irregularly shaped in solution .
the rh values observed for these c60 compounds and the c60/dna mixtures suggests that these reagents also form such irregularly shaped aggregates when in aqueous solutions , including when dissolved in solutions of physiological ionic strength such as dmem .
such qualitative information about the aggregate behavior provides valuable information for understanding the transfection and toxicity results , as discussed below .
representative morphologies of nih 3t3 cells and the c60/dna complexes at r = 0.42 ten minutes after transfection .
the optical images represent complexes formed with compound ( a ) i , ( b ) ii , ( c ) iii , ( d ) iv , ( e ) v , and ( f ) vi . all images are shown at the same magnification .
representative optical microscopy images of nih 3t3 cells incubated with c60/dna complexes formed using compounds ( a ) iii and ( b ) vi at r = 0.42 twenty four hours post - transfection .
all c60 compounds display a broadly distributed aggregate size in each solvent tested ( water , 10 mm tris / edta solution , and serum - free dmem ) .
the compounds had aggregate sizes between 47 and 650 nm ( pdi = 0.330.51 ) and between 33 and 446 nm ( pdi = 0.290.46 ) in plain water and 10 mm tris / edta solution , respectively .
in the presence of dna , there is an increase in rh for all formulations . in general
, the rh values decrease in the order v , vi > i > ii > iii , iv in the presence of dna . presumably , this trend is the result of the influence of the various structures of the c60 derivatives on the condensation behavior of the dna in the transfection mixture . in the case of cationic polymers ,
the condensation process with dna has been thought to rely predominantly on electrostatic interactions with minor contributions from other interactions such as hydrophobic interactions , hydrogen bonding , and van der waals forces.(5 ) in case of the positively charged c60 derivatives , the hydrophobicity of the c60 core , and electrostatic interactions between the water - soluble functional groups and the dna are the main contributors to the condensation process .
compound i , with two adducts on the fullerene core , has less positive charge than the hexa - amino c60 adduct of compound ii and the octa - amino adduct of compound iii .
fewer electrostatic interactions between functionalized c60 vectors and the dna sugarphosphate backbone can lead to a decrease in condensation capacity , resulting in larger c60-vector / dna complexes .
compound v , although polar , has no net charge at neutral ph ; while compound vi will possess a negative charge , the same as that of dna .
thus , for compounds v and vi , hydrophobicity is presumably the only interaction contributing to the formation of the dna condensates , and this interaction appears insufficient to fully condense the dna , leading to large c60 vector / dna complex aggregates .
the relatively large sizes for the c60 vector / dna complexes , and in particularly for those formed from compounds i , ii , v and vi , may be a reason for the poor transfection efficiencies observed with these compounds .
this may also be a reason for the low transfection efficiency reported for a similar positively charged amino - c60 compound with a monoadduct on the fullerene core.(17 ) however , increased positive charge - state alone is insufficient to solely predict increased transfection capability of amino - derivatized fullerenes .
variation in transfection protocol and/or cell types can also dramatically affect transfection efficiencies , as positively charged amino - derivatized c60 similar to compound iv have previously been shown to be inefficient at effecting transfection.(17 ) in general , the condensation behavior of nonviral vector / dna complexes is governed by several factors including the molecular weight of the dna condensing agent , the density of the charges on the condensing agent , and the ratio of composition ( r values ) between the condensing vector and the dna.(5 ) in addition , it is now well - documented that the size of the transfection vector / dna complex is one of the most important parameters for efficient dna delivery into cells , with the most efficient uptake observed for complex with sizes < 150200 nm in diameter.(5 ) the rh values shown in table 3 suggest that only dna complexes formed with compounds iii and iv satisfy these criteria .
on addition of the c60-vector / dna complexes to cells , the complexes formed with compounds i and ii immediately aggregated further to form large clusters , even at the lowest r value tested of 0.42 .
figure 4af shows the optical microscopy images of all c60-vector / dna complexes at r = 0.42 ten min after their addition to nih 3t3 cells .
large , irregularly shaped aggregates ( shown as brown ) can clearly be seen for complexes formed with compound i ( figure 4a ) and for complexes formed with compound ii ( figure 4b ) .
the aggregate concentration is greater and more densely distributed for compound i than for compound ii .
no such increased aggregation propensity was seen for the other c60 vector / dna complexes ( figure 4cf ) .
however , time - dependence of aggregation could be observed for compound iii and compound vi .
figure 5a , b shows the optical microscopy images of compound iii / dna , and compound vi / dna complexes at r = 0.42 at 24 h post - transfection time .
the images show the presence of large sized aggregates , more densly distributed for compound vi than for compound iii .
interestingly , addition of compound iii / dna mixtures caused cell morphological changes when added to cultured cells , causing the attached cells to round up from the substratum surface in contrast to the usual planar , extended / elongated shapes the cells assumed in the normal growth state .
this shape change may be attributed to the depolymerization / disruption of either the actin or tubulin networks of the cytoskeleton by the complexes , and/or to the disruption / inhibition of focal adhesion plaques formed on the cell surface . however , this observation at r = 0.42 does not have any noticeable effects on cell proliferation , as flow cytometry measurements indicate no statistically significant difference in the number of cells for those treated with compound iii / dna complexes relative to the controls cells ( no c60/dna complexes added ) .
our results indicate that compounds iii and iv , when complexed with dna , bring about the highest levels of transfection when compared to the other derivatized c60 compounds under evaluation in this study . at the optimal ratio of compound iii or iv to dna ( r = 16.8 ) ,
however , at 24 h post - transfection , there is only approximately 30% viability in cells treated with these two tranfection mixtures .
this result is in contrast to cell viability of greater than 50% in cells treated with the compounds iii or iv alone at equivalent c60 concentrations ( 4g/l ) as that when in complex with dna at r = 16.8 .
the temporal increase in aggregate sizes for these c60/dna complexes ( not seen in the free c60 compounds alone ) leads to precipitation onto the cell surface when these mixtures are left in the culture medium for 24 h ( e.g. , see figure 5 ) .
this effect may explain the higher levels of cell death in the transfection mixture - treated cells .
the overall trend observed in these studies was that increased cell death was observed with an increase in either c60 concentration alone or for increased r values in c60/dna mixtures , irrespective of the chemical nature of the c60 compound , with compounds iii and iv exhibiting the least toxicity when free in solution , but not necessarily when complexed with dna ( under these conditions , compound v seems to exhibit the least cell toxicity ) .
also , higher levels of cytotoxicity seem to correlate more with complex aggregate formation induced by the presence of dna in the mixtures , leading to the formation of visible precipitates . such deposition of aggregates onto the cell surface will impair normal functioning of the plasma membrane and thereby contribute to cell death.(31 ) there may also be other slow processes contributing to cell death for the positively charged c60 vectors in the c60/dna solutions at the longer ( 8 and 24 h ) transfection times .
following cellular entry , the presence of positively charged amino - c60 compounds ( iiv ) ( individuals or as aggregates ) inside the cell or nucleus may lead to disruptions to other cellular functions , such as interfering with histonedna interactions and/or other cellular processes.(7 ) the favorable transfection efficiencies of compounds iii and iv suggest that these compounds have some characteristics in common with other positively charged amino - c60 derivatives previously cited in the literature which are reported to effect transfection in cells .
these similarities include having a flexible linker situated between the fullerene core and the positively charged amino groups .
although the results presented here suggest that the structures of compounds iii and iv will result in higher levels of transfection than those previously reported by others , direct comparison of our results with the results from others using different structurally derivatized positively charged amino c60 vectors is not possible , since the structure of the c60 vectors , the cell lines tested , and the methodologies used are not consistent .
another distinct feature of all the c60 vectors reported here is their water solubility which obviates the need to use organic solvents such as dimethylformamide ( dmf ) in the preparation of the transfection mixture ; the high cytotoxicity observed for some other c60-based transfection agents has been attributed to the toxicity of dmf .
the comparable transfection efficiencies of compounds iii and iv on nih 3t3 cells to those of commercially available nonviral vectors 25 kda pei and cytopure is encouraging .
25 kda pei is a widely used nonviral vector and is quite often used as a standard for comparing new nonviral vectors .
this high cytotoxicity has been attributed more to free pei than to the pei / dna complex . however , in case of compound iii or iv , at the optimal r = 16.8 , their complexes with dna show higher toxicity ( 30% viability ) compared to the free compounds iii or iv alone ( greater than 50% cell viability ) .
since the cytotoxicity seems to correlate more with complex aggregate formation induced by the presence of dna in the mixtures , future refinement of the c60/dna complex preparation protocol , such as disaggregating these complexes with phosphate buffer and/or nacl , should potentially lead to lower cytotoxicity while maintaining the same level of transfection efficiency , or potentially even increase these transfection efficiency levels .
furthermore , although not addressed in this study ( for reasons of maintaining uniformity in c60/dna complex formation and cellular entry ) , the presence of serum proteins on enhanced c60/dna complex uptake , and on subsequent cell viability during extended transfection times , would also be worth examining , and could be expected to lower the observed cellular cytotoxicity levels seen in this study .
the small , but statistically significant , expression of the reporter protein from c60/dna conjugates formed from compounds v and vi was surprising and interesting since these samples do not possess a net positive charge under the physiological conditions used to form the complexes , although there have been limited reports of other nonpositive charged nanoparticles translocating into cells(35 ) and , indeed , also demonstrating gene transfection.(36 ) thus , at least for some derivatized c60 molecules , the hydrophobic nature of the c60 carbon nanostructures may be the common important property in their binding to dna and their subsequent translocation into the interior of the cell .
there is now a growing body of work that suggests that carbon nanomaterials such as fullerenes , metallofullerenes and carbon nanotubes , due the hydrophobicity of their carbon sheaths , have the ability to efficiently translocate into the interior of cells irrespective of the charge of their water - solubilizing moieties .
in addition , carbon nanomaterials such as gd@c60 , gd@c82 , gd@c80 and gd@ultrashort single - walled carbon nanotubes ( gd@us - tubes ) have shown potential as high - performance magnetic resonance imaging ( mri ) contrast agents suitable for advanced applications such as tracking and/or delivery of magnetically labeled cells by mri in vivo .
these reports , along with the favorable c60-based gene transfection results reported here and by others , offer opportunities for development of analogous gd@c60-based vectors to monitor noninvasively the biodistribution and pharmacokinetics of gene therapy vectors in vivo.(49 )
a new class of water - soluble c60 transfecting agents with positively charged , negatively charged , or neutral chemical functionalities under physiological conditions was prepared using hirschbingel chemistry .
transfection , cytotoxicity and biophysical structure / activity studies were performed in an effort to elucidate the relationship between the hydrophobicity of the fullerene core , the hydrophilicity of the water - solubilizing groups , and the overall charge state of the c60 vectors in gene delivery / expression .
however , only two positively charged c60 derivatives , namely , an octa - amino derivatized c60 and a dodeca - amino derivatized c60 vector , showed efficient in vitro transfection .
increased levels of cellular toxicity were observed for positively charged c60 vectors relative to the negatively charged and neutral vectors .
structural analyses using dynamic light scattering and optical microscopy confirmed that higher positive charge on c60 compounds is a dominant physical attribute necessary for optimal c60/dna structural features leading to increased transfection efficiency , and that aggregation is the major factor that negatively affected the cytotoxicity profiles of the c60 vector / dna complexes .
aggregation is presumably also the dominant reason for increased cytotoxicity of certain specific derivatized c60 compounds ( most notable for compound i ) even in the absence of dna , at least when analyzed under the serum - free conditions utilized in this study .
future studies should be able to capitalize on this information and enable the design of additional c60 derivatives that address these issues specifically and help lower the propensity of these reagents to aggregate in the presence of dna .
such reagents would be expected to enhance transfection of cells and tissues while simultaneously lowering toxicity levels .
future studies using these reagents will also more precisely indicate the cellular uptake and endosomal release mechanisms that these compounds inherently possess , thereby lending further insight into potential enhanced chemical design of future generations of these carbon - based nanomaterials specifically for dna delivery into the cell nucleus .
the successful demonstration of intracellular dna uptake , intracellular transport , and gene expression from dna using c60 vectors suggests the possibility of developing analogous gd@c60-based vectors to serve simultaneously as both therapeutic and diagnostic agents .
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a new class of water - soluble c60 transfecting agents has been prepared using hirschbingel chemistry and assessed for their ability to act as gene - delivery vectors in vitro . in an effort to elucidate the relationship between the hydrophobicity of the fullerene core , the hydrophilicity of the water - solubilizing groups , and the overall charge state of the c60 vectors in gene delivery and expression , several different c60 derivatives were synthesized to yield either positively charged , negatively charged , or neutral chemical functionalities under physiological conditions .
these fullerene derivatives were then tested for their ability to transfect cells grown in culture with dna carrying the green fluorescent protein ( gfp ) reporter gene .
statistically significant expression of gfp was observed for all forms of the c60 derivatives when used as dna vectors and compared to the ability of naked dna alone to transfect cells .
however , efficient in vitro transfection was only achieved with the two positively charged c60 derivatives , namely , an octa - amino derivatized c60 and a dodeca - amino derivatized c60 vector .
all c60 vectors showed an increase in toxicity in a dose - dependent manner .
increased levels of cellular toxicity were observed for positively charged c60 vectors relative to the negatively charged and neutral vectors .
structural analyses using dynamic light scattering and optical microscopy offered further insights into possible correlations between the various derivatized c60 compounds , the c60 vector / dna complexes , their physical attributes ( aggregation , charge ) and their transfection efficiencies .
recently , similar gd@c60-based compounds have demonstrated potential as advanced contrast agents for magnetic resonance imaging ( mri ) .
thus , the successful demonstration of intracellular dna uptake , intracellular transport , and gene expression from dna using c60 vectors suggests the possibility of developing analogous gd@c60-based vectors to serve simultaneously as both therapeutic and diagnostic agents .
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Introduction
Experimental Section
Results and Discussion
Conclusion
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generalized anxiety disorder ( gad ) is a chronic disorder mainly characterized by pathological worry .
gad also presents with a variety of somatic and psychological symptoms such as restlessness , muscle tension , sleep disturbance , irritability , difficulty concentrating , and fatigue .
gad is one of the most prevalent anxiety disorders , with its lifetime prevalence estimated to be of 5.7% in the united states and 2.8% in europe [ 2 , 3 ] .
patients with gad have a higher likelihood of developing other mood disorders in their lifetime ; one of the most common is major depressive disorder , present in 62% of gad patients .
since gad affects normal functioning , these patients are usually frequent users of healthcare resources ; in fact , studies aimed to explore gad clinical prevalence have estimated it to be 7.3% in primary care and up to 13% in the psychiatric outpatient setting [ 5 , 6 ] .
gad is difficult to diagnose by primary care physicians and psychiatrists . in people who suffer gad , commonly occurring comorbid conditions ( e.g. , depression , alcoholism , other anxiety disorders ) often mask the underlying anxiety and result in a missed diagnosis of gad [ 7 , 8 ] .
other factors complicating the diagnosis of gad are patients tendency to complain about the somatic symptoms , such as sleeplessness and fatigue , rather than the psychic symptoms ( e.g. , nervousness , apprehension , chronic worry ) , and physicians lack of education in the diagnosis of gad .
diagnostic criteria have been modified in each edition of the diagnostic and statistical manual of mental disorders ( dsm ) [ 1 , 11 , 12 ] .
duration of excessive worry criterion and the number of associated symptoms are still questioned today . the duration criterion was increased to 6 month in the dsm - iii - r to distinguish gad from adjustment disorders or situational stress reactions .
however , recent studies have suggested that this duration excludes significantly impaired patients from diagnosis .
dsm - v , which is expected to get released in may 2013 , will revise the wording and organization of gad diagnostic criteria in order to simplify them .
meanwhile , several groups are conducting studies aiming to assess the need for 6 months duration of excessive anxiety and worry criterion [ 13 , 15 - 17 ] . using data from the us national comorbidity survey replication , ruscio et al . have demonstrated that broadening the gad diagnosis criteria , more than doubles its prevalence .
this broadening of the criteria consists on a shortening of anxiety duration from at least 6 months to at least 1 month ( but less than 6 months ) , relaxing excessiveness to include excessive and non - excessive worry , and reducing the associated symptoms from a minimum of 3 to a minimum of 2 .
most of the increase in prevalence comes from reducing the minimum duration to 1 month and , to a lesser extent , from eliminating the excessive worry requirement .
prevalence is minimally affected by requiring 2 rather than 3 associated symptoms . in a recent study conducted
both in developed and developing countries , the 6-month duration criterion was not found to influence symptom severity , age of onset , persistence , impairment or comorbidity when compared to shorter criterion durations ( 1 - 2 months and 3 - 5 months ) . to our knowledge , there is no study aimed to evaluate prospectively the evolution of anxiety symptoms in patients diagnosed with broad dsm - iv criteria under medical practice . before considering changing dsm - iv criteria , full consequences of these changes should be explored .
broadening of gad criteria could lead to earlier diagnosis of gad patients and to diagnosis of otherwise under diagnosed patients .
earlier diagnosis could translate into a sooner start of treatment and this could lead to an improvement in the course of the illness and the quality of life .
in fact , gad patients have reported lower perceived quality of life than non - anxious controls and a lower degree of social functioning than patients with arthritis or diabetes . in a recent study by lee et al . , those patients with more than 12 months duration of symptoms were more severely impaired and had a lower rate of recovery than the groups with shorter duration of symptoms , suggesting that an earlier diagnosis could also benefit patient response and recovery . in an attempt to elucidate the consequences of broadening dsm - iv criteria for generalized anxiety disorder ,
the current study examined prospectively the evolution of gad symptoms in two groups of diagnosed patients in public and private mental healthcare settings ; one group according to the existing dsm - iv criteria and the other according to broader criteria ( at least 1 month but less than 6 months of excessive or non - excessive worry and 2 associated symptoms listed on dsm - iv for gad diagnosis ) . by using the total ham - a scores , changes in evolution of anxiety symptoms
were studied in both criteria groups under the basis of routine medical care in a 6 month period .
finally , self - reported quality of life , disability and other patient - reported - outcomes were studied as a measure of the overall well - being of gad patients .
this was a multicentre , prospective and observational study carried out in outpatient psychiatric clinics between october 2007 and january 2009 .
the study was approved by the local ethics committee of the hospital clnico de san carlos ( madrid ) and was conducted according to the helsinki declaration for research in the human being .
functional and clinical outcome measures were completed in all three visits and included the following instruments : hamilton anxiety rating scale , montgomery - asberg depression rating scale ( baseline and 6 month visit only ) , clinical global impression - severity of illness scale , sleep scale from medical outcomes study ( mos - sleep ) , world health organization disability assessment schedule ii ( baseline and 6 month visit only ) , and the quality of life questionnaire , eq-5d ( all described in detail below ) . at baseline , socio - demographic data ,
current therapy and psychiatric and medical illnesses information were collected . for the 3 and 6 months visits ,
psychiatrists participating in the study made a confirmatory diagnosis of gad in 6 consecutive patients , 3 according to dsm - iv criteria and 3 according to broad criteria .
although patients could have symptoms and receive treatment before entering the study , they had nt been diagnosed before by the psychiatrist .
eligible patients were men and women over 18 years of age that were diagnosed with gad by a trained psychiatrist at the baseline visit .
exclusion criteria included previous gad diagnosis , inability or difficulty to understand patient - reported - outcomes questionnaires written in spanish .
diagnosis was carried out by a trained psychiatrist , familiar with the study procedures , and who worked in an outpatient setting .
sampling frame included all public and private healthcare settings within the 17 regions of spain .
first stage consisted on a selection of the outpatient psychiatry clinics ( mental health centres ) in each healthcare region .
the number of clinics selected in each region was proportional to the region s population , being the probability of choosing each clinic relative to the population in the area covered by the clinic . in the second stage ,
one psychiatrist per clinic was chosen randomly within those with previous experience in clinical and epidemiological research in psychiatry , and with at least 5 year experience in mental health diseases diagnosis .
if a selected psychiatrist refused to participate , he or she was replaced by another from the same clinic ( also selected randomly ) .
participant psychiatrists were asked to invite in a consecutive manner those patients from the daily list of appointments that fulfilled the inclusion criteria .
psychiatrists were encouraged to maintain the above mentioned proportion of 3 subjects diagnosed following dms - iv criteria and 3 subjects diagnosed following the broad criteria .
sample size was calculated taking into account study s main variable : evolution of anxiety symptoms according to the total ham - a scores for each patient group .
a sample of 3400 evaluable patients was estimated assuming a 2-tailed 95% confidence interval for the total ham - a scores lower than 0.25 points from the observed mean .
based on the results of a previous study , a standard deviation lower than 8 was assumed .
gad was diagnosed following the dsm - iv criteria ( dsm - iv criteria group ) in one group .
the other group ( broad criteria group ) was diagnosed according to broader criteria consisting on shortening of anxiety duration from at least 6 months to at least 1 month ( but less than 6 months ) , including excessive or non - excessive worry and reducing the associated symptoms from a minimum of 3 to a minimum of 2 .
hamilton anxiety rating scale ( ham - a ) : the validated spanish version of the ham - a scale was used to evaluate the evolution of anxiety symptoms during all 3 visits of the study .
the ham - a scales includes 14 items , each of them rates from 0 ( absence ) to 4 ( severe ) .
the total score comes from the addition of all 14 items with a maximum of 56 .
scores higher than 24 were considered as severe anxiety ; between 16 and 24 , moderate ; between 10 and 15 , mild ; and below 9 , no anxiety . a psychic anxiety sub domain ( addition of items 1 - 6 and 14 ) and a somatic anxiety sub domain ( addition of items 7 - 13 )
montgomery - asberg depression rating scale ( madrs ) : the validated spanish version of the madrs scale was used to measure the intensity of depression symptoms .
this observer - rated depression scale consists on 10 items regarding different depressive symptoms , each item being scored from 0 to 6 depending on the severity of symptoms ; the total score results from adding the score of each item , obtaining a minimum of 0 ( no symptoms ) and a maximum of 60 ( very severe ) .
clinical global impression - severity of illness scale ( cgi - s ) : participant psychiatrists evaluated patient s global severity with the cgi - s scale , which rates illness severity from 0 to 7 , where 0 was considered as not evaluated
, 1 as no disease , 2 borderline ill , 3 mildly ill
, 4 moderately ill , 5 markedly ill , 6 severely ill , and 7 extremely ill .
sleep scale from medical outcomes study ( mos - sleep ) : mos - sleep scale evaluates the impact or interference with sleep of any disease or treatment .
the 7 subscales are sleep disturbance ( 4 items ) , snoring ( 1 item ) , awaken short of breath or with headache ( 1 item ) , quantity of sleep ( 1 item ) , optimal sleep ( 1 item ) , sleep adequacy ( 2 items ) , and somnolence ( 3 items ) .
this scale also has a sleep problems index , rated from 0 ( no interference ) to 100 ( maximum interference ) , and a sleep problems subscale .
each item is rated independently with more interference scoring higher , except for the sleep adequacy and quantity and optimal sleep subscales [ 21 , 22 ] .
world health organization disability assessment schedule ii ( who - das ii ) : the who - das ii is a psychometric instrument that measures day to day functioning and disability in the following six activity domains : understanding and communicating , moving and getting around , self care , getting along with people , work activities , and participation in society . the 12-item who - das ii scale
is rated from 1 ( none or nothing ) to 5 ( extreme or inability to perform a given task ) . the total calculated score ranges from 0 to 100 , with higher scores indicating higher degrees of disability , for the total scale as well as for each subscale .
eq-5d : this is a standard self - reported quality of life questionnaire developed in several european countries and validated for spain .
this questionnaire consists of two parts , the first one aims to evaluate patient s health profile in five dimensions ( mobility , self - care , daily living activities , pain , and anxiety / depression ) .
the patient rates his / her current status as no problems , some / moderate problems , and severe / extreme problems for each dimension .
results are then converted into 1 of the 243 different eq-5d health state descriptions which are used to calculate a unique index or tariff , between 1 ( as healthy as possible ) and 0 ( usually equivalent to death ) .
this index is used to calculate the time - trade off tariff which allows calculating quality - adjusted - life - year ( qaly ) gain ( further explanation below ) .
the second part consists on a visual analogue scale ( vas or thermometer ) in which the patient has to rate his health status between 0 ( equivalent to death ) and 100 ( the best possible healthy status ) .
for statistical analysis , last observation carried forward approach was used for those patients for whom data from the 6 months visit were missing . if the only missing data was that from the 3 months visit , this was estimated proportionally to the change observed at the 6 months visit , given that the missing data at 3 month visit were completely at random .
patients were considered to respond to treatment when a higher than 50% reduction was observed for the total ham - a scale score at the study final visit when compared to that of the baseline visit .
descriptive statistics were prepared for the continuous variables in the study , including the assessment of central position and dispersion ( two - tailed 95% confidence interval ) .
the kolmogorov - smirnov test was applied to check adjustment of data to a gaussian distribution .
student t tests and chi - square test were used for continuous and categorical variables , respectively , to test homogeneity of baseline data between groups .
analysis of covariance ( ancova ) or binary logistic regression models were fitted comparing dsm - iv criteria versus broad criteria groups in most variables adjusting by baseline scoring , sex , age , body mass index , educational level , marital status and percentage of patients with comorbid major depression or depressive disorder .
analysis of variance ( anova ) was applied for quantitative measures and chi square ( mcnemar s test ) for qualitative measures in case of paired comparisons .
the clinical relevance of observed differences in baseline characteristics was ascertained by means of the effect size that was calculated using the difference of means for each group , divided by the combined standard deviation of the variables .
qalys gain after the 6-months follow - up was calculated from patients responses to the eq-5d as described previously using tariff calculated with this instrument .
gained qalys were estimated by using the trapezoidal approach , with baseline and 6- month visit values used as reference values .
all statistical tests were two - tailed , and error of < 0.05 was accepted as statistically significant .
this was a multicentre , prospective and observational study carried out in outpatient psychiatric clinics between october 2007 and january 2009 .
the study was approved by the local ethics committee of the hospital clnico de san carlos ( madrid ) and was conducted according to the helsinki declaration for research in the human being .
functional and clinical outcome measures were completed in all three visits and included the following instruments : hamilton anxiety rating scale , montgomery - asberg depression rating scale ( baseline and 6 month visit only ) , clinical global impression - severity of illness scale , sleep scale from medical outcomes study ( mos - sleep ) , world health organization disability assessment schedule ii ( baseline and 6 month visit only ) , and the quality of life questionnaire , eq-5d ( all described in detail below ) . at baseline , socio - demographic data ,
current therapy and psychiatric and medical illnesses information were collected . for the 3 and 6 months visits ,
psychiatrists participating in the study made a confirmatory diagnosis of gad in 6 consecutive patients , 3 according to dsm - iv criteria and 3 according to broad criteria .
although patients could have symptoms and receive treatment before entering the study , they had nt been diagnosed before by the psychiatrist .
eligible patients were men and women over 18 years of age that were diagnosed with gad by a trained psychiatrist at the baseline visit .
exclusion criteria included previous gad diagnosis , inability or difficulty to understand patient - reported - outcomes questionnaires written in spanish .
diagnosis was carried out by a trained psychiatrist , familiar with the study procedures , and who worked in an outpatient setting .
sampling frame included all public and private healthcare settings within the 17 regions of spain .
first stage consisted on a selection of the outpatient psychiatry clinics ( mental health centres ) in each healthcare region .
the number of clinics selected in each region was proportional to the region s population , being the probability of choosing each clinic relative to the population in the area covered by the clinic . in the second stage ,
one psychiatrist per clinic was chosen randomly within those with previous experience in clinical and epidemiological research in psychiatry , and with at least 5 year experience in mental health diseases diagnosis .
if a selected psychiatrist refused to participate , he or she was replaced by another from the same clinic ( also selected randomly ) .
participant psychiatrists were asked to invite in a consecutive manner those patients from the daily list of appointments that fulfilled the inclusion criteria .
psychiatrists were encouraged to maintain the above mentioned proportion of 3 subjects diagnosed following dms - iv criteria and 3 subjects diagnosed following the broad criteria .
sample size was calculated taking into account study s main variable : evolution of anxiety symptoms according to the total ham - a scores for each patient group .
a sample of 3400 evaluable patients was estimated assuming a 2-tailed 95% confidence interval for the total ham - a scores lower than 0.25 points from the observed mean .
based on the results of a previous study , a standard deviation lower than 8 was assumed .
gad was diagnosed following the dsm - iv criteria ( dsm - iv criteria group ) in one group .
the other group ( broad criteria group ) was diagnosed according to broader criteria consisting on shortening of anxiety duration from at least 6 months to at least 1 month ( but less than 6 months ) , including excessive or non - excessive worry and reducing the associated symptoms from a minimum of 3 to a minimum of 2 .
hamilton anxiety rating scale ( ham - a ) : the validated spanish version of the ham - a scale was used to evaluate the evolution of anxiety symptoms during all 3 visits of the study .
the ham - a scales includes 14 items , each of them rates from 0 ( absence ) to 4 ( severe ) .
the total score comes from the addition of all 14 items with a maximum of 56 .
scores higher than 24 were considered as severe anxiety ; between 16 and 24 , moderate ; between 10 and 15 , mild ; and below 9 , no anxiety . a psychic anxiety sub domain ( addition of items 1 - 6 and 14 ) and a somatic anxiety sub domain ( addition of items 7 - 13 )
montgomery - asberg depression rating scale ( madrs ) : the validated spanish version of the madrs scale was used to measure the intensity of depression symptoms .
this observer - rated depression scale consists on 10 items regarding different depressive symptoms , each item being scored from 0 to 6 depending on the severity of symptoms ; the total score results from adding the score of each item , obtaining a minimum of 0 ( no symptoms ) and a maximum of 60 ( very severe ) .
clinical global impression - severity of illness scale ( cgi - s ) : participant psychiatrists evaluated patient s global severity with the cgi - s scale , which rates illness severity from 0 to 7 , where 0 was considered as not evaluated
, 1 as no disease , 2 borderline ill , 3 mildly ill , 4 moderately ill , 5 markedly ill , 6 severely ill , and 7 extremely ill .
sleep scale from medical outcomes study ( mos - sleep ) : mos - sleep scale evaluates the impact or interference with sleep of any disease or treatment .
the 7 subscales are sleep disturbance ( 4 items ) , snoring ( 1 item ) , awaken short of breath or with headache ( 1 item ) , quantity of sleep ( 1 item ) , optimal sleep ( 1 item ) , sleep adequacy ( 2 items ) , and somnolence ( 3 items ) .
this scale also has a sleep problems index , rated from 0 ( no interference ) to 100 ( maximum interference ) , and a sleep problems subscale .
each item is rated independently with more interference scoring higher , except for the sleep adequacy and quantity and optimal sleep subscales [ 21 , 22 ] .
world health organization disability assessment schedule ii ( who - das ii ) : the who - das ii is a psychometric instrument that measures day to day functioning and disability in the following six activity domains : understanding and communicating , moving and getting around , self care , getting along with people , work activities , and participation in society .
the 12-item who - das ii scale is rated from 1 ( none or nothing ) to 5 ( extreme or inability to perform a given task ) . the total calculated score ranges from 0 to 100 , with higher scores indicating higher degrees of disability , for the total scale as well as for each subscale .
eq-5d : this is a standard self - reported quality of life questionnaire developed in several european countries and validated for spain .
this questionnaire consists of two parts , the first one aims to evaluate patient s health profile in five dimensions ( mobility , self - care , daily living activities , pain , and anxiety / depression ) .
the patient rates his / her current status as no problems , some / moderate problems , and severe / extreme problems for each dimension .
results are then converted into 1 of the 243 different eq-5d health state descriptions which are used to calculate a unique index or tariff , between 1 ( as healthy as possible ) and 0 ( usually equivalent to death ) .
this index is used to calculate the time - trade off tariff which allows calculating quality - adjusted - life - year ( qaly ) gain ( further explanation below ) .
the second part consists on a visual analogue scale ( vas or thermometer ) in which the patient has to rate his health status between 0 ( equivalent to death ) and 100 ( the best possible healthy status ) .
for statistical analysis , last observation carried forward approach was used for those patients for whom data from the 6 months visit were missing .
if the only missing data was that from the 3 months visit , this was estimated proportionally to the change observed at the 6 months visit , given that the missing data at 3 month visit were completely at random .
patients were considered to respond to treatment when a higher than 50% reduction was observed for the total ham - a scale score at the study final visit when compared to that of the baseline visit .
descriptive statistics were prepared for the continuous variables in the study , including the assessment of central position and dispersion ( two - tailed 95% confidence interval ) .
the kolmogorov - smirnov test was applied to check adjustment of data to a gaussian distribution . for categorical variables
student t tests and chi - square test were used for continuous and categorical variables , respectively , to test homogeneity of baseline data between groups .
analysis of covariance ( ancova ) or binary logistic regression models were fitted comparing dsm - iv criteria versus broad criteria groups in most variables adjusting by baseline scoring , sex , age , body mass index , educational level , marital status and percentage of patients with comorbid major depression or depressive disorder .
analysis of variance ( anova ) was applied for quantitative measures and chi square ( mcnemar s test ) for qualitative measures in case of paired comparisons .
the clinical relevance of observed differences in baseline characteristics was ascertained by means of the effect size that was calculated using the difference of means for each group , divided by the combined standard deviation of the variables .
qalys gain after the 6-months follow - up was calculated from patients responses to the eq-5d as described previously using tariff calculated with this instrument .
gained qalys were estimated by using the trapezoidal approach , with baseline and 6- month visit values used as reference values .
all statistical tests were two - tailed , and error of < 0.05 was accepted as statistically significant .
a total of 3,549 patients were finally recruited by 618 psychiatrists in spanish psychiatric clinics for this observational study .
four hundred and fifty three patients ( 12.8% ) were excluded because they did not meet all inclusion criteria .
remaining patients were diagnosed of gad according to dsm - iv criteria ( 1,815 patients ) or to broad criteria ( 1,281 patients ) as described on the methods section .
a total of 529 ( 17.0% ) patients drop - out from study , with no significant differences between study groups ( dsm - iv criteria : 313 patients , broad criteria : 216 patients ; p=0.899 ) ( fig .
however , main reasons for drop - outs were a somewhat different between groups with 52.7% lost to follow - up in dsm - iv group , compared with 67.1% in broad criteria group ( p=0.033 ) , and unknown reasons in 22.0% of withdrawals in dsm - iv group compared with 13.6% in broad criteria group ( p=0.023 ) .
the mean age was 45.5 years for the dsm - iv group and slightly lower for the broad criteria group ( 42.9 years , p<0.001 vs. dsm - iv group ) .
regarding gad , all features studied were slightly , but significantly higher in the standard dsm - iv criteria group .
thus , the mean number of anxiety symptoms was 4.7 for the dsm - iv group compared with 4.3 for the broad criteria group ( p<0.001 ) .
significant differences were also observed in the percentage of patients presenting with each individual gad symptom . all ham - a scores indicated severe anxiety on both patient groups , being the scores in the dsm - iv group significantly higher ( table 1 ) .
likewise , statistical differences were observed at baseline in the cgi and madrs scales between the two groups .
however , the magnitude of the effect was low for all quantitative variables at baseline ( effect size < 0,50 ) so the statistically significant differences observed were of small or negligible clinical relevance .
most patients in both groups ( 87.9% for dsm - iv ; 82.0% for broad criteria ) were following a pharmacological treatment in the six months previous to the baseline visit .
( 2 ) , benzodiazepines were the most commonly used drugs followed by antidepressants and antiepileptics . at the end of the study
, there was an increase in the number of patients under pharmacological treatment ( 97% in both groups , p<0.001 versus baseline in all cases ) .
the use of antiepileptic drugs increased by 3.2 and 3.8 fold in the dsm - iv and broad criteria groups , respectively , by the end - of - trial ( p<0.001 in all cases ) .
the percentage of antiepileptic drugs users in dsm - iv group at baseline and at 6 months was significantly higher , albeit only slightly ( p=0.002 in both cases , fig .
both groups showed a significant reduction in the percentage of benzodiazepines users after 6 months of follow - up ( p<0.001 ) , being this reduction even higher in the broad criteria group ( p=0.011 ) .
also a slight but statistically significant increase was observed in the percentage of anti - depressive users in both groups ( p<0.001 , see fig .
the percentage of subjects in dsm - iv and broad criteria groups that had at least one psychiatric comorbidity was 51.4% and 43.7% , respectively , ( p<0.001 ) . table 2 describes the most frequent psychiatric comorbidities and medical disorders in both study groups .
however , neither the mean number of comorbid psychiatric disorders nor the medical illnesses were different between both criteria groups , except for comorbid major depression and other depressive disorders which were slightly more frequent , but statistically significant in dsm - iv group ( p<0.01 and p=0.05 , respectively , table 2 ) . regarding psychiatric disorders , most of them were present around 5 months before the diagnosis of gad , without significant differences between groups in disease evolution . major depression and panic disorders were the most frequent comorbid psychiatric disorders in both study groups . for medical illnesses ,
chronic pain was present in more than half of patients in both groups , although a higher percentage was seen in dsm - iv group ; 64.3% versus 52.0% , p<0.001 ( table 2 ) .
anxiety severity was assessed with ham - a and cgi - s scales at 3 and 6 month visits .
( 3 ) shows the evolution of ham - a total and sub - scores over study time in the dsm - iv and broad criteria groups .
all ham - a scores improved after 3 and 6 months in both criteria groups .
moreover , the percentage of patients without symptoms of anxiety , showing ham - a<9 , increased up to 22.3% and 31.4% at the 3 month visit for the dsm - iv and broad criteria groups ( p<0.001 vs. basal visit in both groups , and p=0.006 between groups ) , and up to 49% and 58% at the 6 month visit ( p<0.001 vs. basal visit in both groups , and p=0.261 between groups ) , indicating similar results for both groups at the end of the study . also , patients responding to anxiety treatment increased when considering the responder criteria ( > 50% reduction of baseline scores ) ; 30.7% and 38.2% for the dms - iv and broad criteria , respectively , at the 3 months visit ( p= 0.004 ) ; increasing up to 59.7% in the dms - iv and 67.7% at the 6 month visit ( p=0.103 ) .
4 ) , differences between criteria groups were statistically significant at the study time points . regarding each gad symptom , fig .
( 5 ) shows the reduction in the percentage of patients presenting with each anxiety symptom .
however , higher significant reductions were observed in dsm - iv group for the following symptoms : fatigue , irritability and sleep disturbance ( p<0.05 in all cases , fig . 5 ) . in this study
, depression symptoms were evaluated using the madrs . when compared to scores at baseline visit ( table 1 )
, patients in both criteria groups improved their madrs scores with a mean reduction of 12.1 and 12.5 points for the dms - iv and broad criteria groups , respectively ( p=0.264 dsm - iv vs. broad criteria ) .
mos - sleep scale scores revealed an overall improvement in sleep patterns over study time in both criteria group .
as described in table 3 , all sleep domains in the mos - sleep scale changed significantly after 6 months when compared to basal visit scores .
differences between the dsm - iv and broad criteria groups were observed for the total score ( p= 0.018 ) , sleep disturbance domain ( p=0.012 ) , sleep quantity ( p=0.016 ) , sleep adequacy ( p=0.031 ) , and sleep problems ( p=0.019 ) scores , always favouring the broad criteria group .
health - related quality of life was measured by the eq-5d questionnaire . as seen in table 3 , both the health status ( visual analogue scale ) and the frequency of severe problems in each domain had significantly improved at the 6 month visit in both study groups ( p<0.001 in all cases ) . also , comparisons between both criteria groups in quality - of - life gain by domain were significant in favour of the broad criteria group in health status , daily living activities and mood status .
mean qalys gain at the 3 month and 6 month visits were 0.034 and 0.103 years , respectively , for the dsm - iv criteria group and 0.034 ( 3 month ) and 0.101 ( 6 month ) for the broad criteria patients , without statistical significance between groups ( table 3 ) . at the 6 month visit , the percentage of patients reporting an improvement in their health status as compared with that of one year
earlier increased by 13.8% for the dsm - iv criteria group , and 16.6% for the broad criteria group ( differences not significant ) . for measuring the disability degree in both groups of patients ,
, there was a significant improvement in all 7 domains of the who - das ii scale after the 6 month follow - up in both criteria groups .
the improvements were not significantly different in the dsm - iv group when compared to the new criteria group except for household activity domain , in which the improvement in the dsm - iv group was significantly better when compared to the broad criteria group .
nevertheless , a trend toward statistical significance was observed in the total scoring of the disability scale favouring broad criteria group .
a total of 3,549 patients were finally recruited by 618 psychiatrists in spanish psychiatric clinics for this observational study .
four hundred and fifty three patients ( 12.8% ) were excluded because they did not meet all inclusion criteria .
remaining patients were diagnosed of gad according to dsm - iv criteria ( 1,815 patients ) or to broad criteria ( 1,281 patients ) as described on the methods section .
a total of 529 ( 17.0% ) patients drop - out from study , with no significant differences between study groups ( dsm - iv criteria : 313 patients , broad criteria : 216 patients ; p=0.899 ) ( fig .
however , main reasons for drop - outs were a somewhat different between groups with 52.7% lost to follow - up in dsm - iv group , compared with 67.1% in broad criteria group ( p=0.033 ) , and unknown reasons in 22.0% of withdrawals in dsm - iv group compared with 13.6% in broad criteria group ( p=0.023 ) .
the mean age was 45.5 years for the dsm - iv group and slightly lower for the broad criteria group ( 42.9 years , p<0.001 vs. dsm - iv group ) .
regarding gad , all features studied were slightly , but significantly higher in the standard dsm - iv criteria group .
thus , the mean number of anxiety symptoms was 4.7 for the dsm - iv group compared with 4.3 for the broad criteria group ( p<0.001 ) .
significant differences were also observed in the percentage of patients presenting with each individual gad symptom . all ham - a scores indicated severe anxiety on both patient groups , being the scores in the dsm - iv group significantly higher ( table 1 ) .
likewise , statistical differences were observed at baseline in the cgi and madrs scales between the two groups .
however , the magnitude of the effect was low for all quantitative variables at baseline ( effect size < 0,50 ) so the statistically significant differences observed were of small or negligible clinical relevance .
most patients in both groups ( 87.9% for dsm - iv ; 82.0% for broad criteria ) were following a pharmacological treatment in the six months previous to the baseline visit .
( 2 ) , benzodiazepines were the most commonly used drugs followed by antidepressants and antiepileptics . at the end of the study
, there was an increase in the number of patients under pharmacological treatment ( 97% in both groups , p<0.001 versus baseline in all cases ) . the use of antiepileptic drugs increased by 3.2 and 3.8 fold in the dsm - iv and broad criteria groups , respectively , by the end - of - trial ( p<0.001 in all cases ) .
the percentage of antiepileptic drugs users in dsm - iv group at baseline and at 6 months was significantly higher , albeit only slightly ( p=0.002 in both cases , fig .
both groups showed a significant reduction in the percentage of benzodiazepines users after 6 months of follow - up ( p<0.001 ) , being this reduction even higher in the broad criteria group ( p=0.011 ) . also a slight but
statistically significant increase was observed in the percentage of anti - depressive users in both groups ( p<0.001 , see fig .
at the study entry , the percentage of subjects in dsm - iv and broad criteria groups that had at least one psychiatric comorbidity was 51.4% and 43.7% , respectively , ( p<0.001 ) .
table 2 describes the most frequent psychiatric comorbidities and medical disorders in both study groups .
however , neither the mean number of comorbid psychiatric disorders nor the medical illnesses were different between both criteria groups , except for comorbid major depression and other depressive disorders which were slightly more frequent , but statistically significant in dsm - iv group ( p<0.01 and p=0.05 , respectively , table 2 ) . regarding psychiatric disorders , most of them were present around 5 months before the diagnosis of gad , without significant differences between groups in disease evolution .
major depression and panic disorders were the most frequent comorbid psychiatric disorders in both study groups . for medical illnesses ,
chronic pain was present in more than half of patients in both groups , although a higher percentage was seen in dsm - iv group ; 64.3% versus 52.0% , p<0.001 ( table 2 ) .
anxiety severity was assessed with ham - a and cgi - s scales at 3 and 6 month visits . fig .
( 3 ) shows the evolution of ham - a total and sub - scores over study time in the dsm - iv and broad criteria groups .
all ham - a scores improved after 3 and 6 months in both criteria groups .
moreover , the percentage of patients without symptoms of anxiety , showing ham - a<9 , increased up to 22.3% and 31.4% at the 3 month visit for the dsm - iv and broad criteria groups ( p<0.001 vs. basal visit in both groups , and p=0.006 between groups ) , and up to 49% and 58% at the 6 month visit ( p<0.001 vs. basal visit in both groups , and p=0.261 between groups ) , indicating similar results for both groups at the end of the study . also , patients responding to anxiety treatment increased when considering the responder criteria ( > 50% reduction of baseline scores ) ; 30.7% and 38.2% for the dms - iv and broad criteria , respectively , at the 3 months visit ( p= 0.004 ) ; increasing up to 59.7% in the dms - iv and 67.7% at the 6 month visit ( p=0.103 ) .
4 ) , differences between criteria groups were statistically significant at the study time points . regarding each gad symptom , fig .
( 5 ) shows the reduction in the percentage of patients presenting with each anxiety symptom .
however , higher significant reductions were observed in dsm - iv group for the following symptoms : fatigue , irritability and sleep disturbance ( p<0.05 in all cases , fig . 5 ) . in this study ,
depression symptoms were evaluated using the madrs . when compared to scores at baseline visit ( table 1 )
, patients in both criteria groups improved their madrs scores with a mean reduction of 12.1 and 12.5 points for the dms - iv and broad criteria groups , respectively ( p=0.264 dsm - iv vs. broad criteria ) .
mos - sleep scale scores revealed an overall improvement in sleep patterns over study time in both criteria group .
as described in table 3 , all sleep domains in the mos - sleep scale changed significantly after 6 months when compared to basal visit scores .
differences between the dsm - iv and broad criteria groups were observed for the total score ( p= 0.018 ) , sleep disturbance domain ( p=0.012 ) , sleep quantity ( p=0.016 ) , sleep adequacy ( p=0.031 ) , and sleep problems ( p=0.019 ) scores , always favouring the broad criteria group .
health - related quality of life was measured by the eq-5d questionnaire . as seen in table 3 , both the health status ( visual analogue scale ) and the frequency of severe problems in each domain had significantly improved at the 6 month visit in both study groups ( p<0.001 in all cases ) . also , comparisons between both criteria groups in quality - of - life gain by domain were significant in favour of the broad criteria group in health status , daily living activities and mood status .
mean qalys gain at the 3 month and 6 month visits were 0.034 and 0.103 years , respectively , for the dsm - iv criteria group and 0.034 ( 3 month ) and 0.101 ( 6 month ) for the broad criteria patients , without statistical significance between groups ( table 3 ) .
at the 6 month visit , the percentage of patients reporting an improvement in their health status as compared with that of one year earlier increased by 13.8% for the dsm - iv criteria group , and 16.6% for the broad criteria group ( differences not significant ) . for measuring the disability degree in both groups of patients , the who - das scale was used . as shown in table 4 , there was a significant improvement in all 7 domains of the who - das ii scale after the 6 month follow - up in both criteria groups .
the improvements were not significantly different in the dsm - iv group when compared to the new criteria group except for household activity domain , in which the improvement in the dsm - iv group was significantly better when compared to the broad criteria group .
nevertheless , a trend toward statistical significance was observed in the total scoring of the disability scale favouring broad criteria group .
this real world prospective study in usual medical practice has shown that broadening of gad dsm - iv diagnostic criteria , by reducing the duration of excessive or non - excessive worry to one month and associated symptoms to two , does not significantly affect patient s responsiveness to psychiatrist - prescribed therapy .
the patient sample of this study seems to be similar to other gad populations presented in previous studies , with a higher prevalence among women and among those aged above 25 years [ 4 , 18 ] .
the most frequent comorbid psychiatric disorder was major depression ; however , its presence was somewhat smaller than the observed in other studies , if we take into account major depression only .
the main goal when treating a gad patient is to improve the symptoms , hopefully leading in the long - term to their complete remission and to recovery of patient s functionality .
several guidelines recommend antidepressants , such as the selective serotonin reuptake inhibitors and the serotonin noradrenalin reuptake inhibitors , and antiepileptics as first line treatments , and benzodiazepines as second - line treatment [ 28 , 29 ] .
although patients in this study received a confirmatory diagnosis of gad , a high percentage of them were already following a therapy recommended for gad at baseline .
these results suggest that , in our sanitary context , general practitioners and/or family physicians start treatment to control anxiety symptoms , mostly with benzodiazepines , at patients first complaint .
the general practitioner / family physician usually refers those patients with lower treatment response than expected to the psychiatrist .
it should be taken into account that refractory patients may be difficult to manage at the primary care level in the spanish health system .
one of the reasons is the brief period of time devoted by physicians to each patient visit in the spanish primary health care level , as described by duran et al . .
other studies have explored the consequences of broadening dsm - iv diagnostic criteria for gad [ 3 , 13 , 15 - 16 , 31 ] .
most of them have considered a reduction in the duration criteria [ 3 , 16 ] , the excessiveness of worry , and the number of associated symptoms . a possible
learning from all of them is that the socio - demographic characteristics between the populations diagnosed with broad or strict dsm - iv criteria are not substantially different . as expected , the broadening of dsm - iv gad criteria results in an increase in the point - prevalence to more than double [ 15 - 16 ] .
concluded that the increase was mainly due to the reduction of minimum duration to one month . in regards to gad s severity , ruscio
et al . observed a tendency to reduced severity for the broad criteria group , however
we have observed a similar tendency suggesting that patients diagnosed with broader gad criteria present a slightly less severe form of the disease .
every study that has investigated the possibility of broadening the dsm - iv criteria has done it retrospectively [ 13 , 15 , 16 , 27 , 31 , 32 ] .
the present study is the first one to compare two groups of gad patients with dsm - iv and broader criteria following a prospective design .
according to our results , broadening of gad diagnostic criteria does not affect the response to the prescribed therapy , since ham - a scores , total and domains , improved similarly in both groups , and so did the presence of gad symptoms ( fig .
a significant improvement was perceived by physicians ( cgi - s scores ) at the end of the study .
the influence of broader gad diagnostic criteria in patient s disability has been studied previously by lee
et al .
lee et al . observed an increase in the sheehan disability scale score when increasing time for the duration criteria although it was not statistically significant .
the same observation was made by kessler et al . with data from the ncs .
parallel to their clinical improvement , patients in our study experienced an improvement in the studied quality of life variables .
health status improvements in our study were similar to those observed in primary care patients receiving treatment over six months for their gad .
the biggest improvements were observed in the daily living activities and mood domains of the eq-5d .
qalys gain observed corresponded to more than one month of perfect health for both groups ; which were similar in magnitude to that observed in other health conditions such as trigeminal neuralgia , successfully treated with pregabalin .
all other dimensions of functionality included in the who das - ii scale , and sleep scale were substantially ameliorated in both study groups by the end - of - trial ; however , the differences in the magnitude of these improvements for both groups were hardly meaningful from a clinical point of view .
results from this study should be interpreted bearing in mind its observational design with its inherent limitations . since this study
is based on out - patient psychiatric clinics , patients included in this study might not be representative of the whole gad population , since psychiatrists could deal with more severe and refractory cases .
also , due to the high degree of expertise required for participant psychiatrists , diagnosis in our study may have been more accurate than in other settings , such as primary care
. a consequence of broadening gad criteria in other healthcare settings could be the difficulty in distinguishing gad from adjustment disorders or situational stress reactions by other less trained physicians . in this study
there was a high proportion of patients treated with antiepileptics ; that could be explained by the fact that most subjects included in the study were non responders to recommended first line drug treatment according to the european guidelines on the treatment and management of gad patients .
finally , as this was a real world prospective study we did not have a control group so further clinical research with randomized controlled studies and newly diagnosed patients needs to be carried out to confirm the consequences of broadening the criteria .
broadening criteria for gad diagnosis , by reducing excessive and non - excessive anxiety and worry duration to one month and the number of associated symptoms to two , captures a similar population of patients compared to the one identified by applying standard dsm - iv criteria .
broadening diagnostic criteria is able to identify the core symptoms of gad according to the dsm - iv criteria and could lead to an earlier diagnosis .
funding for this study was provided by pfizer spain ; pfizer spain participated in the idea and design of the study .
enrique lvarez , jose l. carrasco and jos m. olivares declare not to have any conflict of interests as a consequence of participating in this study .
inma vilardaga was employed of the european biometric institute , a consulting agency which was responsible for logistic of study and analysis of data .
mara perez and vanessa lpez - gmez are employed by pfizer spain , the body funding the study .
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objective : to elucidate the consequences of broadening dsm - iv criteria for generalized anxiety disorder ( gad ) , we examined prospectively the evolution of gad symptoms in two groups of patients ; one group diagnosed according to dsm - iv criteria and the other , according to broader criteria.method:multicentre , prospective and observational study conducted on outpatient psychiatric clinics .
patients were selected from october 2007 to january 2009 and diagnosed with gad according to dsm - iv criteria ( dsm - iv group ) or broader criteria .
broader criteria were considered 1-month of excessive or non - excessive worry and only 2 of the associated symptoms listed on dsm - iv for gad diagnosis .
socio - demographic data , medical history and functional outcome measures were collected three times during a 6-month period.results:3,549 patients were systematically recruited ; 1,815 patients in dsm - iv group ( dg ) and 1,264 in broad group ( bg ) ; 453 patients did not fulfil inclusion criteria and were excluded .
most patients ( 87.9% in dg , 82.0% in bg ) were currently following pharmacological therapies ( mainly benzodiazepines ) to manage their anxiety symptoms .
the changes observed during the study were : 49.0% and 58.0% , respectively of patients without anxiety symptoms as per ham - a scale at the 6 month visit ( p=0.261 ) and 59.7% and 67.7% , respectively ( p=0.103 ) of responder rates ( > 50% reduction of baseline scoring).conclusion : broadening of gad criteria does not seem to affect psychiatric care results in subjects with gad , is able to identify the core symptoms of the disease according to the dsm - iv criteria and could lead to an earlier diagnosis .
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INTRODUCTION
MATERIALS AND METHODOLOGY
Study Design
Study Sample
Diagnostic Assessments
Clinical and Functional Outcome Measures
Statistical Analysis
RESULTS
Patient Characteristics
Treatment
Comorbidity
Reduction of Anxiety Symptoms After 6-Month Treatment and Severity of Illness
Functional Outcome Measures
DISCUSSION
CONCLUSION
CONFLICT OF INTEREST
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the generation of expressed sequence tags ( ests ) was originally proposed as a strategy for cdna characterization over a decade ago ( 1 ) .
subsequent improvements in sequencing methods and dramatically reduced unit costs have increased the attractiveness of the est - based research , such that it is now one of the most widely employed methods used for gene discovery and genome characterization .
consequently , the number of organisms with est sequences deposited in the genbank dbest database is increasing rapidly ( ) . to maximize the value of these ests , ncbi has built unigenes that incorporated est data for a number of species ( ) , and the institute for genomics research ( tigr ) has been working on the gene indices for more than 70 species ( ) . annotating est and cdna sequences
two tools that have been designed for locating protein - coding regions in cdna and est sequences are orffinder ( ) and estscan ( ) , respectively .
the estscan server is designed for processing a batch of est sequences for identifying the protein - coding regions with a function for correcting insertions or deletions , but it is only trained for mammals and yeast .
we tested estscan with our aspergillus niger est sequences and found that the results were not satisfactory .
for example , using a full - length cdna sequence encoding glucoamylase , a well - characterized enzyme in a.niger , we found that estscan could not identify its correct coding region and had the undesired side effect of inserting nucleotides even when the test sequence was correct .
we have implemented a web server called orfpredictor for the prediction of protein - coding regions within est - derived sequences .
the algorithm uses the translation reading frames predicted by using blastx ( 2 ) as a guide for the identification of the coding region in sequences that have a hit ( 3 ) and predicts a coding region ab initio for sequences without a hit .
a mature eukaryotic mrna molecule , starting from the 5 end , typically consists of a 5 cap , a 5-untranslated region ( 5-utr ) , a protein - coding region [ open reading frame ( orf ) ] and a 3-utr followed by a poly(a ) tail .
the protein - coding region extends from the start codon aug ( atg in a cdna ) and continues until the reading frame defined by the start codon is terminated by one of three translation stop codons , uga , uaa or uag . most cdna libraries are constructed using oligo(dt ) primers to direct first - strand synthesis by reverse transcriptase .
essentially , all clones in oligo(dt ) primed cdna libraries will therefore include information for the 3 end of the processed transcript and a poly(a ) region .
ests are single - pass sequencing reads obtained from either the 5 or 3 end of the cdna insert .
the high - quality sequence obtained using systems , such as abi 3730xl , is typically 700800 nt per read .
given that the 3- and 5-utrs are typically much shorter than 500 nt , most ests and the consensus sequences ( contigs ) generated by an est assembler are expected to include some coding sequence useful for predicting gene function . in the annotation and analysis of ests ,
overlapping est sequences are often assembled into contigs to remove redundancy , reduce the frequency of sequencing errors and extend the length of sequence derived from each mrna species . assuming the ests are sequenced from the 5 end , sequence information from contigs and individual ests fall into 10 categories ( figure 1 ) as follows :
a full - length sequence that includes the 5-utr with one or more stop codons ( 5 stop codon ) , translation start codon , complete protein - coding region , translation stop codon and the 3-utr .
the protein - coding orf may have internal atg codons and the 3-utr may possess multiple stop codons .
the 5-utr may be truncated.a full - length sequence as defined for category ( a ) , except that it does not contain any 5 stop codons.a partial sequence that has a portion of the 5-utr , one or more 5 stop codons , the start codon and a portion of the coding region.a sequence that contains only 5-utr sequence and there is a 5 stop codon.a sequence that contains a 5-utr sequence , the start codon and a portion of the protein - coding orf.a sequence that contains only 5-utr sequence without a 5 stop codon.a sequence that contains a portion of the protein - coding region , but does not contain the start codon or the stop codon.a sequence with the potential protein - coding region truncated at its 5 end , one or more atg codons in the truncated orf , the stop codon and a 3-utr.a sequence that contains a portion of the potential protein - coding region and the 3-utr sequence.a sequence that contains a portion of the 3-utr and a poly(a ) sequence at its 3 end . a full - length sequence that includes the 5-utr with one or more stop codons ( 5 stop codon ) , translation start codon , complete protein - coding region , translation stop codon and the 3-utr .
the protein - coding orf may have internal atg codons and the 3-utr may possess multiple stop codons .
the 5-utr may be truncated . a full - length sequence as defined for category ( a ) , except that it does not contain any 5 stop codons . a partial sequence that has a portion of the 5-utr , one or more 5 stop codons , the start codon and a portion of the coding region . a sequence that contains only 5-utr sequence and there is a 5 stop codon . a sequence that contains a 5-utr sequence , the start codon and a portion of the protein - coding orf . a sequence that contains only 5-utr sequence without a 5 stop codon .
a sequence that contains a portion of the protein - coding region , but does not contain the start codon or the stop codon . a sequence with the potential protein - coding region truncated at its 5 end , one or more atg codons in the truncated orf , the stop codon and a 3-utr . a sequence that contains a portion of the potential protein - coding region and the 3-utr sequence . a sequence that contains a portion of the 3-utr and a poly(a ) sequence at its 3 end . for sequences generated by sequencing cdna inserts from their 3 ends ,
the categories of their reverse complementary sequences only include ( a ) , ( b ) , ( h ) , ( i ) and ( j ) .
therefore , it is more challenging to predict the coding region within an est than it is to predict the coding region of a fully sequenced cdna . distinguishing the translation start codon from other atg codons remains a difficult task .
identifying start codons is further complicated because there is not a universal consensus sequence surrounding eukaryotic start codons , although the conserved consensus sequence , gccrccaugg ( r : purine ; aug : start codon ) is present in mammals ( 4 ) .
however , blastx using a nucleotide query against a protein database is able to reliably identify protein - coding regions within a dna sequence if sufficient similarity exists between the translated query and an entry in the database ( 3 ) .
sequencing errors may disrupt the conceptual translation of orfs , blastx could also detect frame shifts if there are insertions / deletions in the coding regions of the query sequences . when significant blastx alignments can be generated our algorithm uses them as a guide to identify the translation reading frames and coding regions . for est - derived sequences without a database match , their frames and coding sequences are predicted based on the presence and the location of intrinsic signals in a sequence that include start codons , 5 or / and 3 stop codons and stretches of poly(a ) ( figure 1 ) .
our algorithm uses the following rules to locate protein - coding regions and predict the translation reading frame . for cases where blastx identified a significant database match ( e - value lower than a user chosen threshold ) ,
the frame assignment in the blastx output will be used and rules 19 are applied . if there is a conflict , rules 1 and 2 will override the other rules . for sequences that do not produce a significant blastx alignment rules 310
rule 1 : the predicted coding region must contain at least a portion of the translated query aligned by using blastx.rule 2 : if there is a frame shift , the first frame assignment in the blastx alignment is used.rule 3 : when there are no internal stop codons within a potential protein - coding region that is flanked by translation start and stop codons , the predicted coding region extends from the start codon to the stop codon ( figure 1a).rule 4 : a sequence that contains a poly(a ) signature but does not contain a stop codon does not include any portion of the coding region ( figure 1j).rule 5 : if there are one or more atg codons in a sequence and they are all downstream from one or more stop codons , the first atg following the last 5 stop codon is selected as the start codon ( figure 1a and c).rule 6 : to be considered a potential coding region , an orf that is flanked by a 5 stop codon and a 3 stop codon must be at least 90 nt ( code for a protein that has at least 30 amino acids).rule 7 : if a sequence includes a poly(a ) signature preceded by one or more 3 stop codons , but does not include a 5 stop codon , the sequence upstream of the stop codons is considered the coding region ( figure 1b , h and i).rule 8 : if a sequence lacks a poly(a ) signature and encodes an orf without any stop codons , it is assumed that the entire sequence is the coding region ( figure 1e , f and g ) . although in rare cases ( such as in figure 1f ) , the 5-utr will be considered as a coding sequence.rule 9 : for cases like that presented in figure 1d , it is impossible to know if the stop codon is a 5 stop codon or a 3 stop codon , if it lacks a poly(a ) signature .
however , because cdna clones are more likely to be truncated at their 5 end , the program assumes in this case that the sequence upstream of the stop codon is the coding sequence.rule 10 : the longest stretch of orf present in the six possible reading frames is selected as the coding region .
rule 1 : the predicted coding region must contain at least a portion of the translated query aligned by using blastx . rule 2 : if there is a frame shift , the first frame assignment in the blastx alignment is used .
rule 3 : when there are no internal stop codons within a potential protein - coding region that is flanked by translation start and stop codons , the predicted coding region extends from the start codon to the stop codon ( figure 1a ) .
rule 4 : a sequence that contains a poly(a ) signature but does not contain a stop codon does not include any portion of the coding region ( figure 1j ) .
rule 5 : if there are one or more atg codons in a sequence and they are all downstream from one or more stop codons , the first atg following the last 5 stop codon is selected as the start codon ( figure 1a and c ) .
rule 6 : to be considered a potential coding region , an orf that is flanked by a 5 stop codon and a 3 stop codon must be at least 90 nt ( code for a protein that has at least 30 amino acids ) .
rule 7 : if a sequence includes a poly(a ) signature preceded by one or more 3 stop codons , but does not include a 5 stop codon , the sequence upstream of the stop codons is considered the coding region ( figure 1b , h and i ) .
rule 8 : if a sequence lacks a poly(a ) signature and encodes an orf without any stop codons , it is assumed that the entire sequence is the coding region ( figure 1e , f and g ) .
although in rare cases ( such as in figure 1f ) , the 5-utr will be considered as a coding sequence .
rule 9 : for cases like that presented in figure 1d , it is impossible to know if the stop codon is a 5 stop codon or a 3 stop codon , if it lacks a poly(a ) signature .
however , because cdna clones are more likely to be truncated at their 5 end , the program assumes in this case that the sequence upstream of the stop codon is the coding sequence .
rule 10 : the longest stretch of orf present in the six possible reading frames is selected as the coding region .
the server provides a user interface for copy and paste , or for loading the users ' sequences and blastx outputs .
these various inputs are summarized as follows :
a sequence file in the fasta format .
the poly(a ) or poly(t ) signatures that identify mrna poly(a ) tails should be retained , as they are used to determine the strand to be used for coding region and reading frame prediction.blastx output for all the est - derived query sequences .
although it is optional , the user is encouraged to provide a pre - run blastx output .
the user can choose a cut - off e - value when setting up the blastx run .
if a blast output file is provided by a user , the frame used in blastx for alignments will be used for the prediction of the protein - coding region . for query sequences without a blastx hit or for which the blastx output is not provided , predictions will be performed based on the intrinsic signals of the query sequences using the rules described above .
users can also use our targetidentifier server ( ) to obtain blastx outputs for their query sequences .
targetidentifier server uses the uniprot / swiss - prot protein database.e - value : the user can also set a threshold e - value for their blastx file .
if the e - value in the blastx file is larger than the user selected threshold , the query sequence will be taken as
no hit. the default threshold is 1 10.strand : the user can choose which strand will be used for prediction . if the sequences were obtained by sequencing cdnas from the 5 ends , the + strand should be chosen .
if the file contains sequences obtained by sequencing from both ends , both strands should be used for prediction . in this case , the default setting ,
users can select download or use email for receiving their results . a sequence file in the fasta format .
the poly(a ) or poly(t ) signatures that identify mrna poly(a ) tails should be retained , as they are used to determine the strand to be used for coding region and reading frame prediction .
although it is optional , the user is encouraged to provide a pre - run blastx output .
the user can choose a cut - off e - value when setting up the blastx run .
if a blast output file is provided by a user , the frame used in blastx for alignments will be used for the prediction of the protein - coding region . for query sequences without a blastx hit or for which the blastx output is not provided , predictions will be performed based on the intrinsic signals of the query sequences using the rules described above .
users can also use our targetidentifier server ( ) to obtain blastx outputs for their query sequences .
e - value : the user can also set a threshold e - value for their blastx file .
if the e - value in the blastx file is larger than the user selected threshold , the query sequence will be taken as
strand : the user can choose which strand will be used for prediction . if the sequences were obtained by sequencing cdnas from the 5 ends , the + strand should be chosen . if the sequences were obtained by sequencing cdnas from their 3 ends , the strand should be chosen .
if the file contains sequences obtained by sequencing from both ends , both strands should be used for prediction . in this case , the default setting ,
an identifier for the sequence , the reading frame for the predicted coding region , the location of the beginning and end of the predicted coding region , a flag shown as fs that locates any translation frame shifts detected in the blastx alignment and the predicted protein sequence .
the other file contains the query identifiers for those sequences that do not have predicted protein - coding regions ( figure 1j ) .
most ests encompass only a portion of the mrna sequence . therefore , it is more challenging to predict the coding region within an est than it is to predict the coding region of a fully sequenced cdna . distinguishing the translation start codon from other atg codons
identifying start codons is further complicated because there is not a universal consensus sequence surrounding eukaryotic start codons , although the conserved consensus sequence , gccrccaugg ( r : purine ; aug : start codon ) is present in mammals ( 4 ) .
however , blastx using a nucleotide query against a protein database is able to reliably identify protein - coding regions within a dna sequence if sufficient similarity exists between the translated query and an entry in the database ( 3 ) .
sequencing errors may disrupt the conceptual translation of orfs , blastx could also detect frame shifts if there are insertions / deletions in the coding regions of the query sequences . when significant blastx alignments can be generated our algorithm uses them as a guide to identify the translation reading frames and coding regions . for est - derived sequences without a database match , their frames and coding sequences are predicted based on the presence and the location of intrinsic signals in a sequence that include start codons , 5 or / and 3 stop codons and stretches of poly(a ) ( figure 1 ) .
our algorithm uses the following rules to locate protein - coding regions and predict the translation reading frame . for cases where blastx identified a significant database match ( e - value lower than a user chosen threshold ) ,
the frame assignment in the blastx output will be used and rules 19 are applied . if there is a conflict , rules 1 and 2 will override the other rules . for sequences that do not produce a significant blastx alignment rules 310
rule 1 : the predicted coding region must contain at least a portion of the translated query aligned by using blastx.rule 2 : if there is a frame shift , the first frame assignment in the blastx alignment is used.rule 3 : when there are no internal stop codons within a potential protein - coding region that is flanked by translation start and stop codons , the predicted coding region extends from the start codon to the stop codon ( figure 1a).rule 4 : a sequence that contains a poly(a ) signature but does not contain a stop codon does not include any portion of the coding region ( figure 1j).rule 5 : if there are one or more atg codons in a sequence and they are all downstream from one or more stop codons , the first atg following the last 5 stop codon is selected as the start codon ( figure 1a and c).rule 6 : to be considered a potential coding region , an orf that is flanked by a 5 stop codon and a 3 stop codon must be at least 90 nt ( code for a protein that has at least 30 amino acids).rule 7 : if a sequence includes a poly(a ) signature preceded by one or more 3 stop codons , but does not include a 5 stop codon , the sequence upstream of the stop codons is considered the coding region ( figure 1b , h and i).rule 8 : if a sequence lacks a poly(a ) signature and encodes an orf without any stop codons , it is assumed that the entire sequence is the coding region ( figure 1e , f and g ) .
although in rare cases ( such as in figure 1f ) , the 5-utr will be considered as a coding sequence.rule 9 : for cases like that presented in figure 1d , it is impossible to know if the stop codon is a 5 stop codon or a 3 stop codon , if it lacks a poly(a ) signature . however , because cdna clones are more likely to be truncated at their 5 end , the program assumes in this case that the sequence upstream of the stop codon is the coding sequence.rule 10 : the longest stretch of orf present in the six possible reading frames is selected as the coding region .
rule 1 : the predicted coding region must contain at least a portion of the translated query aligned by using blastx . rule 2 : if there is a frame shift , the first frame assignment in the blastx alignment is used .
rule 3 : when there are no internal stop codons within a potential protein - coding region that is flanked by translation start and stop codons , the predicted coding region extends from the start codon to the stop codon ( figure 1a ) .
rule 4 : a sequence that contains a poly(a ) signature but does not contain a stop codon does not include any portion of the coding region ( figure 1j ) . rule 5 : if there are one or more atg codons in a sequence and they are all downstream from one or more stop codons , the first atg following the last 5 stop codon is selected as the start codon ( figure 1a and c ) .
rule 6 : to be considered a potential coding region , an orf that is flanked by a 5 stop codon and a 3 stop codon must be at least 90 nt ( code for a protein that has at least 30 amino acids ) . rule 7 : if a sequence includes a poly(a ) signature preceded by one or more 3 stop codons , but does not include a 5 stop codon , the sequence upstream of the stop codons is considered the coding region ( figure 1b , h and i ) .
rule 8 : if a sequence lacks a poly(a ) signature and encodes an orf without any stop codons , it is assumed that the entire sequence is the coding region ( figure 1e , f and g ) .
although in rare cases ( such as in figure 1f ) , the 5-utr will be considered as a coding sequence .
rule 9 : for cases like that presented in figure 1d , it is impossible to know if the stop codon is a 5 stop codon or a 3 stop codon , if it lacks a poly(a ) signature .
however , because cdna clones are more likely to be truncated at their 5 end , the program assumes in this case that the sequence upstream of the stop codon is the coding sequence .
rule 10 : the longest stretch of orf present in the six possible reading frames is selected as the coding region .
the server provides a user interface for copy and paste , or for loading the users ' sequences and blastx outputs
these various inputs are summarized as follows :
a sequence file in the fasta format .
the poly(a ) or poly(t ) signatures that identify mrna poly(a ) tails should be retained , as they are used to determine the strand to be used for coding region and reading frame prediction.blastx output for all the est - derived query sequences .
although it is optional , the user is encouraged to provide a pre - run blastx output .
the user can choose a cut - off e - value when setting up the blastx run .
if a blast output file is provided by a user , the frame used in blastx for alignments will be used for the prediction of the protein - coding region . for query sequences without a blastx hit or for which the blastx output is not provided , predictions will be performed based on the intrinsic signals of the query sequences using the rules described above .
users can also use our targetidentifier server ( ) to obtain blastx outputs for their query sequences .
targetidentifier server uses the uniprot / swiss - prot protein database.e - value : the user can also set a threshold e - value for their blastx file .
if the e - value in the blastx file is larger than the user selected threshold , the query sequence will be taken as
no hit. the default threshold is 1 10.strand : the user can choose which strand will be used for prediction . if the sequences were obtained by sequencing cdnas from the 5 ends , the + strand should be chosen .
if the file contains sequences obtained by sequencing from both ends , both strands should be used for prediction . in this case , the default setting ,
users can select download or use email for receiving their results . a sequence file in the fasta format .
the poly(a ) or poly(t ) signatures that identify mrna poly(a ) tails should be retained , as they are used to determine the strand to be used for coding region and reading frame prediction .
although it is optional , the user is encouraged to provide a pre - run blastx output .
the user can choose a cut - off e - value when setting up the blastx run .
if a blast output file is provided by a user , the frame used in blastx for alignments will be used for the prediction of the protein - coding region . for query sequences without a blastx hit or for which the blastx output is not provided , predictions will be performed based on the intrinsic signals of the query sequences using the rules described above .
users can also use our targetidentifier server ( ) to obtain blastx outputs for their query sequences .
e - value : the user can also set a threshold e - value for their blastx file . if the e - value in the blastx file is larger than the user selected threshold , the query sequence will be taken as no hit. the default threshold is 1 10 .
strand : the user can choose which strand will be used for prediction . if the sequences were obtained by sequencing cdnas from the 5 ends , the + strand should be chosen . if the sequences were obtained by sequencing cdnas from their 3 ends , the
if the file contains sequences obtained by sequencing from both ends , both strands should be used for prediction . in this case , the default setting ,
it contains the following information for each input sequence . an identifier for the sequence , the reading frame for the predicted coding region , the location of the beginning and end of the predicted coding region , a flag shown as fs that locates any translation frame shifts detected in the blastx alignment and the predicted protein sequence .
the other file contains the query identifiers for those sequences that do not have predicted protein - coding regions ( figure 1j ) .
we evaluated the accuracy of orfpredictor using 2127 arabidopsis cdna sequences that have annotated protein sequences in genbank , and 4289 a.niger and 3065 phanerochaete chrysosporium sequences assembled from ests with phrap ( ) .
we first compared the predicted arabidopsis protein sequences obtained when blastx alignments were used as a guide with the annotated protein sequences in genbank , and confirmed that our program was able to predict the protein - coding regions with 100% accuracy .
then , we compared the ab initio predicted protein sequences with the results obtained by using blastx .
we then examined the prediction accuracy using the a.niger and p.chrysosporium sequences , which had a blastx hit in the ncbi nr database with an e - value 1 10 .
the ab initio predicted frames were then compared with the frames identified by using blastx .
we found that the reading frame predicted ab initio was identical with the frames predicted by using blastx for 3943 ( 91.9% ) of the a.niger sequences and 2867 ( 93.5% ) of the p.chrysosporium sequences .
we implemented a web server , orfpredictor , for predicting protein - coding regions in est - derived sequences .
orfpredictor uses the reading frame predicted by using blastx when a significant alignment is produced , whereas for sequences that do not return a significant blastx alignment protein - coding regions are predicted ab initio .
the predicted protein sequences can then be used as the input for additional annotation tools , such as interproscan ( 5 ) , for identifying protein families , domains and functional sites , the conserved domain search service ( 6 ) for the detection of structural and functional domains , and signalp ( 7 ) for locating potential signal peptides .
categories of information derived from the est sequences . ( a ) a typical full - length cdna sequence including one or more stop codons in the 5-utr , a start codon and a stop codon
. the coding region may contain multiple atg codons encoding methionine and the 3-utr may harbor additional stop codons .
( b ) a full - length cdna without a stop codon in the 5-utr . ( c ) a sequence containing a 5-utr with a stop codon and a portion of the coding region .
( d ) a sequence containing a 5-utr with a stop codon . ( e ) a sequence containing a 5-utr without a 5 stop codon , and a portion of the coding region .
( f ) a sequence containing a portion of 5-utr without a 5 stop codon . (
g ) a sequence containing the internal portion of a coding region with or without internal atg codons .
( h ) a sequence containing a portion of the coding region with an internal atg codon , a 3 stop codon and 3-utr .
( i ) a sequence containing a portion of the coding region with no internal atg codons , a 3 stop codon and a 3-utr .
red star : stop codon at 5 end ; green circle : start codon ; blue circle : internal atg codon ; red hexagon : stop codon ; solid line : sequenced portion of the full - length cdna ; and dashed line : unsequenced or truncated portion of the full - length cdna . the orfpredictor server interface for loading data and choosing other parameters .
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orfpredictor is a web server designed for identifying protein - coding regions in expressed sequence tag ( est)-derived sequences . for query sequences with a hit in blastx ,
the program predicts the coding regions based on the translation reading frames identified in blastx alignments , otherwise , it predicts the most probable coding region based on the intrinsic signals of the query sequences .
the output is the predicted peptide sequences in the fasta format , and a definition line that includes the query i d , the translation reading frame and the nucleotide positions where the coding region begins and ends .
orfpredictor facilitates the annotation of est - derived sequences , particularly , for large - scale est projects .
orfpredictor is available at .
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INTRODUCTION
OVERVIEW OF THE ALGORITHM AND IMPLEMENTATION
Algorithm and implementation
Input
Output
EVALUATIONS OF ACCURACY
SUMMARY
Figures and Tables
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the rostral ventromedial medulla ( rvm ) is an important relay region that contributes to the descending pain control pathway from the periaqueductal gray ( pag ) to the superficial laminae ( laminae i and ii ) of the spinal cord [ 1 , 2 ] .
it is well known that the rvm is closely linked to long - lasting activation of descending control circuits that involve descending facilitation , which significantly contributes to the development of persistent pain induced by tissue and nerve injury .
although many studies have focused on this region , the cellular and molecular mechanisms of descending pain facilitation control remain poorly understood . due to the role of the descending pain facilitation pathway , several types of injuries , such as tissue and nerve injury , often become chronic and persistent , eventually leading to neuropathic pain .
, significant progress has been made in basic and clinical studies ; however , the currently available therapies for neuropathic pain remain inadequate , and the search continues not only for improved treatments but also for novel targets .
the mammalian target of rapamycin ( mtor ) , a conserved serine - threonine protein kinase that is inhibited by the effective clinical immunosuppressant rapamycin , regulates several intracellular processes in response to various extracellular signals and thereby modulates mrna translation .
thus , mtor plays a critical role in the modulation of long - term plasticity and memory processes [ 57 ] .
activation of the mtor complex with the protein raptor ( mtorc1 ) promotes the phosphorylation of mtor downstream targets , including eukaryotic initiation factor 4e - binding protein ( 4e - bp1/2 ) and s6 kinase ( s6k ) , which can further lead to local protein synthesis .
it has been reported that deletion of either the 4e - bp1/2 or the s6k gene in mice results in deficits in synaptic plasticity and long - term memory [ 8 , 9 ] . moreover
, phosphorylated mtor ( p - mtor ) , which is the activated form , is upregulated in the peripheral nervous system as well as at the spinal cord level in several pain models [ 1015 ] .
inhibition of spinal cord mtor by intrathecal administration has proven to be effective in alleviating the nociceptive behaviors of animals under pain conditions [ 10 , 11 , 16 , 17 ] .
synaptic plasticity changes in chronic pain conditions can occur not only at the spinal cord level but also at the supraspinal level , including the rvm .
therefore , considering the important role of the rvm in descending pain facilitation , targeting mtor in the rvm might be a promising way to combat pain . because serotoninergic ( 5-htergic ) neurons are the primary constitutive element in the rvm and can send projections to the superficial spinal dorsal horn ( sdh ) [ 1820 ]
, we thus hypothesize that 5-htergic spinally projecting neurons in the rvm contain mtor , the activation of which could in turn increase the excitability of the 5-htergic neurons and thus may potently potentiate the descending facilitation pain control pathway and exaggerate neuropathic pain conditions .
accordingly , we used a spared nerve injury ( sni ) model to evaluate the role of mtor in the rvm in neuropathic pain in rats .
adult male sprague - dawley ( sd ) rats ( weighing 250290 g ) were used in the present study .
the ethics committee for animal experiments of the fourth military medical university ( xi'an , china ) approved the animal experiments ( permit number : 10071 ) .
briefly , rats were anesthetized with pentobarbital ( 45 mg / kg , i.p . ) , and three terminal branches of the sciatic nerve were exposed by direct incision of the skin and a section of the biceps femoris muscle in the left thigh .
the tibial and common peroneal branches were carefully tight - ligated with 5 - 0 silk sutures and sectioned distal to the ligation , removing 24 mm of the distal nerve stump .
the surgical procedures for the sham - operated group were identical to those for the sni group , except that the nerves were not lesioned .
mechanical allodynia , as a behavioral sign of sni - induced neuropathic pain , was assessed by measuring the 50% paw withdrawal threshold ( pwt ) as described previously .
the 50% pwt in response to a series of ascending von frey filaments ( stoelting , kiel , wi , usa ) was determined by the up - and - down method .
von frey force was delivered perpendicularly to the plantar surface of the hind paw for 2 to 3 seconds .
an abrupt withdrawal of the hind paw during stimulation was recorded as a positive response .
the 50% pwt was calculated using the following formula : 50% pwt = 10 .
mechanical allodynia was assessed by measuring the 50% pwt of the ipsilateral hind paw in sni- or sham - operated rats .
measurements of the paw withdrawal latency ( pwl ) were obtained using a timer that was started by the activation of the heat source and stopped when withdrawal of the paw was detected with a photodetector .
three measurements of the pwl were taken for each hind paw and were averaged as the result of each test session .
the ipsilateral hind paw was tested with intervals of more than 5 min between consecutive tests .
fluoro - gold ( fg ) was used as a retrograde tracer to label the rvm neurons that project to the sdh .
the procedures for fg injection were essentially the same as in our previous studies [ 20 , 27 ] .
briefly , after exposing the lumbar cord , 0.1 l of a 4% solution of fg ( fluorochrome , denver , co , usa ) dissolved in 0.9% saline was stereotaxically injected into the left side of the lumbar dorsal horn with a microsyringe attached to a glass micropipette by pressure injection . due to the transportation period of the tracer ,
after perfusion , the brains and spinal cords of the rats were cut into sections .
the sections were used to evaluate the fg injection sites in the sdh as well as the distribution patterns of the retrogradely fg - labeled neurons in the rvm under an epifluorescence microscope ( bx-60 ; olympus , tokyo , japan ) using an appropriate filter for fg ( excitation 350395 nm ; emission 430 nm ) .
after the rats were anesthetized with pentobarbital ( 45 mg / kg , i.p . ) , a 26-gauge stainless steel guide cannula was stereotaxically implanted into a site above the rvm ( 10.52 mm posterior to bregma , 0 mm lateral from the midline , and 10.20 mm beneath the surface of the skull ) .
intra - rvm microinjections were delivered via a 33-gauge injector needle cannula that was lowered 0.5 mm deeper into the brainstem than the guide cannula . the microinjection apparatus consisted of a hamilton syringe ( 10 l ) connected to an injector ( 33-gauge ) by a thin polyethylene tube and a motorized syringe pump .
rapamycin ( 250 m/1 l , dissolved in a saline / dmso mix comprising 25% dmso , tocris bioscience , minneapolis , mn , usa ) , the specific inhibitor for mtor , was infused into the rvm at a rate of 0.1 l / min ; an equivalent volume of 25% dmso was used as a vehicle .
after injection , the microinjection needle was left in place for at least 2 min .
the injection sites were verified at the end of all of the experiments by nissl staining , and injection sites outside the rvm region were excluded from the study .
these rats were randomly divided into two groups designed for different purposes , as shown in figure 6(c ) : group 1 : to investigate whether rapamycin could influence the induction stage of sni - induced neuropathic pain , behavioral tests were performed before the first drug or vehicle injection , followed by sni ( pre - sni ) , and 30 min after the second injection on day 1 after sni ( sni - d1 ) ( figure 6(c ) , top ) and group 2 : behavioral tests were performed before sni ( pre - sni ) , 6 days after sni ( sni - d6 ) , and 30 min after drug or vehicle injection on day 7 after sni ( sni - d7 ) for the purpose of demonstrating the effect of rapamycin on the maintenance stage of sni - induced neuropathic pain ( figure 6(c ) , bottom ) .
the rats were transcardially perfused with 150 ml of 0.01 m phosphate - buffered saline ( pbs , ph 7.4 ) , followed by 500 ml of 4% paraformaldehyde in 0.1 m phosphate buffer ( pb , ph 7.4 ) .
the brainstems and/or spinal cords were transversely sliced into 25 mm thick coronal sections using a freezing microtome ( cm1950 , leica , heidelberg , germany ) .
double - immunofluorescence staining for p - mtor / neun , p - mtor / fg , or p - mtor/5-ht was performed .
the sections were sequentially incubated at room temperature with primary antisera in 0.01 m pbs containing 5% normal donkey serum ( nds ) , 0.3% triton x-100 , 0.05% nan3 , and 0.25% carrageenan ( pbs - nds , ph 7.4 ) for 24 h. then , the sections were incubated with fluorescein - labeled igg ( secondary antisera ) for 6 h. a negative control experiment , in which the primary antisera were omitted , and a peptide competition assay were both carried out .
after the immunofluorescence histochemical staining , the sections were observed and images were captured using a confocal laser - scanning microscope ( clsm , fv1000 , olympus ) .
micrographs of 1012 sections per rat , which were 150 m apart within bregma 9.30 to 11.60 mm , were analyzed for p - mtor expression . using imagej software , the rvm area , including the nucleus raphe magnus ( rmg ) and the nucleus reticularis gigantocellularis pars ,
the p - mtor - positive cells within the area were counted manually by an observer blinded to the treatment conditions .
the same counting method was used to evaluate the coexpression of 5-ht / p - mtor as well as that of p - mtor / fg within the rvm .
cells with visible green cytoplasmic staining represent 5-htergic or fg - labeled cells , while red staining represents p - mtor - positive cells ; thus , cells with 5-ht or fg double - labeling with p - mtor appear yellow .
the rats were decapitated , and brain slices ( 400 mm ) containing the rvm were cut at 0c with a vibratome ( vt1200s , leica ) in a sucrose cutting solution containing the following ( in mm ) : kcl 2.5 , nah2po4 1.2 , nahco3 26 , sucrose 252 , mgso47h2o 6 , cacl2 0.5 , and glucose 10 , bubbled with 95% o2/5% co2 ( ph 7.4 ) . for the electrophysiology studies ,
the brain slices were transferred to a submerged recovery chamber with oxygenated artificial cerebrospinal fluid ( acsf ) containing the following ( in mm ) : nacl 124 , kcl 2.5 , mgso47h2o 2 , nah2po4 1 , nahco3 25 , cacl2 2 , and glucose 37 for 2 hours at room temperature before recording .
for the biochemical experiments , the slices were slowly brought to a final temperature of 30c in acsf gassed with 95% o2/5% co2 and incubated for at least 1 hour before the experiments .
the brain slices were treated with rapamycin ( 250 m ) and vehicle for 30 min .
subsequently , the rvm regions were microdissected and snap - frozen over dry ice . following the standard western blot protocol ,
rats were anesthetized with an overdose of pentobarbital ( 60 mg / kg , i.p . ) , and the rvm regions were carefully dissected and harvested for western blotting . to obtain total protein extracts ,
the tissues were lysed in 300 l lysis buffer containing 10 mm tris , 150 mm nacl , 1% triton x-100 , 0.5% np-40 , and 1 mm edta at ph 7.4 .
the samples were adequately mixed at a 100 : 1 ( v / v ) ratio of protease inhibitor cocktail and phosphatase inhibitor cocktail ( roche , tucson , az , usa ) .
the procedures for the in vitro - infused brain slices were similar to those of the tissue protocols .
then , 30 g of cell lysis material ( quantitatively measured using the bca protein assay ; thermo scientific , rockford , il , usa ) was resolved by sodium dodecyl sulfate - polyacrylamide gel electrophoresis ( sds - page ) and transferred to pvdf membranes ( immobilon - p , millipore ) . after blocking in nonfat milk for 1 h
, the membranes were incubated overnight at 4c with the following primary antibodies : rabbit anti - mtor ( 1 : 1000 , cell signaling technology ) ; rabbit anti - p - mtor ( 1 : 1000 , cell signaling technology ) ; rabbit anti - s6k ( 1 : 1000 , cell signaling technology ) ; rabbit anti - p - s6k ( 1 : 1000 , cell signaling technology ) ; and mouse anti--actin ( 1 : 5000 , sigma , st .
the immunoblots were then reacted with the corresponding horseradish peroxidase- ( hrp- ) conjugated secondary antibodies ( anti - rabbit 1 : 5000 , anti - mouse 1 : 5000 ; amersham pharmacia biotech , piscataway , nj , usa ) .
all of the reactions were detected by the enhanced chemiluminescence ( ecl ) detection method ( amersham ) and exposure to film .
target protein levels were normalized against -actin levels and expressed as fold changes relative to the nave control group .
neurons in the rvm region were targeted for recording using an upright microscope equipped with zeiss ( oberkochen , germany ) infrared - differential interference contrast ( ir - dic ) optics , a 40 water - immersion objective , and a video - imaging camera .
the patch pipette was filled with intracellular solution containing the following ( in mm ) : k - gluconate 130 , nacl 5 , kcl 15 , egta 0.4 , hepes 10 , mg - atp 4 , and tris - gtp 0.2 , ph 7.257.35 , with an osmotic pressure of 290300 mosm / l . the pipette resistance , as measured in the bath , was typically 4 0.5 m. the voltage was held at 60 mv , and neurons were given at least 3 min to stabilize before data were collected .
spontaneous discharge and the number of action potentials were used to investigate sni - induced changes in neuronal excitability in the rvm .
the excitatory postsynaptic currents ( epscs ) of the rvm 5-htergic neurons , which are mediated by ampa receptors , were voltage - clamped and recorded at 60 mv with an axon 700b amplifier ( molecular devices , sunnyvale , ca , usa ) after blocking gabaergic transmission by picrotoxin ( 100 mm , sigma ) , a gabaa receptor antagonist .
the membrane excitability of the recorded neurons was measured in current - clamp mode by determining the number of action potentials elicited by intracellular injection of 0 , 10 , 20 , 30 , 40 , 50 , and 60 pa depolarizing currents for 400 ms .
the spike number was determined to estimate the influence of rapamycin on the recorded neurons . in all cases ,
biocytin ( 0.5% ) was introduced into the intracellular solution to identify the morphological properties of the recorded neurons .
after recording , the brain slices were immediately fixed in 4% paraformaldehyde in 0.1 m pb for 4 h at room temperature .
then , sections were rinsed with 3% hydrogen peroxide in 0.01 m pbs for 30 min .
after thorough washing with pbs , the tissue was incubated with a goat anti-5-ht ( 1 : 500 , immunostar ) antibody in pbs - nds ( ph 7.4 ) for 24 h , followed by incubation with alexa 594 avidin d ( 1 : 1000 , invitrogen ) and alexa 488 donkey anti - goat ( 1 : 500 , invitrogen ) antibodies in pbs for 6 h at room temperature .
the sections were then observed , and images were captured with a confocal microscope ( olympus ) .
two - way anova with bonferroni multiple comparisons tests or one - way anova with tukey 's multiple comparisons post hoc tests were used for between - groups comparisons ( e.g. , the western blot data with surgery and drug administration as main effects ) .
student 's paired t - test was used to analyze the differences between two groups ( e.g. , the difference in the numbers of p - mtor - positive cells between the sni - induced neuropathic pain group and the sham group ) .
spared nerve injury ( sni ) produced increased nociceptive responses to innocuous mechanical stimulation ( mechanical allodynia ) of the ipsilateral hind paw in rats from as early as post - sni operation day 1 , and this effect was maintained for at least 2 weeks ( figure 1(a ) ) .
however , sni had no impact on the latency of withdrawal to the radiant heat stimulus ( no thermal hyperalgesia ) ( figure 1(b ) ) .
in fact , our present data are consistent with a previous report demonstrating that the low mechanical threshold induced by sni could persist even for 9 weeks after surgery , but there was no reported decrease in the hind paw withdrawal latency to the radiant heat stimulus . by using double - immunofluorescence staining
, we found that the activated form of mtor , p - mtor , was expressed in the rvm , and it was exclusively expressed by neurons based on the observation that p - mtor was almost completely colocalized with neun , a marker for neurons , in rats in both the sham ( figures 2(a)2(c ) ) and post - sni day 7 groups ( figures 2(d)2(f ) ) .
we observed that the number of p - mtor - positive neurons was significantly increased in the rvm on day 7 after sni compared to the sham group ( figures 2(a ) , 2(d ) , and 2(f ) ) , indicating that activation of mtor in the rvm may contribute to sni - induced neuropathic pain .
it has been reported that s6k , a main downstream substrate for mtor , is involved in several intracellular processes , including neuronal plasticity and long - term memory .
therefore , we used western blot analysis to further evaluate the mtor signaling pathway , including mtor and s6k , in the rvm after sni . compared with nave control rats , the expression of both phosphorylated mtor and phosphorylated s6k ( p - mtor and p - s6k ) was not significantly altered in the sham rats ( figures 2(h ) , 2(i ) , and 2(k ) ) . as indicated in figure 2(h ) , p - mtor and p - s6k were significantly elevated in the rvm 3 days after sni , and phosphorylation was maintained for at least 14 days compared to the control group ( figures 2(h ) and 2(i ) ; p - mtor : sni d3 : 1.42 0.25 ; sni d7 : 1.86 0.39 ; sni d14 : 1.49 0.28-fold of naive control , p < 0.05 ; p - s6k : sni d3 : 1.75 0.23 ; sni d7 : 1.96 0.57 ; sni d14 : 1.61 0.28-fold of nave control , p
< 0.05 , n = 4 ) . the change in p - s6k was similar to that in p - mtor , indicating that the mtor signaling pathway in the rvm was activated by sni 3 days after surgery . compared with the nave control group , p - mtor and p - s6k expression
was slightly increased , but no significant difference was detected at 1 day after sni ( figures 2(h ) , 2(i ) , and 2(k ) , p - mtor : sni d1 : 1.17 0.36-fold of nave control , p > 0.05 ; p - s6k : sni d1 : 1.14 0.38-fold of nave control , p > 0.05 , n = 4 ) .
total mtor and s6k were not changed in any of the groups in the present study ( figures 2(h ) , 2(j ) , and 2(l ) ) . in summary , these data suggest that the mtor signaling pathway , including mtor and s6k , was activated in the rvm , which might indicate new protein synthesis and could result in changes in neuroplasticity
. injections of fg into the lumbar sdh ( figure 3(a ) and 3(b ) ) resulted in many retrogradely labeled spinally projecting neurons in the rvm ( figure 3(d ) ) .
although fg was injected unilaterally into the ( left ) lumbar sdh , 81.9% ( 203/248 ) of the retrogradely labeled spinally projecting neurons contained p - mtor - immunoreactive ( ir ) staining ( figures 3(c)3(e ) ) , which indicates that more than three - quarters of the spinally projecting neurons in the rvm express p - mtor .
because 5-htergic neurons have previously been shown to be involved in the descending pain control pathway , particularly those localized within the rvm , we further investigated the relationship between 5-ht and p - mtor .
our immunofluorescence staining results showed that a majority of the p - mtor - ir neurons contained 5-ht ( figures 4(a)4(f ) ) .
the number of 5-htergic neurons was slightly increased within the rvm in the sni rats , but no statistical significance was detected compared to the sham control group ( 25.52 5.13 per section in the sni d7 group versus 20.8 4.02 in the sham control group , n = 3 rats / group , p > 0.05 ) .
interestingly , 5-ht - positive p - mtor - ir neurons were remarkably increased in the rvm 7 days after sni compared to sham rats ( figure 4(g ) ) .
in contrast , 5-ht - negative p - mtor - ir neurons were not significantly altered 7 days after sni ( figure 4(h ) ) .
these results indicate that sni - induced neuropathic pain caused a substantial upregulation of p - mtor , which primarily occurred in 5-htergic neurons in the rvm . as reported previously , rapamycin is a specific and effective inhibitor of mtor . to investigate the inhibitory effect of rapamycin in vitro
, we incubated brain slices containing the rvm with rapamycin ( 250 m ) for 30 min .
we found that sni significantly increased the expression of both p - mtor and its downstream substrate p - s6k ( figure 5(a ) , p - mtor : sni d7 + vehicle : 1.80 0.48-fold of sham + vehicle , p < 0.05 ; p - s6k : sni d7 + vehicle : 1.77 0.45-fold of sham + vehicle , n
= 4 , p < 0.05 ) , which is inconsistent with our tissue western blot data . moreover , rapamycin ( 250 m ) significantly reversed the upregulation of p - mtor as well as p - s6k after 30 min of drug infusion ( p - mtor : sni d7 + rapamycin : 1.11 0.27-fold of nave control versus sni d7 + vehicle : 1.80 0.48-fold of nave control , p < 0.05 ; p - s6k : sni 7d + rapamycin : 1.17 0.29-fold of nave control versus sni d7 + vehicle 1.77 0.45-fold of nave control , n = 4 , p < 0.05 ) , indicating that rapamycin could rapidly inhibit the activation of mtor after sni in vitro . to further investigate the neuronal excitability of 5-ht and the effect of rapamycin on 5-htergic neurons in the rvm
a total of sixty 5-ht - positive neurons ( n = 60 ) from 12 rats were identified by biocytin introduction in combination with 5-ht immunofluorescence staining ( figure 5(b ) ) . moreover ,
they showed a relatively higher membrane capacitance ( cm ) and a smaller membrane resistance ( rm ) compared to other small neurons in the rvm , indicating that they have a larger membrane surface and smaller electrical resistance .
we first investigated the frequency and amplitude of spontaneous excitatory postsynaptic currents ( sepscs ) in the rvm neurons .
most of the 5-htergic neurons recorded in the rvm that were responsive to rapamycin showed increased activity .
inconsistent with previous reports , the frequency and amplitude of sepscs were significantly increased after sni ( figures 5(c)5(f ) ) , which indicates that the probability of presynaptic transmitter release and the postsynaptic neuronal excitability , respectively , was elevated .
subsequently , we used rapamycin ( 250 m ) to evaluate whether the enhanced presynaptic and postsynaptic excitability could be reversed .
we found that the amplitude ( baseline , 45.26 1.89 pa ; rapamycin , 35.83 2.58 pa .
p < 0.05 , n = 15 , paired t - test ) , but not the frequency ( baseline , 3.41 0.12 hz ; rapamycin , 3.29 0.33 , p > 0.05 , n = 15 , paired t - test ) , of the sepscs was inhibited by rapamycin application in rats with sni but not in the sham control rats ( frequency : baseline , 1.22 0.11 hz ; rapamycin , 1.20 0.19 hz .
p > 0.05 , n = 15 , paired t - test ; amplitude : baseline , 29.79 1.80 pa ; rapamycin , 28.14 1.28 pa .
p > 0.05 , n = 15 , paired t - test ) ( figures 5(c)5(f ) ) .
these results suggest that rapamycin can decrease the postsynaptic excitability of 5-htergic neurons in the rvm after sni .
we next compared the effects of rapamycin on the action potentials of the 5-htergic neurons and determined that the spike number of the recorded neurons was obviously increased after sni ( figures 5(g ) and 5(h ) ) .
rapamycin ( 250 m ) did not change the spike number in recorded neurons from the sham group ( f(1,62 ) = 2.25 , p > 0.05 , n = 15 , two - way repeated measures anova ) ( figure 5(g ) ) .
however , in the presence of rapamycin , the spike number of 5-htergic neurons in the sni group was significantly reduced ( f(1,48 ) = 8.56 , p < 0.05 , n = 15 , two - way repeated measures anova ) ( figure 5(h ) ) .
these results indicate that rapamycin inhibited the excitability of 5-htergic neurons in the rvm under neuropathic pain conditions .
we preliminarily investigated nociceptive behaviors in the sni rats mentioned above . in agreement with previous reports
, significant mechanical allodynia rather than thermal hyperalgesia was observed in the present study ( figure 1 ) .
based on this observation , we next used mechanical pwt instead of heat pwl to assess the nociceptive behaviors . to determine whether rapamycin could prevent the development of the induction stage of mechanical allodynia , we microinjected rapamycin via a cannula implanted into the rvm ( figures 6(a ) and 6(b ) ) immediately before sni surgery and at day 1 after sni , which was followed by behavioral testing 30 min later ( figure 6(c ) ) .
after treatment with rapamycin , the pwt in the sni + rapamycin group showed no significant change compared to that in the sni + vehicle control group at day 1 after sni ( figure 6(d ) ) , indicating that rapamycin could not alleviate the neuropathic pain induced by sni in the induction stage .
subsequently , we investigated whether rapamycin could reverse established neuropathic pain . on day 6 after sni ,
the rats demonstrated typical increased nociceptive responses to nonnoxious mechanical stimulation ( figure 6(e ) ) . compared with the vehicle control group ,
the mechanical allodynia was significantly reduced after microinjection of rapamycin into the rvm on day 7 after sni ( figure 6(e ) ) . because our biochemical results showed that the amount of both p - mtor and p - s6k was increased after sni ( figures 2(h ) , 2(i ) , and 2(k ) ) and because rapamycin infusion into the brain slices could effectively reverse the upregulated level of p - mtor and p - s6k ( figure 5(a ) )
, we concluded that the effect of rapamycin in partially reversing the mechanical allodynia was exerted via inactivation of the mtor signaling pathway , thus decreasing the excitability of 5-htergic spinally projecting neurons in the rvm .
in the current study , we provide the first demonstration of the following : ( 1 ) mtor is expressed in the rvm region and can be activated in nerve injury - induced neuropathic pain ; ( 2 ) this mtor is largely expressed in 5-htergic neurons , which mainly comprise the descending pain control pathway ; ( 3 ) inhibition of the activated mtor restores the overexcitability of the 5-htergic neurons to normal ; and ( 4 ) inactivation of mtor by intra - rvm rapamycin microinjection alleviates established hyperalgesia ( abolished at the maintenance stage of neuropathic pain ) rather than influencing the beginning priming ( induction stage ) of neuropathic pain .
these findings suggest that the mtor signaling pathway in the rvm is involved in the maintenance of nerve injury - induced neuropathic pain and that inhibition of mtor in the rvm could effectively alleviate neuropathic pain in sni rats . due to the importance of the descending pain control pathway in mammals ,
it has been demonstrated that blockade of rvm activity with lidocaine produced conditioned place preference ( cpp ) , which is linked to pain relief , in nerve injury models , indicating that descending pain facilitation pathways modulate injury - induced spontaneous tonic pain .
wei et al . have reported that selectively depleting functional 5-ht phenotypes in rvm neurons with shrna interference ( rnai ) of tryptophan hydroxylase-2 ( tph-2 , the rate - limiting enzyme in the synthesis of neuronal 5-ht ) attenuated tissue or nerve injury - induced allodynia and hyperalgesia .
this finding provides strong evidence that descending 5-ht from the rvm is an important contributor to pain facilitation during the development of persistent pain .
recently , by taking advantage of optogenetic methods , optogenetic stimulation in tph2-channelrhodopsin 2 ( chr2 ) transgenic mice was shown to decrease both mechanical and thermal pain thresholds .
however , in contrast , several studies showed the opposite results [ 3235 ] , that 5-ht from the rvm is important for the descending inhibitory pathway .
these controversial arguments regarding whether rvm 5-ht plays a facilitatory or inhibitory role might be explained by the different subtypes of 5-ht receptors located in the sdh , according to the reports .
for example , 5-ht3 receptors are reported to mediate descending facilitation and to contribute to pain hypersensitivity , whereas the activation of 5-ht2 receptors can potentiate glycine release in the sdh to inhibit pain transmission . in addition , a previous study also provided evidence that , under conditions of experimental pain , activation of 5-ht7 receptors leads to antinociceptive effects in the spinal cord . in the present study
, we found that 5-htergic neurons were slightly , although not significantly , increased after nerve injury .
however , the excitability of these 5-htergic neurons was elevated after sni ( figure 5 ) .
rapamycin could effectively inhibit 5-ht overexcitability and thus attenuate hyperalgesia ( figure 6 ) , which indicates that 5-ht in the rvm is probably involved in the descending pain facilitation pathway under nerve injury - induced neuropathic pain conditions .
mtor has been extensively studied in tumors , cardiovascular diseases , and neurodegenerative disorders [ 40 , 41 ] .
recently , emerging evidence has indicated that mtor plays a role in pain processing , and it is becoming clear that mtor is important in the regulation of nociception , at both the peripheral and spinal cord levels [ 1017 ] . to date , however , no report has investigated mtor at the supraspinal level and its role in nociceptive modulation . here , we provide potent evidence that mtor contributes to neuropathic pain by increasing the neuronal excitability of 5-htergic neurons in the rvm , thus potentiating descending pain facilitation .
4e - bp1/2 and s6k are involved in the regulation of cell physiology through the modulation of protein synthesis .
4e - bp1/2 inhibits the interaction of the cap - binding translation initiation factor eif4e with other elongation factors , which is a key regulatory process in translation .
mtor - mediated phosphorylation of 4e - bp1/2 releases this inhibition , allowing translation initiation to proceed .
s6k - mediated phosphorylation of s6 promotes the unwinding and initiation of translation of a subgroup of mrnas called 5-terminal oligopyrimidine tract ( top ) mrnas .
top mrnas encode ribosomal proteins and elongation factors 1a and 2 , which are important in translational control . in the present study
, we detected that p - mtor and p - s6k levels were significantly elevated in the rvm after sni ( figure 2 ) , which suggests that mtor - mediated protein translation and synthesis are increased .
in contrast , the number of 5-htergic neurons was slightly increased within the rvm in the sni rats , but no statistical significance was detected compared to the sham control group .
however , by using whole - cell patch recording , we found that the 5-htergic neurons were overexcited , with significant increases in the amplitude and frequency of sepscs as well as the number of action potentials ( figure 5 ) .
in addition , rapamycin inhibited only the amplitude and not the frequency of sepscs ( figure 5 ) ; we thus propose that the postsynaptic overexcitability of the 5-htergic neurons , which primarily depends on an increase in glutamate receptors , is mainly due to the activation of mtor .
it has been reported that mtor signaling can potentiate the insertion of ampa ( -amino-3-hydroxy-5-methyl-4-isoxazole - propionic acid ) receptors into the postsynaptic membrane and lead to long - term potentiation ( ltp ) [ 44 , 45 ] .
thus , the data collected from our immunofluorescence staining and electrophysiology are consistent with previous reports and suggest that the activation of mtor might lead to an increase in ampa receptors and their insertion into the postsynaptic membrane , resulting in the elevated neuronal excitability of the 5-htergic neurons in the rvm . as an effective immunosuppressant , rapamycin is widely used to prevent transplant rejection .
chronic treatment of patients with mtor inhibitors is associated with an increased incidence of pain [ 46 , 47 ] , including the possible development of complex regional pain syndrome ( crps ) [ 48 , 49 ] .
these conflicting results might be due to the following reasons : ( 1 ) the drug concentration of rapamycin or its analogues was not the same as in the other reports in which the inhibition of mtor produces antinociception ; ( 2 ) long - term treatment might lead to feedback activation of other pronociceptive signal proteins or molecules ; and ( 3 ) intrathecal administration of rapamycin ( at the spinal cord level ) might play a very complicated role in pain transmission with unknown mechanisms .
the present study used intra - rvm , instead of intrathecal , administration of rapamycin ( 250 m , 30 min before the behavioral tests ) , and this treatment remarkably attenuated the nociceptive behaviors induced by sni ( figure 6 ) .
moreover , intra - rvm rapamycin treatment was effective on day 7 after sni ( the maintenance stage of neuropathic pain ) but not on day 1 after sni ( the induction stage ) .
the behavioral pharmacological data suggest that inhibition of mtor in the rvm at the late phase ( maintenance stage ) of neuropathic pain might be effective , even though pain has already been well established . by contrast , inhibition of mtor in the rvm had no effect on the development ( induction stage ) of neuropathic pain .
all of these results are consistent with our biochemical data showing that rvm p - mtor was not greatly enhanced at day 1 but showed a significant increase at 7 days after sni ( figure 2 ) . combining our current results with previous findings , we conclude that the specific inhibition of mtor by rapamycin in the rvm is a promising avenue for the management of neuropathic pain .
this effect probably occurs via deactivation of 5-htergic spinally projecting neurons in the rvm , which are required for descending pain facilitation .
due to the lack of a specific mtor activator , reverse experiments involving the activation of mtor in the rvm , which should produce or enhance nociception , are difficult to achieve .
moreover , optogenetic methods as well as transgenic animals should be further introduced to confirm the role of mtor in the rvm , not only in neuropathic pain but also in inflammatory pain .
through the deactivation of 5-htergic spinally projecting neurons in the rvm and thus the weakening of descending pain facilitation , specific targeting of the activation of mtor in the rvm is a promising avenue for the management of neuropathic pain .
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the mammalian target of rapamycin ( mtor ) , a serine - threonine protein kinase , integrates extracellular signals , thereby modulating several physiological and pathological processes , including pain .
previous studies have suggested that rapamycin ( an mtor inhibitor ) can attenuate nociceptive behaviors in many pain models , most likely at the spinal cord level .
however , the mechanisms of mtor at the supraspinal level , particularly at the level of the rostral ventromedial medulla ( rvm ) , remain unclear .
thus , the aim of this study was to elucidate the role of mtor in the rvm , a key relay region for the descending pain control pathway , under neuropathic pain conditions .
phosphorylated mtor was mainly expressed in serotonergic spinally projecting neurons and was significantly increased in the rvm after spared nerve injury- ( sni- ) induced neuropathic pain .
moreover , in sni rat brain slices , rapamycin infusion both decreased the amplitude instead of the frequency of spontaneous excitatory postsynaptic currents and reduced the numbers of action potentials in serotonergic neurons . finally , intra - rvm microinjection of rapamycin effectively alleviated established mechanical allodynia but failed to affect the development of neuropathic pain .
in conclusion , our data provide strong evidence for the role of mtor in the rvm in nerve injury - induced neuropathic pain , indicating a novel mechanism of mtor inhibitor - induced analgesia .
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1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusion
|
recent progress in our understanding of brain development has significantly altered concepts for treating neurodegenerative diseases , including those that affect the retina to cause blindness .
contrary to previous thought , it is now recognized that neurons are generated in the sub ventricular zone ( svz ) of the lateral ventricle , and the sub - granular zone ( sgz ) in the hippocampus from neural stem cells ( nsc ) of glial origin throughout life ( 1 ) . outside these discrete regions in the mammalian cns , including the retina , active neurogenesis has not been reproducibly demonstrated under normal conditions ( 2 ) .
however , rare neurogenic changes are observed in the injured adult mammalian retina ; the source of injury - induced neurogenesis is traced to mller glia ( mg ) ( 35 ) .
recent observations that mammalian mg possess nsc properties and are able to generate retinal neurons in vitro and upon transplantation in vivo posit these cells as the potential target for regenerating diseased or injured photoreceptors and retinal ganglion cells ( 6 ) . while growing evidence confirms that mg possess evolutionarily conserved neurogenic potential , studies from various labs have observed that , unlike their lower vertebrate counterparts , the injury or disease - activated mammalian mg proliferate , but that they convert to neurons rather infrequently ( 4 , 7 , 8) . whether or not the converted neurons are functional , make synaptic connections , and survive for the long term remains unknown .
the challenge that remains is how to unlock the neurogenic potentials of the mammalian mg in vivo .
this may essentially involve approaches to reprogram these cells to dedifferentiate and regain their lost ability to generate neurons or trans - differentiate them into neurons .
to address this challenge , the neurogenic potentials of mg need to be evaluated against the backdrop of two models , with the understanding that examples from lower vertebrates would constitute a framework , but that solutions will be unique to the mammalian retina : ( 1 ) svz / sgz model , where mg have nsc properties and thus the capacity for neuronal differentiation like radial glia - derived nscs in svz and sgz .
given the observations that the adult nscs are responsive to environmental cues for neuronal differentiation ( 1 ) , mg in this model should be amenable to directed differentiation along neuronal lineage through the manipulation of niche - based signals by recombinant growth factors and/or small molecules .
( 2 ) extra svz / sgz model , where mg may have neurogenic potential like parenchymal astrocytes but incapable of neuronal differentiation , presumably under the influence of the non - neurogenic niche . for example
, both mg and astrocytes express nsc regulators such as sox2 ( mg ) and pax6 ( astrocytes ) and when cultured in the presence of mitogens tend to differentiate along neuronal lineage ( 2 , 6 , 9 , 10 ) .
however , in vivo , although they proliferate and express neural progenitor markers such as nestin in response to injury , they maintain their glial phenotype , with rare expression of neuronal markers ( 2 , 5 , 10 ) .
given their stable glial identity for supporting neurons despite expressing some of the nsc - specific genes , a trans - differentiation approach for the lineage conversion of mg has emerged as a practical option in this model .
this notion is supported by recent observations that injury - activated resident astrocytes differentiate into functional neurons upon enforced expression of nsc regulator , sox2 and/or proneural gene , ascl1 ( 11 ) .
regardless , information gained in deciphering the two models will be useful . in both cases ,
knowledge of the developmental mechanism for the lineage switch , the reaction of mg to different kinds of injuries , phenotypes of the activated mg in spatial and temporal contexts , transcriptional and epigenetic status , and niche - related signals will be critical .
these approaches will reveal whether the roadblock to regeneration is cell - intrinsic or cell - extrinsic or both , information that will be essential in formulating strategies for making mg - dependent therapeutic regeneration practical and clinically applicable . toward this effort ,
the recent success of reprogramming to induce pluripotency in somatic cells and lineage - specific differentiation of pluripotent cells , whether through directed differentiation ( svz / sgz model ) or trans - differentiation ( extra svz / sgz model ) , is owed directly to the knowledge of developmental mechanisms ( 12 ) .
there is a significant knowledge gap in our understanding of how neurogenesis shifts to the generation of mg in the mammalian retina , information essential to navigate the cellular or molecular roadblocks to neuronal differentiation .
for example , although we know that notch signaling regulates the generation of mg , how it is incorporated into the gliogenic program and to what extent it is involved in suppressing neuronal differentiation remains rather unknown ( 4 ) .
given the contextual role of notch signaling ( 4 ) , its complexity due to the oscillatory expression of its effector hes1 ( 13 ) , and its gliogenic interactions with the eye field gene lhx2 ( 14 ) , this information will be essential to formulate niche - based approaches for facilitating neuronal differentiation of mg .
along this line , it will also be advantageous to know whether genes involved in neurogliogenesis elsewhere in the cns , such as lin28 , a heterochronic gene regulating the developmental timing ( 15 ) and ezh2 , an epigenetic regulator of gene expression ( 16 ) participate in mg differentiation and coordinate the influence of the niche .
recent observations that the decision of retinal progenitors during late histogenesis to differentiate along glial or neuronal lineage is influenced by lin28 ( 17 ) and that ezh2 plays a role in constraining the generation of mg ( 18 ) posit these genes as potential targets for pivoting mg torwards neuronal differentiation .
regardless of whether or not mg possesses dormant stem cell properties according to the aforementioned models they must initiate and complete an intertwined program of activation and neuronal conversion for successful regeneration ( 4 ) .
given that injuries in general lead to proliferation of mg across species , but not to their neuronal differentiation in higher vertebrates , it is likely that the cross - talk between transcriptional networks sub - serving the activation and neuronal differentiation are either not connected to the process , or the network components are in place but are not epigenetically primed for optimal expression in the mammalian mg .
these probabilities could be examined by a genome - wide screening of prospectively enriched mg in select animal models in quiescent and activated states ( facilitated by lineage reporters and other enrichment protocols such as the side population ( sp ) cell profiling by microarrays , rna seq , and chip seq analyses . a similar approach to characterizing mg in different states of activation and differentiation in controlled conditions in vitro
will yield corroborative evidence and facilitate the means to test putative mechanism(s ) by manipulating gene expression .
together , these approaches may provide insight into molecular axes that can be tested against the background of those operational in zebrafish ( 7 , 8 , 19 ) .
for example , the molecular axis defined by lin28 and ascl1 , which facilitates mg - mediated regeneration in zebrafish , is a valid target for study in mammals .
a variety of approaches in higher vertebrates have demonstrated important regulatory roles for lin28 in the maintenance of neural progenitors and neuroglial decision ( 20 ) .
it is likely that lin28 influences different developmental function by contextual recruitment of hmga2 , a gene encoding a dna architecture protein ( 21 , 22 ) and ascl1 ( 23 ) by directly regulating the heterochronic mirna let-7 ( 17 , 21 , 23 ) .
let-7 targets hmga2 ( 17 , 21 ) and ascl1 ( 23 ) ; therefore , the lin28-let-7-hmga2/ascl1 axis has emerged as an evolutionary conserved axis that could be a candidate for unlocking neurogenic potential of mg .
for example , overexpression of lin28a ( 24 ) in the enriched mg ( fig .
2a ) prompted these cells to acquire neuronal morphology and to express immunoreactivities and transcripts corresponding to neuronal genes ( fig .
more importantly , neuronal differentiation was accompanied by an increase in levels of mir-124 , a proneural mirna ( 25 ) , hmga2 , and ascl1 , and a decrease in those of rest , a global inhibitor of neuronal differentiation ( 26 ) , thus demonstrating the capacity of lin28a in activating the neurogenic program in mg . however , targeting the lin28-let-7-hmga2/ascl1 axis alone may not be sufficient for the functional reprogramming of mg along neuronal lineage , given that its influence may not be able to completely counter the inhibitory resistance of the rest axis .
rest , by suppressing the expression of proneural mirnas , mir-124 and mir-9 - 9 * , whose targets are proglial genes encoding sox9 and he s family of regulators , may keep mg non - neuronal and therefore non - regenerative ( 25 ) .
the existence of the rest - mir-124 - 9 - 9 * -sox9/hes1 axis in mg suggested that either inhibiting rest or ectopically expressing mir-124/mir-9 - 9 * or both may direct mg along the neuronal lineage .
it is likely that the ectopic expression of mir-124/mir-9 - 9 * in itself might be effective , as they directly or indirectly influence the expression and function of rest ( 25 ) .
for example , over expression of mir-124 - 9 - 9 * ( 27 ) in mg ( fig .
2a ) facilitated neuronal morphology with accompanied expression and suppression of neuronal- and glial - specific genes , respectively ( fig .
expression of both ascl1 and lin28a was up regulated and that of rest was down regulated in the perturbed groups , compared to controls .
however , no significant difference in hmga2 transcript levels was observed , suggesting a threshold requirement of lin28a levels for influencing hmga2 expression . that mir-124 and mir-9 - 9 * could facilitate proneural function of ascl1
was demonstrated by the improvement of ascl1-mediated reprogramming of mg by ectopically expressed mirnas ( 28 ) .
together , these observations posit these molecular axes as valid intrinsic targets for studying and unlocking the dormant regenerative potential of mg .
mg , the guardian of homeostasis in the retina , respond to injuries by proliferating and migrating out of the inner nuclear layer ( 5 ) .
however , despite proliferation and migration as in zebrafish retina , as mentioned , mammalian mg do not initiate regeneration effectively .
this raises the possibility that , in addition to internal constraints discussed above , the environment in the mammalian retina might not be conducive for neurogenic conversion of mg . a niche - based approach to unlock neurogenic potential of mg will involve examining the relationship of mg with neighboring retinal cells , microglia , immigrant astrocytes , and endothelial cells in the context of signaling pathways and their capacity to engage the molecular axes involved in reprograming along neuronal lineage .
for example , the understanding of the role of microglia , one of the first responders to injury , and which may mediate regeneration through inflammatory signals , is still evolving in the retina ( 29 ) .
studies have demonstrated that notch ( 8 , 30 ) , wnt ( 5 , 6 ) , fgf ( 6 , 8) , insulin and insulin - like growth factor-1 ( 8) , shh ( 31 ) , and cytokines such as tnf ( 8 , 19 ) , leptin , and il-11 ( 32 ) may play important roles in mediating the influence of the niche in reprogramming mg .
however , the identity of cells delivering the signals and whether or not these signals and their associated pathways act in concert , which would determine niche - based strategies to promote regeneration , remain largely unknown .
however , studies in zebrafish have begun to shed light on these issues . for example , it has been observed that tnf released by dying photoreceptors in mechanically damaged zebrafish retina may constitute one of the early signals for activating mg for regeneration ( 19 ) .
also , it was reported that zebrafish mg respond to retinal injury by secreting leptin and il-11 , which help reprogram cells in an autocrine fashion ( 32 ) . in both cases , these cytokines facilitated injury - dependent induction of ascl1 , a key step in reprogramming of mg ( 8) . whether or not signaling - mediated by these factors are similarly involved in the mammalian retina remains to be demonstrated .
although the identity of cells delivering notch signaling in mg remains speculative , emerging evidence from zebrafish and higher vertebrates posit it as an important niche - based target for mg - dependent regeneration .
it remains active long after facilitating the differentiation of mg , enabling them to launch the proliferative response when injury takes place ( 4 , 5 ) .
however , persistence of notch signaling in activated mg , which are poised for neuronal conversion , might be deleterious for regenerative process , given the inhibitory influence of notch signaling on the activation of proneural genes ( 33 ) . the inability to confine the activation of notch signaling in mg to a narrow temporal window following injury
may be one of the reasons for their anemic neuronal conversion in mammals , compared to that in zebrafish ( 30 ) .
emerging evidence , once again from the zebrafish model , suggests that these pathways , including notch signaling , may act in concert and mg integrate the diverse niche - based influence of a variety of signaling molecules for a calibrated regenerative response to injury ( 8) .
therefore , niche - related information is crucial for understanding the temporal and spatial modulation of signaling required for activating mg and shifting them from the state of activation to neuronal differentiation .
this is fundamental information missing from studies of mg - dependent regeneration , without which the underlying mechanisms remain difficult to grasp and the therapeutic target rather illusory .
currently , the identity of the activated mg represents cells in the inner nuclear layer of the retina , which have incorporated brdu in response to injury and express mg - specific markers .
given the transition of mg from a quiescent to a proliferative state and the generation of their progenies in different stages of development , the identity based on brdu incorporation , though important , is of limited value .
attempts have been made to prospectively enrich activated mg by hoechst dye efflux assays and to characterize them as sp cells , a phenotype shared by the majority of stem cells ( 4 , 6 ) .
however , since sp cells are also heterogeneous , this functional approach has limitations in the absence of specific cellular markers .
although several signaling pathways ( e.g. notch signaling ) and intrinsic factors ( e.g. , ascl1 ) have been observed to regulate mg activation in fish , birds , and rodents ( 8) , their status as markers of activated mg in different stages of regeneration remain unspecified .
progress in understanding the regenerative mechanisms in tissues like blood ( 34 ) , intestine ( 35 ) , and skin ( 36 ) has been facilitated by the identification and characterization of markers of the resident stem cells and their progenies . within the cns ,
insight into regeneration in the svz has come from the characterization of b1 quiescent and active progenitors and resulting neuroblasts ( 37 ) .
therefore , to follow mg - mediated regeneration , spatially and temporally , and to identify the stage - specific intrinsic players , a concerted effort is needed to search for reliable and reproducible markers . currently , there is no consensus on reliable and reproducible mammalian models to study the activation and neurogenic potential of mg .
rodent models representing retinal injuries , ranging from those caused by the exposure to light , neurotoxins , and genetic mutations have been used .
given the observations that glia respond differently to different types and durations of injuries ( 2 ) , that their levels of activation differ from species to species ( ahmad et al .
, unpublished observations ) , and that within the same species there are strain differences in responses ( 38 ) , a case may be made for developing injury- and species / strain - specific models for reproducible and unambiguous examination of mg neurogenic potentials .
these models will be valuable in incorporating transgenic technology for reliable lineage tracing , and more importantly for molecular characterization of activated mg .
characterization of these cells for gene regulatory network and epigenetic signature is essential to shed light on the status of their neurogenic potential and approaches to unlock it .
furthermore , cell type specific injury models would test the ability of mg to replace specific neuronal types , which will be helpful in designing approaches for mg - dependent therapeutic regeneration .
for example , the loss of vision in two of the intractable blinding diseases , age - related macular degeneration ( amd ) and glaucoma , is due to selective loss of photoreceptors and retinal ganglion cells ( rgcs ) , respectively .
the neurotoxin injury models where exposure of the retina to n - methyl - n - nitrosourea ( mnu ) ablates photoreceptors ( 39 ) and nmda causes the degeneration of rgcs ( 40 ) would reveal whether or not mg could differentiate along specific neuronal types . also , it may shed light on if mg - dependent therapeutic regeneration would be a practical approach to address degenerative changes in amd and/or glaucoma .
the recent success of reprogramming to induce pluripotency in somatic cells and lineage - specific differentiation of pluripotent cells , whether through directed differentiation ( svz / sgz model ) or trans - differentiation ( extra svz / sgz model ) , is owed directly to the knowledge of developmental mechanisms ( 12 ) .
there is a significant knowledge gap in our understanding of how neurogenesis shifts to the generation of mg in the mammalian retina , information essential to navigate the cellular or molecular roadblocks to neuronal differentiation .
for example , although we know that notch signaling regulates the generation of mg , how it is incorporated into the gliogenic program and to what extent it is involved in suppressing neuronal differentiation remains rather unknown ( 4 ) .
given the contextual role of notch signaling ( 4 ) , its complexity due to the oscillatory expression of its effector hes1 ( 13 ) , and its gliogenic interactions with the eye field gene lhx2 ( 14 ) , this information will be essential to formulate niche - based approaches for facilitating neuronal differentiation of mg . along this line
, it will also be advantageous to know whether genes involved in neurogliogenesis elsewhere in the cns , such as lin28 , a heterochronic gene regulating the developmental timing ( 15 ) and ezh2 , an epigenetic regulator of gene expression ( 16 ) participate in mg differentiation and coordinate the influence of the niche .
recent observations that the decision of retinal progenitors during late histogenesis to differentiate along glial or neuronal lineage is influenced by lin28 ( 17 ) and that ezh2 plays a role in constraining the generation of mg ( 18 ) posit these genes as potential targets for pivoting mg torwards neuronal differentiation .
regardless of whether or not mg possesses dormant stem cell properties according to the aforementioned models they must initiate and complete an intertwined program of activation and neuronal conversion for successful regeneration ( 4 ) . given that injuries in general lead to proliferation of mg across species , but not to their neuronal differentiation in higher vertebrates , it is likely that the cross - talk between transcriptional networks sub - serving the activation and neuronal differentiation are either not connected to the process , or the network components are in place but are not epigenetically primed for optimal expression in the mammalian mg .
these probabilities could be examined by a genome - wide screening of prospectively enriched mg in select animal models in quiescent and activated states ( facilitated by lineage reporters and other enrichment protocols such as the side population ( sp ) cell profiling by microarrays , rna seq , and chip seq analyses . a similar approach to characterizing mg in different states of activation and differentiation in controlled conditions in vitro
will yield corroborative evidence and facilitate the means to test putative mechanism(s ) by manipulating gene expression .
together , these approaches may provide insight into molecular axes that can be tested against the background of those operational in zebrafish ( 7 , 8 , 19 ) .
for example , the molecular axis defined by lin28 and ascl1 , which facilitates mg - mediated regeneration in zebrafish , is a valid target for study in mammals .
a variety of approaches in higher vertebrates have demonstrated important regulatory roles for lin28 in the maintenance of neural progenitors and neuroglial decision ( 20 ) .
it is likely that lin28 influences different developmental function by contextual recruitment of hmga2 , a gene encoding a dna architecture protein ( 21 , 22 ) and ascl1 ( 23 ) by directly regulating the heterochronic mirna let-7 ( 17 , 21 , 23 ) .
let-7 targets hmga2 ( 17 , 21 ) and ascl1 ( 23 ) ; therefore , the lin28-let-7-hmga2/ascl1 axis has emerged as an evolutionary conserved axis that could be a candidate for unlocking neurogenic potential of mg .
for example , overexpression of lin28a ( 24 ) in the enriched mg ( fig .
2a ) prompted these cells to acquire neuronal morphology and to express immunoreactivities and transcripts corresponding to neuronal genes ( fig .
more importantly , neuronal differentiation was accompanied by an increase in levels of mir-124 , a proneural mirna ( 25 ) , hmga2 , and ascl1 , and a decrease in those of rest , a global inhibitor of neuronal differentiation ( 26 ) , thus demonstrating the capacity of lin28a in activating the neurogenic program in mg . however , targeting the lin28-let-7-hmga2/ascl1 axis alone may not be sufficient for the functional reprogramming of mg along neuronal lineage , given that its influence may not be able to completely counter the inhibitory resistance of the rest axis .
rest , by suppressing the expression of proneural mirnas , mir-124 and mir-9 - 9 * , whose targets are proglial genes encoding sox9 and he s family of regulators , may keep mg non - neuronal and therefore non - regenerative ( 25 ) .
the existence of the rest - mir-124 - 9 - 9 * -sox9/hes1 axis in mg suggested that either inhibiting rest or ectopically expressing mir-124/mir-9 - 9 * or both may direct mg along the neuronal lineage .
it is likely that the ectopic expression of mir-124/mir-9 - 9 * in itself might be effective , as they directly or indirectly influence the expression and function of rest ( 25 ) .
for example , over expression of mir-124 - 9 - 9 * ( 27 ) in mg ( fig .
2a ) facilitated neuronal morphology with accompanied expression and suppression of neuronal- and glial - specific genes , respectively ( fig .
expression of both ascl1 and lin28a was up regulated and that of rest was down regulated in the perturbed groups , compared to controls .
however , no significant difference in hmga2 transcript levels was observed , suggesting a threshold requirement of lin28a levels for influencing hmga2 expression .
that mir-124 and mir-9 - 9 * could facilitate proneural function of ascl1 was demonstrated by the improvement of ascl1-mediated reprogramming of mg by ectopically expressed mirnas ( 28 ) .
together , these observations posit these molecular axes as valid intrinsic targets for studying and unlocking the dormant regenerative potential of mg .
mg , the guardian of homeostasis in the retina , respond to injuries by proliferating and migrating out of the inner nuclear layer ( 5 ) .
however , despite proliferation and migration as in zebrafish retina , as mentioned , mammalian mg do not initiate regeneration effectively .
this raises the possibility that , in addition to internal constraints discussed above , the environment in the mammalian retina might not be conducive for neurogenic conversion of mg . a niche - based approach to unlock neurogenic potential of mg will involve examining the relationship of mg with neighboring retinal cells , microglia , immigrant astrocytes , and endothelial cells in the context of signaling pathways and their capacity to engage the molecular axes involved in reprograming along neuronal lineage .
for example , the understanding of the role of microglia , one of the first responders to injury , and which may mediate regeneration through inflammatory signals , is still evolving in the retina ( 29 ) .
studies have demonstrated that notch ( 8 , 30 ) , wnt ( 5 , 6 ) , fgf ( 6 , 8) , insulin and insulin - like growth factor-1 ( 8) , shh ( 31 ) , and cytokines such as tnf ( 8 , 19 ) , leptin , and il-11 ( 32 ) may play important roles in mediating the influence of the niche in reprogramming mg . however , the identity of cells delivering the signals and whether or not these signals and their associated pathways act in concert , which would determine niche - based strategies to promote regeneration , remain largely unknown .
for example , it has been observed that tnf released by dying photoreceptors in mechanically damaged zebrafish retina may constitute one of the early signals for activating mg for regeneration ( 19 ) .
also , it was reported that zebrafish mg respond to retinal injury by secreting leptin and il-11 , which help reprogram cells in an autocrine fashion ( 32 ) . in both cases , these cytokines facilitated injury - dependent induction of ascl1 , a key step in reprogramming of mg ( 8) . whether or not signaling - mediated by these factors are similarly involved in the mammalian retina remains to be demonstrated .
although the identity of cells delivering notch signaling in mg remains speculative , emerging evidence from zebrafish and higher vertebrates posit it as an important niche - based target for mg - dependent regeneration .
it remains active long after facilitating the differentiation of mg , enabling them to launch the proliferative response when injury takes place ( 4 , 5 ) .
however , persistence of notch signaling in activated mg , which are poised for neuronal conversion , might be deleterious for regenerative process , given the inhibitory influence of notch signaling on the activation of proneural genes ( 33 ) . the inability to confine
the activation of notch signaling in mg to a narrow temporal window following injury may be one of the reasons for their anemic neuronal conversion in mammals , compared to that in zebrafish ( 30 ) .
emerging evidence , once again from the zebrafish model , suggests that these pathways , including notch signaling , may act in concert and mg integrate the diverse niche - based influence of a variety of signaling molecules for a calibrated regenerative response to injury ( 8) .
therefore , niche - related information is crucial for understanding the temporal and spatial modulation of signaling required for activating mg and shifting them from the state of activation to neuronal differentiation .
this is fundamental information missing from studies of mg - dependent regeneration , without which the underlying mechanisms remain difficult to grasp and the therapeutic target rather illusory .
currently , the identity of the activated mg represents cells in the inner nuclear layer of the retina , which have incorporated brdu in response to injury and express mg - specific markers .
given the transition of mg from a quiescent to a proliferative state and the generation of their progenies in different stages of development , the identity based on brdu incorporation , though important , is of limited value .
attempts have been made to prospectively enrich activated mg by hoechst dye efflux assays and to characterize them as sp cells , a phenotype shared by the majority of stem cells ( 4 , 6 ) . however , since sp cells are also heterogeneous , this functional approach has limitations in the absence of specific cellular markers .
although several signaling pathways ( e.g. notch signaling ) and intrinsic factors ( e.g. , ascl1 ) have been observed to regulate mg activation in fish , birds , and rodents ( 8) , their status as markers of activated mg in different stages of regeneration remain unspecified .
progress in understanding the regenerative mechanisms in tissues like blood ( 34 ) , intestine ( 35 ) , and skin ( 36 ) has been facilitated by the identification and characterization of markers of the resident stem cells and their progenies . within the cns ,
insight into regeneration in the svz has come from the characterization of b1 quiescent and active progenitors and resulting neuroblasts ( 37 ) .
therefore , to follow mg - mediated regeneration , spatially and temporally , and to identify the stage - specific intrinsic players , a concerted effort is needed to search for reliable and reproducible markers .
currently , there is no consensus on reliable and reproducible mammalian models to study the activation and neurogenic potential of mg .
rodent models representing retinal injuries , ranging from those caused by the exposure to light , neurotoxins , and genetic mutations have been used .
given the observations that glia respond differently to different types and durations of injuries ( 2 ) , that their levels of activation differ from species to species ( ahmad et al .
, unpublished observations ) , and that within the same species there are strain differences in responses ( 38 ) , a case may be made for developing injury- and species / strain - specific models for reproducible and unambiguous examination of mg neurogenic potentials .
these models will be valuable in incorporating transgenic technology for reliable lineage tracing , and more importantly for molecular characterization of activated mg .
characterization of these cells for gene regulatory network and epigenetic signature is essential to shed light on the status of their neurogenic potential and approaches to unlock it .
furthermore , cell type specific injury models would test the ability of mg to replace specific neuronal types , which will be helpful in designing approaches for mg - dependent therapeutic regeneration .
for example , the loss of vision in two of the intractable blinding diseases , age - related macular degeneration ( amd ) and glaucoma , is due to selective loss of photoreceptors and retinal ganglion cells ( rgcs ) , respectively .
the neurotoxin injury models where exposure of the retina to n - methyl - n - nitrosourea ( mnu ) ablates photoreceptors ( 39 ) and nmda causes the degeneration of rgcs ( 40 ) would reveal whether or not mg could differentiate along specific neuronal types . also , it may shed light on if mg - dependent therapeutic regeneration would be a practical approach to address degenerative changes in amd and/or glaucoma .
the discipline of mg - based regeneration is a recent one , particularly in mammals . with information on the nature of
the activated mg and the molecular axes mediating their response to the niche , aided by reproducible animal models and lineage reporters , a pharmacological recruitment of these endogenous progenitors for therapeutic regeneration is a near possibility . in addition , information emerging from these studies will help us understand how the neurogenic potential in the adult mammalian brain is constrained relative to that of lower vertebrates , currently a significant knowledge gap .
|
mller glia ( mg ) are the primary support cells in the vertebrate retina , regulating homeostasis in one of the most metabolically active tissues . in lower vertebrates such as fish
, they respond to injury by proliferating and reprogramming to regenerate retinal neurons . in mammals
, mg may also react to injury by proliferating , but they fail to initiate regeneration .
the barriers to regeneration could be intrinsic to mammalian mg or the function of the niche that can not support the mg reprogramming required for lineage conversion or both .
understanding these mechanisms in light of those being discovered in fish may lead to the formulation of strategies to unlock the neurogenic potential of mg and restore regeneration in the mammalian retina .
|
Introduction
Neurogenic potentials of MG in mammals
Mechanisms underlying the neuroglial switch in the mammalian retina
Molecular axes involved in MG-mediated regeneration
Influences of the niche and how they are mediated
Markers of activated MG
Animal models for MG-mediated therapeutic regeneration
Conclusions
|
the field of utility organometallics is increasingly turning to more complex mixed - metal systems to deliver superior results , whether this be for metal halogen exchange , alkylation , insertion , or the trapping of reactive intermediates . in theory
, mixed - metal reagents can offer more flexible , tunable solutions to any problem compared to more traditional monometallic alternatives due to the increased number of components which can be varied .
furthermore , the different metal centers and ligand sets can exhibit cooperativity ( or synergism ) to produce new reactivities inaccessible to the monometallic components .
hydrogen abstraction is an area in which the lure of such mixed - metal systems is proving to be irresistible .
the combination of an alkali metal with a range of different subordinates ( i.e. , metals that would be expected to be less reactive than the alkali metals - zn , mg , mn , cr , fe , cu , cd , al ) have been integrated with a diverse array of ligands ( alkyl , amido , alkoxy , halide ) and have been efficiently deployed in both ethereal and hydrocarbon media to overcome some of the great challenges facing deprotonation chemistry such as the incapability to metalate low brnsted acidity substrates , lack of compatibility with sensitive functionalities , low selectivity and the need to perform reactions under cryogenic conditions .
recently our own group has started to investigate the use of diamines in a bimetallic context , reporting in a communication the synthesis of the diazaethene zincate complex ( tmeda)li[(ipr)nch = chn(ipr)]zn(tbu ) 1 .
1,3-diazabutadiene ligands , from which diazaethene ligands are often prepared , are increasingly important within coordination chemistry due to their flexible coordination modes and redox properties .
the synthesis of this complex from the fully saturated ( ipr)nhch2ch2nh(ipr ) ( diisopropylethylenediamine ) 2 ( scheme 1 ) displayed a new mode of hydrogen activation previously unseen within zincate chemistry .
we now report a detailed mechanistic overview of this surprising transformation from both experimental and theoretical perspectives .
as reported previously , the cocomplexation of the monolithiated diamine li[(ipr)nch2ch2nh(ipr ) ] with ( tbu)2zn(tmeda ) at 0 c gives the expected product ( tmeda)li[(ipr)nch2ch2nh(ipr)]zn(tbu)23 ( scheme 1 ) . in a fashion similar to that of the two - step mechanism for monoamido zincates originally proposed by uchiyama et al .
, the amino n h group of 3 is rapidly deprotonated by a tbu ligand to form the dimetalated diamido ( tmeda)li[(ipr)nch2ch2n(ipr)]zn(tbu ) 4 with the evolution of ibuh .
although diamido 4 has thus far not crystallized , it has been characterized by multinuclear nmr spectroscopy , and we have succeeded in the crystallographic identification of several analogous structures . using the homologous ( ipr)nhch2ch2ch2nh(ipr ) ( diisopropylpropylenediamine ) , the dimetalated diamido ( tmeda)li[(ipr)nch2ch2ch2n(ipr)]zn(tbu ) 5 ( scheme 2 and figure 1 ) could be crystallized from the corresponding reaction of li[(ipr)nch2ch2ch2nh(ipr ) ] with ( tbu)2zn(tmeda ) .
unfortunately , the crystal data are of insufficient quality to allow discussion of the geometric parameters of the structure , but the connectivity is definite .
zincate 5 contains an ncccnzn six - membered ring in a boat - type conformation .
the diamido ligand thus chelates the zn center while its two ipr arms are oriented out of the plane of the ring .
the ( tmeda)li cation is coordinated to the hull of the boat , anchored exclusively to the two secondary amido groups to produce a puckered linznn four - membered ring .
h nmr spectroscopic studies confirm the fully saturated nature of the propylene bridge with methylene resonances at 3.33 , 2.95 , and 1.93 ppm , while no characteristic diazaethene resonances were present in the 56 ppm region .
molecular structure of the diamido zincate ( tmeda)li[(ipr)nch2ch2ch2n(ipr)]zn(tbu ) 5 . hydrogen atoms and disordered tbu / tmeda components have been omitted for clarity .
on utilizing thf as a donor ligand in the absence of tmeda , in a repeat of the synthesis of 4 , the dimeric thf - solvated product { ( thf)li[(ipr)nch2ch2n(ipr)]zn(tbu)}26 was isolated and subsequently crystallographically characterized ( scheme 2 and figure 2 ) .
lithium zincate 6 has a centrosymmetric molecular structure consisting of two [ ( ipr)nch2ch2n(ipr)]zn(tbu ) complex anions , juxtaposed , and bridged by two ( thf)li cations to produce an eight - membered ( linznn)2 ring .
the cyclic conformation of 6 allows for the symmetrical coordination of each amido group to one lithium and one zinc center . both sets of metal centers occupy distorted trigonal planar geometries .
the ipr groups on the amido ligands lie syn with respect to the znnccn ring , while the nonplanar nature of the diamide ( n1c4c5n2 torsion angle of 50.0(2) ) as well as its n c [ 1.471(3 ) ] and c c [ 1.530(3 ) ] bond distances , which are comparable with those found in the ruthenium complex [ ( ipr)nch2ch2n(ipr)]ruh2(co)5 [ n c , 1.48(1 ) ; c c , 1.52(1 ) ] , establish that the ch2ch2 backbone remains saturated .
thermal ellipsoids are shown at the 50% probability level . selected bond lengths [ ] and angles [ deg ] : zn1n1 , 2.019(2 )
; zn1n2 , 1.986(2 ) ; zn1c9 , 2.019(9 ) ; li1n1 , 2.001(4 ) ; li1n2(a ) , 2.041(4 ) ; li1o1 , 2.004(4 ) ; n1c4 , 1.471(3 ) ; n2c5 , 1.470(3 ) ; c4c5 , 1.530(3 ) ; n1zn1n2 , 91.20(7 ) ; n1zn1c9 , 127.5(2 ) ; n2zn1c9 , 141.2(2 ) ; n1c4c5n2 , 50.0(2 ) ; n1li1n2(a ) , 127.0(2 ) ; n1li1o1 , 117.7(2 ) ; n3li1o1 , 114.9(2 ) . to gain mechanistic insight a detailed nmr spectroscopic study
was carried out on the reaction between the monometallic compounds li[(ipr)nch2ch2nh(ipr ) ] and ( tbu)2zn(tmeda ) .
a li nmr spectrum of isolated crystals of the cocomplexation product ( tmeda)li[(ipr)nch2ch2nh(ipr)]zn(tbu)23 in c6d6 solution reveals two major resonances at 0.37 ppm and 0.63 ppm , suggesting a mixture of products .
when this solution is gently warmed and reanalyzed , the resonance at 0.37 ppm is absent , intimating that the product associated with this low - frequency resonance has been fully consumed , and only the resonance at 0.63 ppm remains ( see figure s24 in supporting information ) .
this is consistent with the disappearing resonance at 0.37 ppm representing the bisalkyl zincate 3 which , via an intramolecular deprotonation and concomitant release of ibuh , forms the diamido complex 4 .
further support for this interpretation can be found when considering how close the signal assigned to complex 4 ( 0.63 ppm ) comes to that of the structurally analogous propylene derivative 5 ( 0.75 ppm , i.e. a difference of only 0.12 ppm ) .
when the reaction was repeated in situ at 0 c and an aliquot removed and analyzed by li nmr spectroscopy , two pertinent resonances at 2.39 ppm , and 1.71 ppm , as well as the resonance belonging to the previously identified intermediate 4 , are present ( see figure s24 in supporting information ) .
li dosy nmr experiments reveal that the intermediate responsible for the resonance at 1.71 ppm has a molecular weight similar to that of 4 ( see figure s25 in supporting information ) , presumably ruling out an aggregation process for the formation of this , as yet , unidentified species . comparison with the li nmr spectra of the diazaethene 1 in c6d6 reveals that the signal at 2.39 ppm is the result of a trace amount of this final product . due to the highly reactive , transient nature of the bis - alkylzincate 3 , its presence is not detected in this in situ nmr study .
another aliquot of the solution was removed after a 2-day stir at room temperature , but the recorded li nmr spectrum indicated no significant further compositional change had taken place .
aliquots were then removed after 5 min , 1 h , and 2 h reflux time , and the recorded spectra revealed a gradual transformation from the saturated intermediate 4 to the unsaturated final product 1 .
after 2 h reflux , decomposition of the reaction mixture is evidenced by the deposition of a black solid .
any further intermediates along the reaction pathway must be too short lived to be visible on the nmr time scale .
the next step in the reaction pathway 4 1 has been shown to likely proceed through a hydride intermediate as we successfully trapped a hydride by using the electrophilic ketone ( tbu)2co .
the hydride produced can then deprotonate the now allylic proton on the ethylene bridge to give the final product .
the system must then find a way of solubilizing and activating the produced lih , which is normally insoluble in hydrocarbon solvents . a more typical dehydrogenation pathway involving redox active transition metals , such as those reported by brookhart ,
can be ruled out as there are no redox - active metals present in our alkali metal
instead , the reaction could be envisaged to proceed through a ligand - sited oxidation followed by a reduction ( scheme 3 ) .
this reaction can thus be said to exploit a unique three - way cooperation , where the chemical properties of the two metals and the ethylenediamide ligand combine to allow a type of chemistry otherwise unavailable . in order to explore the possible pathways by which this reaction could proceed we carried out detailed mechanistic investigations using density functional theory ( dft ,
see the supporting information for full details on the level of theory employed ) in order to explore the potential energy surface ( pes ) associated with the proposed mechanisms .
we initially inspected the structure and stability of the monomer species 4 at the dft level of theory . in agreement with the three - carbon bridge analogue
( 5 , figure 1 ) , 4 is found to be a stable minimum on the pes with the diamido ligand coordinated to the zn atom ( n zn : 2.04 and 1.99 ) to form a five - membered ring ( nccnzn ) in a slightly twisted ( nccn torsion of 31.4 ) envelope conformation with the zn atom below the nccn plane , ( figure 3 ) . the ( tmeda)li cation is coordinated directly above the two n atoms of the five - membered ring by two essentially equivalent li
based on the optimized structure of 4 two different mechanisms by which the proposed zinc hydride species could be formed are possible .
all h atoms , except those involved in the reaction , are omitted for clarity .
the h atom could be initially abstracted by the li cation to form a zn h bond below the plane of the ring , or direct abstraction by the zn to form the zn h bond above the ring plane .
optimisation of the two hydride intermediate structures revealed that both were stable minima on the pes but were formed in endothermic reactions relative to 4 ( figure 4 ) .
the intermediate with the h atom above the nccn plane ( inta ) is destabilized by 14.7 kcal / mol ( g ) relative to 4 , while the intermediate with the h atom below the plane ( intb ) is destabilized by 8.6 kcal / mol ( g ) , relative to 4 .
while both reactions are endothermic , the relative stabilities of the intermediates are not sufficiently different to determine which pathway is more favorable .
therefore , we characterized the transition states leading to the intermediate structures in order to determine whether there was a kinetic preference toward either species . the activation barrier
associated with the formation of inta is 23.2 kcal / mol ( g * ) , compared to a barrier of 31.8 kcal / mol ( g * ) for the formation of intb .
an activation barrier of 31.8 kcal / mol is still accessible under the experimental conditions employed in the reactivity of 4 ; thus , while inta is expected to be preferentially formed as an initial hydride intermediate in the formation of the diazaethene product ( 1 ) , we also considered intb as a potential intermediate from which the second hydrogen abstraction could occur .
abstraction of the second h atom from inta occurs with a barrier of 17.4 kcal / mol , relative to inta , ( g * ) in an exothermic reaction ( g = 13.2 kcal / mol ) to generate h2 and the final product 1 . however , despite exhaustive sampling , no transition state could be located that corresponded to the formation of h2 and 1 starting from intb .
therefore , the overall reaction 4 1 + h2 , which is essentially thermoneutral ( g = 1.5 kcal / mol , figure 4 ) , appears to preferentially occur via the hydride inta .
free energy ( g ) profile for the formation of 1 from 4 via a hydride intermediate .
the path in red corresponds to the formation of the hydride intermediate above the ring plane ( inta ) , while the path in blue corresponds to the formation of the hydride intermediate below the ring plane ( intb ) .
the relative stabilities of ts(4-inta ) and ts(4-intb ) ( figure 4 ) are dictated by the different roles that the li and zn centers play in the two different mechanisms . in both transition states , in order for the zn h bond to be formed , one zn
n bond must be broken such that the zn atom is able to accommodate the incoming h and the unsaturated n = c bond can form . within the ts(4-inta )
n bond breaking is facilitated by the formation of a stabilizing interaction between the n atom and the li .
this is evidenced by the rotation around the c c bond [ increase in the dihedral angle from 31.4 in 4 ( figure 3 ) to 56.3 ( figure 5a ) ] , which decreases the li
n distance from 2.02 ( figure 3 ) to 1.98 ( figure 5a ) and increases the zn
n distance from 2.04 ( figure 3 ) to 3.39 ( figure 5a ) .
in contrast , the rotation around the c c bond in ts(4-intb ) is not as great ( 34.1 , figure 5b ) .
in addition , in ts(4-intb ) the li atom is forced to migrate toward the second n atom in order to facilitate the transfer of the h atom beneath the plane of the ring .
n distance to 3.60 for the primary n ( figure 5b ) , and consequently , the breaking of the zn
n bond length is only slightly increased from 2.04 in 4 ( figure 3 ) to 2.12 in ts(4-intb ) ( figure 5b ) .
thus , the formation of the hydride intermediate is truly the result of the synergic interplay between the zn ( stabilizing the formation of the zn h bond ) and li ( stabilizing the breaking of the zn
returning to experimental studies , a molecular hydride species [ ( tmeda)li]2[(ipr)nch2ch2n(ipr)]zn(tbu)h 7 has been isolated by using a variation of this mixed - metal approach with the ethylenediamide ligand , demonstrating that this system has the ability to encapsulate lih ( scheme 4 and figure 6 ) .
dilithium hydridozincate 7 was synthesized while attempting to generate [ ( tmeda)li]2[(ipr)nch2ch2n(ipr)]zn(tbu)2 by the n
h deprotonation of ( tmeda)li[(ipr)nch2ch2nh(ipr)]zn(tbu)23 with a molar equivalent of nbuli(tmeda ) ( scheme 4 ) .
[ ( tmeda)li]2[(ipr)nch2ch2n(ipr)]zn(tbu)2 facilitates the -hydride formation and elimination of ibuh to generate the zinc hydride species 7 .
dft calculations have confirmed that such a process [ 4 + ( tmeda)lih 7 ] would be exothermic ( g = 28.4 kcal / mol ) .
molecular hydride 7 can also be produced via the alternative reaction of the usually kinetically challenged base [ ( tmeda)li]2zn(tbu)4 with ( ipr)nhch2ch2nh(ipr ) with an improved yield of 65% relative to 35% .
molecular structure of the zincate hydride complex [ ( tmeda)li]2[(ipr)nch2ch2n(ipr)]zn(tbu)h 7 with hydrogen atoms ( except for the hydride ion ) omitted for clarity .
thermal ellipsoids are shown at the 50% probability level . selected bond lengths [ ] and angles [ deg ] : li1h100 , 1.96(2 ) ; li2h100 , 2.07(2 ) ; zn1h100 , 2.14(2 ) ; li1n2 , 1.979(4 ) ; li2n1 , 1.989(4 ) ; zn1n1 , 2.043(2 ) ; zn1n2 , 2.074(2 ) ; zn1c1 , 2.045(3 ) ; n1c9 , 1.481(3 ) ; n2c8 , 1.448(3 ) ; c8c9 , 1.523(4 ) ; n1zn1h100 , 92.8(6 ) ; n1zn1n2 , 89.66(8 ) ; n1zn1c1 , 133.1(1 ) ; n2zn1c1 , 125.19(9 ) ; n2zn1h100 , 92.8(5 ) ; h100zn1c1 , 113.0(6 ) ; n2li1n5 , 121.8(2 ) ; n2li1n6 , 129.6(2 ) ; n2li1h100 , 101.5(6 ) ; n5li1n6 , 85.7(2 ) ; n5li1h100 , 115.9(6 ) ; n6li1h100 , 101.6(6 ) .
mixed - metal zincate hydrides have repeatedly demonstrated their utility in organic synthesis as reagents for chemoselective , diastereoselective , and catalytic reductions .
highly soluble , stable , and well - defined zincate hydride species are thus highly desirable . despite this , however , structurally characterized alkali metal zincate hydrides are extremely rare . to date the only examples on the ccdb are the zinc zinc bonded arzn(-h)(-na)znar ( ar = c6h3 - 2,6-[c6h3-(ipr)2]2 ) , the heterocubanes [ ( tbuoznh)4n(liotbuthf)n ] ( n = 03 ) , and the higher - order zincates na2[(et)2znh]2 and na3[(ipr)3zn(-h)zn(ipr)3 ] .
although metal hydride complexes are notoriously insoluble , a reputation perpetuated by common hydride reagents such as lih and cah2 , the hydride carrier 7 is soluble in the nonpolar solvent hexane .
the structure of zinc hydride 7 bears a partial resemblance to that of the thf solvated dimer 6 , with a [ ( ipr)nch2ch2n(ipr)]zn(tbu ) complex anion having a lithium cation bound to each nitrogen atom . the li
n amido bond distances in 7 ( average 1.984 ) are close to those in dimer 6 ( average 2.021 ) . likewise the zn
n bond distances in hydride 7 [ 2.074(2 ) and 2.043(2 ) ] are only marginally longer than those in alkylamido 6 ( average 2.002 ) , owing to the increased number of anions bound to the zinc .
the tbu group has been pushed out of the plane of the amido ligand to allow the zinc center to enter a distorted tetrahedral geometry by binding to the hydride ligand . as in the lithium zincate 6 , the n
c [ 1.481(3 ) and 1.448(3 ) ] and c c [ 1.523(4 ) ] bond lengths in 7 are consistent with an unactivated ethylene bridge .
the trapped 3-hydride was successfully located and freely refined in the x - ray diffraction study .
it caps a li2zn triangle asymmetrically with unequal li h bond distances [ 1.96(2 ) and 2.07(2 ) ] and a longer zn h bond [ 2.14(2 ) ] .
the ccdb at the time of writing contains only 45 compounds exhibiting a zn hydride bond . excluding an anomalously long contact within a zinc borohydride compound ( 2.409 ) , zn hydride bond lengths range from 1.4092.167 with an average distance of 1.771 .
the zn hydride contact in 7 is thus long - range to the best of our knowledge , the longest zn hydride contact in a non - borohydride complex to date ( the value of 2.167 mentioned above also comes from a borohydride complex ) .
there are currently 147 compounds in the ccdb containing li hydride contacts with their lengths spanning the wide range 1.6072.802 .
the average distance ( 2.044 ) is comparable with those found in 7 . to the best of our knowledge
however , there have been various dilithio 3-hydride compounds reported with other metals such as ga , al , zr , w , sm , ta , and fe .
variable - concentration multinuclear ( h , c and li ) nmr spectroscopic studies of 7 , as well as exchange spectroscopic ( exsy ) experiments , confirm the presence of a dynamic equilibrium in c6d6 solution . as well as resonances consistent with the solid state structure of 7 ,
mixed - metal zincate complex 4 can thus be described as a molecular scaffold for the molecularization of the usually insoluble polymeric lih .
an examination of the li nmr spectrum reveals a doublet with a jli h coupling constant of 13.3 hz , confirming the retention of the li
only a few examples have been detected since the first measurement by bergman of ( cp*)irh2sime3li(pmdeta ) ( cp * = c5me5 , pmdeta = n , n , n,n,n-pentamethyldiethylenetriamine ) in 1985 .
those detected have coupling constants ranging from 6.414.7 hz , placing our example at the upper end of the literature examples .
surprisingly , two independent h resonances , at 3.27 and 3.19 ppm , were detected coupling with the doublet in the li nmr spectrum .
this result can be tentatively assigned to the li resonance for the zincate hydride 7 sharing the same chemical shift as dissociated ( tmeda)lih , in agreement with the presence of a dynamic equilibrium in solution .
given the equilibrium between 4 and 7 , the formation of a diazaethene compound , analogous to 1 , may also occur using 7 as the starting point .
the optimized structure of 7 closely matches the experimental x - ray structure , with a mean unsigned error of 0.07 between the measured and calculated bond lengths .
the largest deviation between the calculated and experimental values occurs for the interactions involving the hydride ion ( e.g. , zn h : 1.94 calculated vs. 2.14 experimental , see table s1 in supporting information for details ) .
the deviations in the hydride ion distances may be due to the uncertainty inherent in the crystallographic determination of hydride positions ; however , even with the shorter calculated zn
h interaction , at 1.94 the zn h contact is clearly long - range .
the introduction of the ( tmeda)lih ligand to 4 to form 7 results in a different mechanism for the formation of the diazaethene compound .
one of the ethylene c8 hydrogen atoms from 7 ( figure 7 ) is aligned for abstraction by the dilithio hydride to form the intermediate ( intc ) containing a ( lih)2 unit .
the formation of intc occurs in an endothermic step ( g = 21.7 kcal / mol ) , relative to 7 , with an associated barrier of 30.1 kcal / mol ( g * , figure 7 ) .
the subsequent abstraction of the h atom from c9 ( figure 7 ) to form h2 and the diazaethene compound 8 , occurs with a barrier ( g * ) of 11.8 kcal / mol , relative to intc , in an exothermic reaction ( g = 16.7 kcal / mol ) .
overall , the reaction from 7 to form 8 is slightly endothermic ( g = 5.0 kcal / mol , figure 7 ) . from the intermediate structure ( intc )
the barriers to the forward and reverse reaction ( i.e. , the formation of 8 + h2 and 7 , respectively ) are comparable in energy ( g * = 3.4 kcal / mol ) . therefore , although the equilibrium from intc will be in the direction of 7 , the formation of 8 + h2 from this structure should also occur to some extent .
free energy ( g ) profile for the formation of 8 from 7 via a dihydride intermediate .
the possible formation of a zn hydride intermediate , in analogy to the mechanisms explored for the formation of 1 ( via inta ) was also investigated .
the barrier associated with the formation of this hydride intermediate ( g * = 28.4 kcal / mol ) is accessible under the experimental reaction conditions and the intermediate produced in this reaction is destabilized by 21.1 kcal / mol , relative to 7 .
however , the barrier to the subsequent second hydrogen abstraction ( i.e. , the formation of 8 + h2 ) is 34.4 kcal / mol , which results in a strong preference for the reverse reaction back to the reactant 7 ( g * = 6.0 kcal / mol ) . as such
, the mechanism resulting in the formation of dihydride intermediate ( intc , figure 7 ) is expected to be the dominant mechanism for the formation of 8 + h2 , from 7 . despite the comparable energetics for the formation of 8 from 7 ( via intc ) and 1 from 4 ( via inta ) , the mechanisms and the synergic interaction of the two metals are distinctly different .
the presence of the second li atom in 7 ( relative to 4 ) allows the zn to stabilize the formation of the c = n bond ( the zn
n bond increases from 2.07 in 7 to 2.18 in ts(7-intc ) , figure 8) , while the li atom is able to facilitate the c h bond breaking step .
finally , the free energy associated with the loss of a ( tmeda)lih ligand from 8 to form 1 is slightly less ( g = 24.9 kcal / mol ) than the equivalent association / dissociation reaction between 4 and 7 . therefore , given the similar overall kinetic barriers associated with the generation of the diazaethene species starting from 4 and 7 , both reaction mechanisms described in figures 5 and 8 , should be competitive and operate in a complementary fashion to generate the observed product 1 .
the closest analogy to the synthesis of 1 was reported by veith who formed 1,3-diaza-2-silacyclopentene by the double lithiation of ( tbu)nhch2ch2nh(tbu ) ( di - tert - butylethylenediamine ) with nbuli followed by an electrophilic quench with cl2si(me)n(h)tbu .
it was acknowledged at the time that the process leading to this dehydrogenation was unclear but it was noted that the reaction was highly concentration dependent . for dehydrogenation to occur the lithiation step needed to be carried out in a highly concentrated nbuli hexane solution ( 2 m ) .
this concentration dependence infers a different mechanism of hydrogen activation from that which results in the synthesis of 1 , possibly involving either larger aggregates or an intermolecular process .
large aggregates of liotbu have previously been found to activate and retain lih in solution .
the diamido species was never characterized in veith s study . we have confirmed by nmr spectroscopy that ( ipr)nhch2ch2nh(ipr ) , like its homologue ( tbu)nhch2ch2nh(tbu ) , can be activated when refluxed in a concentrated nbuli solution . when the reaction was carried out at concentrations akin to that used in the synthesis of 1
, then the dilithiated species could be crystallized as a tmeda solvate ( figure 9 ) .
this product , [ ( tmeda)li(ipr)nch2ch2n(ipr)li]29 , has a distorted ladder structure in the solid state with the diamido ligands adopting a trans bent conformation allowing them to bridge between opposite corners of alternate rungs .
n bond distances of 2.227(2 ) and 1.971(2 ) between the outer rungs and 2.057(2 ) between the inner rungs . maintaining the c2 axis of symmetry , the long and short edges of the ladder alternate as the ladder is ascended .
both outer lithium centers adopt distorted tetrahedral geometries , while the internal lithium atoms are three - coordinate .
this trapping of a ladder segment instigated by the lewis base tmeda supports gardiner s assertions .
selected bond lengths [ ] and angles [ deg ] : li1n1 , 2.227(2 ) ; li1n2 , 1.977(3 ) ; li1n3 , 2.209(3 ) ; li1n4 , 2.199(3 ) ; li2n1 , 2.025(2 ) ; li2n2 , 1.971(2 ) ; li2n1(a ) , 2.057(2 ) ; n1c2 , 1.460(2 ) ; n2c1 , 1.458(2 ) ; c1c2 , 1.521(2 ) ; n1li1n2 , 81.23(9 ) ; n1li1n3 , 125.9(1 ) ; n1li1n4 , 129.1(1 ) ; n2li1n3 , 117.3(1 ) ; n2li1n4 , 125.26(1 ) ; n3li1n4 , 83.34(1 ) ; n1(a)li2n1 , 109.6(1 ) ; n1(a)li2n2 , 151.4(1 ) ; n1li2n2 , 86.7(1 ) .
the activation of ( ipr)nhch2ch2nh(ipr ) has also been achieved starting from the sodium congener na[(ipr)nch2ch2nh(ipr ) ] and ( tbu)2zn(tmeda ) to generate the sodium diazaethene zincate ( tmeda)na[(ipr)nch = chn(ipr)]zn(tbu ) 10 . although the initial cocomplexation adduct ( tmeda)na[(ipr)nch2ch2nh(ipr)]zn(tbu)2 with one n
h bond still remaining is apparently too short - lived to isolate , when the reaction was carried out at 0 c and the resulting solution stored at 30 c , it was possible to isolate and fully characterize the dimeric species { ( tmeda)na[(ipr)nch2ch2n(ipr)]zn(tbu)}211 with no n h bond but containing the saturated ethylene diamido backbone ( figure 10 ) which must exist further along the reaction pathway that ultimately produces 10 .
two independent centrosymmetric molecules of dimer 11 as well as a hexane molecule form the asymmetric unit .
sodium zincate 11 is constructed from a 545 fused ring system in a distorted ladder conformation ; the two znnccn rings lie anti about the strictly planar central znnznn core .
the n c [ 1.452(6 ) and 1.463(6 ) ] and c c [ 1.494(7 ) ] bond distances are indicative of a fully saturated bisamide . contrary to binding in the lithium monomer 5 , the heavier alkali metal binds to only one of the available amido groups , with the core nitrogen atoms ( n1 , n1a ) coordinating exclusively to zinc . despite the variety of amide environments around zinc , the zn
n bond distances show little variation ( average 2.11 ) . due to the narrow bite angle of the diamido ligand ( 85.9 )
the zinc center adopts a distorted tetrahedral geometry . a highly distorted tetrahedral geometry around sodium is also completed by a weak agostic interaction [ 2.977(6 ) ] to a methyl arm of the tertiary butyl ligand on zinc .
this is moderately longer than a similar interaction found in the bis - alkylamido zincate ( tmeda)na(tmp)zn(tbu)2 ( 2.75 ) .
545 fused ring systems have been found before in metallate complexes containing saturated nccn diamido ligands .
examples include the lithium lanthanide complexes [ ( thf)3li[1,2-{n(sime3)}2c6h10]mcl2]2 ( m = nd , yb ) and the dimeric gallium species [ { ( bz)nch2ch2n(bz)}li{(bz)nch2ch2n(bz)}gao]2 ( bz = benzyl , ch2c6h5 ) . molecular structure of one of the independent molecules of the dimeric sodium zincate [ ( tmeda)na[(ipr)nch2ch2n(ipr)]zn(tbu)]211 . a weak agostic nac contact is denoted by a broken bond .
hydrogen atoms , ipr groups , and disordered hexane of crystallization have been omitted for clarity .
selected bond lengths [ ] and angles [ deg ] : na1n1 , 3.720(4 ) ; na2n2 , 2.326(5 ) ; na1n3 , 2.490(6 ) ; na1n4 , 2.490(5 ) ; na1c4 , 2.977(6 ) ; zn1n1 , 2.105(4 ) ; zn1n2 , 2.103(4 ) ; zn1c1 , 2.072(5 ) ; zn1n1(a ) , 2.122(4 ) ; n1c8 , 1.463(6 ) ; n2c9 , 1.452(6 ) ; c8c9 , 1.494(7 ) ; n4na1n3 , 75.4(2 ) ; n4na1n2 , 121.9(2 ) ; n4na1c4 , 147.7(2 ) ; n3na1n2 , 137.7(2 ) ; n3na1c4 , 91.8(2 ) ; n2na1c4 , 87.4(2 ) ; n2zn1c1 , 117.1(2 ) ; n2zn1n1 , 85.9(2 ) ; n2zn1n1(a ) , 110.5(2 ) ; n1zn1c1 , 127.6(2 ) ; n1zn1n5 , 88.8(2 ) ; n5zn1c1 , 120.0(2 ) .
as might be expected from polarity considerations , the sodium reagent proves more reactive than the lithium analogue , providing after a 30 min reflux in hexane , the sodium diazaethene 10 in 61% isolated yield .
although the tmeda solvate 10 could be isolated as a crystalline solid , to obtain crystallographic data of adequate precision , the donor ligand thf had to be used in place of tmeda to produce the analogous thf solvate ( thf)3na[(ipr)nch = chn(ipr)]zn(tbu ) 10a ( figure 11 ) . here , the zinc atom is chelated symmetrically [ zn n bond distances 1.957(2 ) and 1.971(2 ) ] by the diamide ligand , lying below the plane defined by the nccn core of the diazaethene ligand .
the methine carbons of the isopropyl groups lie in the plane of the diazaethene ligand while their methyl groups protrude above and below this plane .
the sodium atom lies above the nccn bridge , though slightly off center , coordinating to both pairs of nitrogen and carbon atoms .
three thf molecules complete the coordination sphere of the sodium center . comparing the c n [ 1.398 ( average ) ] and c c [ 1.353(4 ) ] bond distances of the diazaethene core with those found in the lithium analogue ( tmeda)li[(ipr)nch = chn(ipr)]zn(me ) [ 1.400 ( average ) and 1.349(3 ) ] indicates that the change in alkali metal has little effect on the diazaethene ligand .
dissolution of 10a in c6d6 to perform an nmr spectroscopic analysis was achieved by the addition of a drop of d8-thf . comparing the h nmr spectra of the thf
solvate 10a with the tmeda solvate 10 reveals only marginal shift changes of the resonances associated with the diazaethene ligand . comparing the h nmr spectra of the homologues ( tmeda)li[(ipr)nch = chn(ipr)]zn(tbu ) 1 and ( tmeda)na[(ipr)nch = chn(ipr)]zn(tbu ) 10 reveals a 0.24 ppm downfield shift of the signal attributable to the ch = ch backbone of the diazaethene ligand on moving from lithium to sodium .
interestingly , there are two sets of doublets associated with the methyl groups of the isopropyls of the lithium compound but only one set in the sodium congener .
this would suggest sterically induced hindered rotation of the isopropyl groups in the lithium compound which is relieved on switching to the larger sodium cation .
the synthesis of several alkali metal zincate diazaethene complexes has been achieved previously . however , to the best of our knowledge , all previous examples were synthesized by the reduction of unsaturated bis - imino ligands .
therefore , the synthesis of the sodium zincate 10 , along with the lithium zincates detailed in our recent communication , appear to represent the first such complexes to be synthesized via a double ch activation of a saturated diamido ligand . molecular structure of the diazaethene sodium zincate ( thf)3na[(ipr)nch = chn(ipr)]zn(tbu ) 10a . hydrogen atoms and minor disordered thf components have been omitted for clarity .
thermal ellipsoids shown at the 50% probability level . selected bond lengths [ ] and angles [ deg ] : na1o1 , 2.350(2 ) ; na1o2 , 2.312(2 ) ; na1o3 , 2.312(2 ) ; na1n1 , 2.587(2 ) ; na1n2 , 2.662(2 ) ; na1c8 , 2.612(2 ) ; na1c9 , 2.649(2 )
; zn1n1 , 1.957(2 ) ; zn1n2 , 1.971(2 ) ; zn1c1 , 2.009(2 ) ; n1na1n2 , 60.81(6 ) ; n1zn1n2 , 85.14(8 ) ; n1zn1c1 , 139.88(10 ) ; n2zn1c1 , 134.97(10 ) .
conversions of saturated diamines into unsaturated diazaethenes are usually associated with redox - amenable transition metal catalysts .
however here we show through a combination of experimental ( nmr spectroscopic , x - ray crystallographic ) and theoretical ( dft ) studies that working co - operatively in a molecular mixed - metal system , an alkali metal ( lithium or sodium ) and zinc can bring about such transformations under challenging redox - inactive conditions .
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the surprising transformation of the saturated diamine ( ipr)nhch2ch2nh(ipr ) to the unsaturated diazaethene [ ( ipr)nch = chn(ipr)]2- via the synergic mixture nbum , ( tbu)2zn and tmeda ( where m = li , na ; tmeda = n , n , n,n-tetramethylethylenediamine ) has been investigated by multinuclear nmr spectroscopic studies and dft calculations .
several pertinent intermediary and related compounds ( tmeda)li[(ipr)nch2ch2nh(ipr)]zn(tbu)2 ( 3 ) , ( tmeda)li[(ipr)nch2ch2ch2n(ipr)]zn(tbu ) ( 5 ) , { ( thf)li[(ipr)nch2ch2n(ipr)]zn(tbu)}2 ( 6 ) , and { ( tmeda)na[(ipr)nch2ch2n(ipr)]zn(tbu)}2 ( 11 ) , characterized by single - crystal x - ray diffraction , are discussed in relation to their role in the formation of ( tmeda)m[(ipr)nch = chn(ipr)]zn(tbu ) ( m = li , 1 ; na , 10 ) . in addition , the dilithio zincate molecular hydride [ ( tmeda)li]2[(ipr)nch2ch2n(ipr)]zn(tbu)h 7 has been synthesized from the reaction of ( tmeda)li[(ipr)nch2ch2nh(ipr)]zn(tbu)23 with nbuli(tmeda ) and also characterized by both x - ray crystallographic and nmr spectroscopic studies .
the retention of the li h bond of 7 in solution was confirmed by 7li1h hsqc experiments . also , the 7li nmr spectrum of 7 in c6d6 solution allowed for the rare observation of a scalar 1jli h coupling constant of 13.3 hz .
possible mechanisms for the transformation from diamine to diazaethene , a process involving the formal breakage of four bonds , have been determined computationally using density functional theory .
the dominant mechanism , starting from ( tmeda)li[(ipr)nch2ch2n(ipr)]zn(tbu ) ( 4 ) , involves the formation of a hydride intermediate and leads directly to the observed diazaethene product .
in addition the existence of 7 in equilibrium with 4 through the dynamic association and dissociation of a ( tmeda)lih ligand , also provides a secondary mechanism for the formation of the diazaethene .
the two reaction pathways ( i.e. , starting from 4 or 7 ) are quite distinct and provide excellent examples in which the two distinct metals in the system are able to interact synergically to catalyze this otherwise challenging transformation .
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Introduction
Results
Conclusion
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sterilization in orthodontics has repeatedly been discussed and emphasized in the dental literature.[16 ] the orthodontic personnel are at a great risk of various contagious diseases and hepatitis b , acquired immuno deficiency syndrome ( aids ) and herpes infections are some of the serious conditions encountered in an orthodontic office .
therefore , the center for disease control ( cdc ) specifies that all instruments which are not expected to penetrate into soft tissue or bone but will certainly contact oral tissues must be sterilized , too . in cases in which barrier techniques are not used , cross - infection between the orthodontic personnel and patients is possible .
in addition , since orthodontic clinics have a large number of patients during the day compared to other dental clinics , each clinic requires its own custom - made sterilization processes .
that 12% of unused orthodontic wires are not sterile and produce bacterial colonies in a culture . therefore , the instructions on the packages generally recommended sterilization before use . at present ,
orthodontic wires are selected in different stages of the treatment procedure not only based on wire diameter , but also biomechanical and deactivation properties of wires in order to achieve the best treatment results .
-titanium ( tma ) wires are alloys of titanium and molybdenum , which were introduced in the early 1979 by goldberg and burstone for use in orthodontic treatment .
these wires are becoming more popular because of their excellent balance of mechanical properties and absence of nickel , resulting in better tolerance by many patients ; some of these mechanical properties include :
lower elastic modulus in comparison to stainless steelhigh formabilityability of direct weldinggood spring - backlow stiffnesslow force delivery[1116 ]
lower elastic modulus in comparison to stainless steel ability of direct welding low force delivery[1116 ] the limitation of -ti wires is their high coefficient of friction
. however , some manufacturers have used different methods to solve this problem , including incorporation of a titanium nitride and titanium oxide layer on the wire surface through ion implantation , changing their composition , highly polishing of the surface and increasing the surface smoothness.[71724 ] some previous studies have evaluated the effect of sterilization on wires with various alloy combinations ( -ti , niti , ss),[82528 ] with contradictory results in relation to -ti wires . in addition , after the expiration of patent on -ti wires by ormco ( glendora , california , usa ) various manufacturers produce these wires with different compositions and processing methods , resulting in different mechanical properties and different effects as a result of sterilization processes .
the aim of the present study was to evaluate the effect of sterilization with dry heat and steam on load - deflection characteristics of -ti wires , with different brands .
in the present study five different types of -titanium wires with a cross - section of 0.43 0.64 mm ( 0.017 0.025 inch ) were evaluated [ table 1 ] .
the wires were of the straight type . in cases in which only the preformed arch forms were available their straight posterior segments
a total of 30 wire segments from each type were provided ; each segment measured 30 mm in length .
-titanium wires tested in the present study group i was assigned as the control group .
the wire samples in group ii were sterilized by dry heat in an oven ( 12 bahman digital sterilizer , dsl 40 , isfahan , iran ) at 160c for 1 hour .
the wire samples in group iii were sterilized by steam in an autoclave ( melag euroclav , 23 v - s , berlin , germany ) at 121c and 15 psi for 15 minutes .
the wire samples were left to gradually cool to temperatures near the ambient temperature in the same sterilizers .
both process indicators and test strips were used to ensure the sterilization cycles and processes .
then the samples underwent a three - point load - deflection test in the dry state as described by miura et al .
the test was carried out using a universal mechanical testing machine ( model 1122 , instron corporation , canton , ma , usa ) using a specially designed setup , consisting of two metallic half - cylindrical structures placed parallel to each other .
a single maxillary first premolar bracket ( american orthodontics , master series , wis , usa ) , with a 0.022 0.028-inch slot size with zero angulation and zero torque was bonded on each of the half - cylinders [ figure 1 ] , with a distance of 14 mm between the brackets .
the wire sample to be tested was fixed on the brackets using 0.012-inch elastomeric ligatures ( ortho organizers , carlsbad , california , usa ) .
the center of each wire was deflected by moving a metallic pole adjusted on the upper head of the testing machine at a crosshead speed of 0.5 mm / min .
the load was recorded at 0.1-mm intervals from the inactive position up to 1.5 mm of activation and then returning to the zero point to achieve load - deflection characteristics of each wire .
load - deflection apparatus with the wire in place data was analyzed using repeated measures anova to compare load - deflection properties of the five types of -ti wires and determine the changes in characteristics during loading and unloading separately after sterilization of each wire type .
scheffe is a post - hoc test , done after anova test , to evaluate the differences between each two groups .
tables 2 and 3 present means and standard deviations of loading and unloading forces of different types of -ti wires after 1.5 mm of deflection in the three groups under study at 0.1-mm intervals .
figures 26 show load - deflection curves . means and standard deviations ( sd ) for loading forces at 0.1 mm intervals of deflection for wire samples in groups i - iii means and standard deviations ( sd ) for unloading forces at 0.1 mm intervals of defl ection for wire specimens in groups i - iii load - deflection graph of beta wires in the control group and after sterilization with dry and moist heat load - deflection graph of cna wires in the control group and after sterilization with dry and moist heat load - deflection graph of res wires in the control group and after sterilization with dry and moist heat load - deflection graph of hone wires in the control group and after sterilization with dry and moist heat load - deflection graph of tmal wires in the control group and after sterilization with dry and moist heat repeated measures anova showed that during loading , hone wire had the least force level ( p < 0.05 ) and res wire had the highest force level .
however , there were no statistically significant differences in force levels between res , cna and beta wires ( p > 0.05 ) .
no significant differences were observed in force levels between tmal and beta wires ( p > 0.05 ) .
the force levels of res and cna wires were significantly higher than that of tmal wire ( p < 0.05 ) . during unloading , tmal and hone wires exhibited the lowest force levels ( p < 0.05 ) ; although the force of tmal wire was a little less than that of hone wire
res wire exhibited the highest force level , which was significantly higher than those of beta , tmal and hone wires ( p < 0.05 ) .
although res wires exhibited force levels slightly higher than those of cna wires , there were no significant differences between the two wires ( p > 0.05 ) .
there were no significant differences in force levels between beta and cna wires ( p > 0.05 ) .
repeated measures anova revealed the following [ figures 26 ] : dry heat sterilization group ( ii ) exhibited the highest force levels during loading and unloading ( p < 0.05 ) .
the force levels in the group sterilized by steam ( iii ) were significantly higher than those in the control group ( i ) during both loading and unloading ( p < 0.05 ) .
the force levels of various groups of beta wires during loading and unloading were as follows : i < iii < ii .
the dry heat sterilization group ( ii ) exhibited the highest force levels during loading and unloading ( p < 0.05 ) .
there were more differences between groups i ( control ) and ii in the higher deflection ranges [ figures 3 and 4 ] .
there were no significant differences in force levels between the control ( i ) and steam sterilization groups during loading and unloading ( p > 0.05 ) .
the force levels of various res and cna groups during loading and unloading were as follows : iiii < ii . during loading ,
the dry heat sterilization group ( ii ) exhibited the highest force level ( p < 0.05 ) .
there were no significant differences in force levels between the control ( i ) and steam sterilization ( iii ) groups ( p > 0.05 ) . during unloading , the steam sterilization group ( iii ) had the highest force levels ( p < 0.05 ) .
there were no significant differences in force levels between the dry heat ( ii ) and control ( i ) groups ( p > 0.05 ) .
the force levels of different hone wire groups during loading and unloading had the following relationships , respectively : iiii < ii and iii < iii .
there were no significant differences in force levels during loading and unloading between groups i , ii and iii ( p > 0.05 ) ( iiiiii ) .
according to hysteresis evaluation ( energy loss upon unloading ) of wires before and after sterilization showed that only sterilization with dry heat resulted in an increase in hysteresis of tmal and hone wires ; however , autoclave sterilization did not have any effect on hysteresis of the wires under study .
repeated measures anova showed that during loading , hone wire had the least force level ( p < 0.05 ) and res wire had the highest force level .
however , there were no statistically significant differences in force levels between res , cna and beta wires ( p > 0.05 ) .
no significant differences were observed in force levels between tmal and beta wires ( p > 0.05 ) .
the force levels of res and cna wires were significantly higher than that of tmal wire ( p < 0.05 ) . during unloading , tmal and hone wires exhibited the lowest force levels ( p < 0.05 ) ; although the force of tmal wire was a little less than that of hone wire
res wire exhibited the highest force level , which was significantly higher than those of beta , tmal and hone wires ( p < 0.05 ) .
although res wires exhibited force levels slightly higher than those of cna wires , there were no significant differences between the two wires ( p > 0.05 ) .
there were no significant differences in force levels between beta and cna wires ( p > 0.05 ) .
repeated measures anova revealed the following [ figures 26 ] : dry heat sterilization group ( ii ) exhibited the highest force levels during loading and unloading ( p < 0.05 ) .
the force levels in the group sterilized by steam ( iii ) were significantly higher than those in the control group ( i ) during both loading and unloading ( p < 0.05 ) .
the force levels of various groups of beta wires during loading and unloading were as follows : i < iii < ii .
the dry heat sterilization group ( ii ) exhibited the highest force levels during loading and unloading ( p < 0.05 ) .
there were more differences between groups i ( control ) and ii in the higher deflection ranges [ figures 3 and 4 ] .
there were no significant differences in force levels between the control ( i ) and steam sterilization groups during loading and unloading ( p > 0.05 ) .
the force levels of various res and cna groups during loading and unloading were as follows :
the dry heat sterilization group ( ii ) exhibited the highest force level ( p < 0.05 ) .
there were no significant differences in force levels between the control ( i ) and steam sterilization ( iii ) groups ( p > 0.05 ) . during unloading ,
the steam sterilization group ( iii ) had the highest force levels ( p < 0.05 ) .
there were no significant differences in force levels between the dry heat ( ii ) and control ( i ) groups ( p > 0.05 ) .
the force levels of different hone wire groups during loading and unloading had the following relationships , respectively : iiii < ii and iii < iii .
there were no significant differences in force levels during loading and unloading between groups i , ii and iii ( p > 0.05 ) ( iiiiii ) . according to hysteresis evaluation ( energy loss upon unloading ) of wires before and after sterilization showed that only sterilization with dry heat resulted in an increase in hysteresis of tmal and hone wires ; however , autoclave sterilization did not have any effect on hysteresis of the wires under study .
dry heat sterilization group ( ii ) exhibited the highest force levels during loading and unloading ( p < 0.05 ) .
the force levels in the group sterilized by steam ( iii ) were significantly higher than those in the control group ( i ) during both loading and unloading ( p < 0.05 ) .
the force levels of various groups of beta wires during loading and unloading were as follows : i < iii < ii .
the dry heat sterilization group ( ii ) exhibited the highest force levels during loading and unloading ( p < 0.05 ) .
there were more differences between groups i ( control ) and ii in the higher deflection ranges [ figures 3 and 4 ] .
there were no significant differences in force levels between the control ( i ) and steam sterilization groups during loading and unloading ( p > 0.05 ) .
the force levels of various res and cna groups during loading and unloading were as follows : iiii < ii .
during loading , the dry heat sterilization group ( ii ) exhibited the highest force level ( p < 0.05 ) .
there were no significant differences in force levels between the control ( i ) and steam sterilization ( iii ) groups ( p > 0.05 ) . during unloading ,
the steam sterilization group ( iii ) had the highest force levels ( p < 0.05 ) .
there were no significant differences in force levels between the dry heat ( ii ) and control ( i ) groups ( p > 0.05 ) .
the force levels of different hone wire groups during loading and unloading had the following relationships , respectively : iiii < ii and iii < iii .
there were no significant differences in force levels during loading and unloading between groups i , ii and iii ( p > 0.05 ) ( iiiiii ) . according to hysteresis evaluation ( energy loss upon unloading ) of wires before and after sterilization showed that only sterilization with dry heat resulted in an increase in hysteresis of tmal and hone wires ; however , autoclave sterilization did not have any effect on hysteresis of the wires under study .
-ti wires have gained great popularity in orthodontics due to their favorable nature to tooth and supporting tissues .
multi - stage processing of orthodontic wires by manufacturers has a significant influence on their mechanical properties .
high autoclave or dry heat sterilizer temperatures , too , might influence the mechanical properties of wires .
therefore , it is important for the manufacturers to evaluate the effect of sterilization procedures on -ti wires .
heat sterilization techniques were selected in the present study because they are the most popular type of sterilization technique used in dental offices ; in addition , it is the most typical method recommended until now .
the load - deflection properties are significant parameters in determining the biologic nature of tooth movement .
a modified version of the three - point bending test was used in the present study to evaluate load - deflection properties of orthodontic wires .
this technique provides a simple model by determining the nature of forces applied during orthodontic treatment and closely simulates clinical situations . in the clinic ,
when a wire undergoes deflection to be placed within a bracket on a malaligned tooth , in fact , it undergoes loading . when a wire is inclined to return to its original shape , unloading takes place , which provides the necessary force to elicit a biologic tissue response , resulting in the alignment of teeth . however , laboratory tests do not necessarily reflect the clinical situations ; rather , they provide a basis for the comparison of various brands of wires .
of the five brands of wires under study [ tables 2 and 3 ] , the wires manufactured by ormco ( tmal and hone ) exhibited the lowest force during unloading , with tmal forces being higher than those of hone during loading , statically equal to those of beta wires .
therefore , hone wires , compared to the other types tested , are more easily engaged in the brackets of malaligned teeth due to lower force levels needed to deflect .
res and cna wires required higher force levels for deflection ; therefore , these wires exhibited greater stiffness in comparison to tmal and hone wires , which might be attributed to differences in composition and processing of the alloys used in the manufacture of each of these orthodontic wires .
given the curves and steepness of load - deflection graphs of the control wires in the present study [ figures 26 ] , it appears res , beta and cna wires have different behaviors compared to the two products from the ormco company ( tmal and hone ) .
these results are supported by those of kusy et al . who reported that the same wire manufacturer due to similar composition and surface roughness could have manufactured the -ti new products of res , beta iii and cna .
the original -ti alloy from the ormco company could have been manufactured with the same characteristics .
the results of the present study showed that dry heat sterilization significantly increases force levels during loading and unloading of cna , beta and res and loading of hone wires , making them stiffer .
as indicated by the increased slope of the graphs [ figures 25 ] dry heat sterilization influences the clinical performance of cna , res , beta and hone wires .
it also results in increased force delivery by cna , res and beta to teeth .
the differences in the effect of sterilization with dry heat and steam might be attributed to the use of longer times and higher temperatures in the sterilization process by dry heat .
however , sterilization with dry heat did not result in significant changes in force levels during loading and unloading of tmal wires [ figure 6 ] .
previous studies on the effect of dry heat sterilization on -ti wires have evaluated only tma wires ( ormco , glendora , california , usa ) with a size of 0.016 inch .
reported that use of dry heat sterilization with a temperature of 375f ( 191c ) for 10 minutes on tma archform wires ( ormco , glendora , california , usa ) which had already been used clinically did not exert any influence on tensile and load - deflection test results of the wires .
the results of the present study with tmal wires are consistent with the results of a study by smith et al . with tma wires
evaluated the effect of dry heat sterilization at 375f for 20 minutes on tma wires ( ormco , glendora , california , usa ) and reported a significant increase in the tensile strength of wires .
in addition to the type and size of the wires , other factors , which might have resulted in differences between the results of the present study and other studies , mentioned above might be the differences in the sterilization protocols and the type of the mechanical tests applied .
the results of the present study did not show any effect of steam sterilization ( autoclave ) [ figures 26 ] on load - deflection characteristics of the majority of -ti wires tested ; however , it increased force levels during loading and unloading of beta and unloading of hone wires , which is reflected by an increased slope of loading and unloading of beta and unloading of hone wires [ figures 2 and 5 ]
. therefore , these wires will deliver greater forces to teeth after sterilization in an autoclave .
regarding the low force levels of tmal wire , especially during unloading both in the control group and after sterilization , it appears ion implantation process is only related to surface characteristics and a decrease in friction , with no detrimental effects on load - deflection characteristics of the wire even after sterilization .
the different effects of sterilization on hone and tmal wires might be attributed to differences in the type and concentration of ions or other factors during the manufacturing processes of these wires . it is suggested that clinicians sterilize tmal , res and cna wires in an autoclave before using them without any worries because their favorable load characteristics will not undergo any changes after sterilization , which is consistent with the results of a study carried out by pernier et al .
on the effect of autoclave sterilization at 134c or 274f for 18 minutes on tma , tmal and res wires .
they reported no differences in load - displacement curves after sterilization of any wire tested .
reported no effect of autoclave sterilization ( 250f for 20 minutes ) on tensile strength of tma wires .
in addition , a study by smith et al . did not show any perceptible clinical differences between new wires and clinically used wires , which are later , sterilized in an autoclave ( 274f for 10 minutes ) .
the results of the present study showed differences in force levels during loading and unloading between the five brands of -ti wires under study after 1.5 mm of deflection ; res , cna and beta wires exhibited different force deflection behaviors in comparison with the two products of ormco company ( tmal and hone).there are different changes in load - deflection characteristics of various -ti wires after sterilization with dry heat and heated steam.the clinicians who want to provide maximum safety for their patients can sterilize res , tmal and cna wires in an autoclave without any worries before placing them in the oral cavity .
the results of the present study showed differences in force levels during loading and unloading between the five brands of -ti wires under study after 1.5 mm of deflection ; res , cna and beta wires exhibited different force deflection behaviors in comparison with the two products of ormco company ( tmal and hone ) .
there are different changes in load - deflection characteristics of various -ti wires after sterilization with dry heat and heated steam .
the clinicians who want to provide maximum safety for their patients can sterilize res , tmal and cna wires in an autoclave without any worries before placing them in the oral cavity .
however , further in vivo studies are required to substantiate the results of the present study .
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background : sterilization techniques could affect the characteristics of orthodontic wires .
the aim of the present study was to evaluate the effect of steam and dry heat sterilization techniques on load - deflection behavior of five types of -titanium alloy wires.materials and methods : the samples consisted of 30 straight lengths of five types of -titanium alloy wires : titanium molybdenum alloy ( tma ) low friction ( tmal ) , tma low friction colored ( hone ) , resolve ( res ) , betaforce ( beta ) , and beta cna ( cna ) .
thirty wire segments were divided into three groups of 10 .
group 1 was the control group and the group 2 samples were sterilized by dry heat in an oven ( 60 minutes at 160c ) and group 3 by steam in an autoclave ( 15 minutes at 121c ) . then all the wire samples underwent a three - point bending test in a testing machine to evaluate load - deflection properties .
data was analyzed by repeated measures anova and scheff 's test ( = 0.05).results : the results showed that dry heat sterilization significantly increased force levels during both loading and unloading of cna , beta and res and during loading of hone ( p < 0.05 ) .
steam sterilization significantly increased force levels during both loading and unloading of beta and during unloading of hone ( p < 0.05 ) , with no effects on the load - deflection characteristics of tmal , cna and res ( p > 0.05).conclusion : it appears dry heat sterilization increases stiffness of res , beta , cna and hone but autoclave sterilization did not have any effect on load - deflection characteristics of most of the -titanium wires tested , indicating that clinicians who want to provide maximum safety for their patients can autoclave tmal , res and cna before applying them .
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INTRODUCTION
MATERIALS AND METHODS
RESULTS
Load-deflection characteristics of the five types of -Ti wires
Effect of sterilization process on loading and unloading characteristics of -Ti wires of various brands
BETA wires
RES and CNA wires
HONE wire
TMAL wires
DISCUSSION
CONCLUSIONS
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the american marten ( martes americana ) is an arboreal mesocarnivore that ranges from the boreal forests of northern north america into coniferous and mixed coniferous / deciduous forests of the northern and northeastern united states , including the great lakes region ( clark et al . , 1987 ) .
the american marten was reintroduced to michigan 's upper peninsula ( up ) and northern lower peninsula ( nlp ) in the mid-20th century after regional extirpation due to habitat loss and over - harvest ( cooley et al . , 2004 ) .
over 200 animals were reintroduced to the up over the course of several reintroductions . many fewer animals ( n = 36 ) were reintroduced in the huron - manistee national forest of the nlp and 49 martens were reintroduced to the pigeon river state forest of the nlp .
in contrast , the species is considered a regional forester sensitive species and there is no harvest in the nlp ( usda forest service , 2012 ) .
factors hypothesized to be contributing to the differences in population sustainability between the up and nlp include differences in habitat , genetic diversity , health and others . at the time of the original reintroduction
, american marten were not examined for parasitic or infectious diseases ( spriggs , unpublished data ) .
some parasites are of economic or zoonotic importance and may be introduced with animal translocations .
therefore , reintroduction programs should take into account the presence of parasites which are pathogenic or to which the species of concern is not adapted ( kimber and kollias , 2000 ) .
a collaborative research effort has begun in order to investigate factors that may be contributing to the difference in sustainability between the up and nlp populations .
the aim of this parasite survey was to describe the presence and prevalence of parasites in the huron - manistee national forest of the nlp and to determine whether there are differences in presence or prevalence of parasites between the nlp and up .
should future translocation of animals from the up into the nlp be considered for management of the species , a non - invasive and inexpensive method for screening animals would be desirable . also , american marten are not harvested in the nlp and thus adequate numbers of carcasses are not available for examination .
while some information exists regarding the prevalence of endoparasitism in american marten in north american , relatively little information is available for the species in michigan ( poole et al . , 1983 , veine - smith et al . , 2011 ) .
this study reviews previous parasite prevalence reports from american marten in north america , presents data from the parasitological examination of live - trapped american marten in michigan , and identifies a possible association between hookworm infection and anaemia in affected american marten .
american marten ( n = 49 ) were sampled from the manistee national forest in the nlp ( n = 31 ) , hiawatha national forest in the up ( n = 13 ) and ottawa national forest in the up ( n = 5 ) .
american marten were trapped and immobilized from 2011 to 2015 as described by desmarchelier et al .
nine american marten were recaptured and re - sampled , resulting in a total of 60 faecal samples included in analyses .
blood was collected from the jugular vein and placed into lithium heparin anticoagulant ( bd microtainer tubes , becton dickinson and company ) .
we determined haematocrit using microhematocrit capillary tubes ( safecrit ) and the statspin vt centrifuge ( iris ) .
individuals were identified as anaemic if the haematocrit was < 42% based on a report of normal haematocrit in captive , fed american martens of 47 5% ( nieminen et al . , 2007 ) .
other health parameters were collected as part of a complete health assessment ( spriggs , unpublished data ) .
faecal float and sedimentation examinations were performed at michigan state university 's diagnostic center for population and animal health using standard methods ( zajac and conboy , 2012 ) .
faecal flotation was performed using sheather 's sugar solution ( specific gravity 1.251.27 ) alone ( n = 30 ) from may 2011may 2012 or with both sheather 's sugar solution and zinc sulfate solution ( specific gravity 1.18 , n = 30 ) from june 2012january 2015 and examined by light microscopy .
fifty - nine of the samples , representing 48 individual animals , were sufficient in quantity for faecal sedimentation procedure .
ova were identified based on morphologic characteristics including size and in accordance with parasitologic references and previous reports . upon consultation with a wildlife veterinary parasitologist ( gerhold ) , 2 nematode species ( syphacia muris and aspicularis sp . )
prevalence was calculated as the number of infected hosts divided by the number of hosts examined . because some american marten were sampled more than once , all parasite species found in an individual were considered together for prevalence calculations .
differences between locations ( up and nlp ) and sexes were examined with a pearson 's test with p < 0.05 considered significant .
significant differences between presence or absence of anaemia and hookworm infection were also examined with a pearson 's test .
parasite species richness by host was calculated as the number of parasite species present per host ; species richness by sample was calculated as the number of parasite species present per sample .
an unidentified capillarid species was not included in the prevalence or host species richness if one or more species of capillaria was identified in other samples from the same host .
thus , an individual american marten found with unidentified capillarid at the initial exam and aonchotheca sp . at a subsequent exam
if both samples had unidentified capillaria , then the host was included in the calculation of prevalence of unidentified capillaria .
differences between sex and location in species richness were examined with a wilcoxon rank sum test , with p < 0.05 considered significant . the capture and handling protocol
was approved by the university of tennessee animal care and use committee ( protocol # 2180 ) , and american marten live - trapping and sample collection was an authorized tribal activity under the 2007 inland consent decree between the state of michigan and the little river band of ottawa indians .
sixty samples from 49 individual american marten ( 28 males , 21 females ) were examined , and results are shown in table 1 .
of 49 individual american marten examined , 91.8% were positive for 1 or more parasites and 69.4% were infected with 2 or more parasites .
there was no significant difference in mean species richness by host between sexes or locations ( nlp and up ) .
trematode egg identification was suspected but not confirmed to be alaria sp . during the early part of the study ( may 2011may 2012 ) when sheather 's sugar solution alone was used for flotation , as alaria species may be distorted due to the osmotic pressure of sugar solution . later in the study
( june 2012january 2015 ) , trematode ova found on sedimentation were confirmed to be alaria species when the sample was floated with zinc sulfate solution , in which trematode ova will not be distorted .
once the use of zinc sulfate solution was implemented , no trematode other than alaria species was identified , and the authors concluded that trematode eggs identified in the early samples were most likely alaria species , as suspected . in the up , 3 samples that were positive on sedimentation for trematode ova were suspected to be alaria species and 7 were confirmed via zinc sulfate flotation ; in the nlp , 14 samples positive on sedimentation were suspected to be alaria species and 5 were confirmed via zinc sulfate flotation .
capillaria eggs were seen in 79% and 78.1% of samples from the up and nlp , respectively .
capillaria eggs were further identified as eucoleus aerophila , eucoleus boehmi , or aonchotheca putorii based on size and morphologic characteristics . a hookworm egg is shown in fig . 1 .
there was no significant difference in prevalence of any of the identified parasites between male and female american marten .
of 9 american marten that were sampled more than once , only one had identical results for each time point .
the mean haematocrit was 45.6 8.1 ( range 3068 ; n = 49 ) .
haematocrit was reported to be 47 5% in the only other report of american marten haematocrit ( nieminen et al . , 2007 ) . using < 42% as a cut - off
american marten infected with hookworms were significantly more likely to be anaemic than non - infected american marten ( p = 0.01 ) with an odds ratio of 8.75 ( 95% confidence interval : 1.456.4 ) .
parasite species richness per host was similar to that reported by veine - smith et al .
we identified more parasite species in the nlp than the up , but this result may be a function of the larger sample size from the nlp , and there was no significant difference in richness between the 2 locations .
foreyt and langerquist ( 1993 ) found 2 or more parasites in 35% of american marten from eastern washington . in another survey from washington ,
9 helminth species were found , and 48.4% of hosts had coinfection of 2 or more parasites , with a maximum of 4 ( hoberg et al . , 1990 ) .
overall , parasite prevalences were similar between the up and nlp in our samples and we conclude that the up may be suitable source for a translocation of marten to the nlp from the point of view of parasite translocation .
additional screening of both populations , the use of molecular techniques to definitively speciate parasites , and testing of individual martens marked for translocation are recommended .
parasites in our study were identified to the genus and/or species level as possible by microscopic examination alone .
future research could employ the use of molecular tools such as polymerase chain reaction to provide more specific results .
such techniques were not used in this study because we desired an inexpensive method that could be repeated in any future reintroduction programs that require screening of large numbers of animals .
parasites identified as a concern for reintroduction of north american river otter included alaria canis , strongyloides lutrae , crenosoma goblei , capillaria and coccidia due to potential for pathogenic effects or high prevalence ( kimber and kollias , 2000 ) .
river otter reintroduction programs tested and treated parasitized otters , and similar methods could be considered for reintroduction of american marten ( hoover et al . , 1985 , griess , 1987 , serfass et al . ,
of 9 american marten that were sampled more than once , only one had identical results at subsequent sampling , indicating either a change in infection status or inconsistent shedding of ova in the remaining 8 marten .
alaria species are flukes found in the small intestine of definitive hosts including felids , canids and mustelids .
alaria mustelae is known to infect mink ( mustela vison ) and short - tailed weasels ( mustela erminea ) , as well as american marten ( veine - smith et al . ,
alaria taxidae was identified from 25% ( n = 6 ) of american marten from the district of mackenzie , northwest territories and was identified in manitoba , canada , at prevalences ranging from 36 to 73% depending on the area ( holmes , 1963 , poole et al . , 1983 ) .
a definitive host infected with alaria species in its intestines sheds eggs in its faeces .
after 2 weeks in wet soil or water , the eggs hatch , producing a miracidium .
the miracidium invades a freshwater snail , at which point it develops into a cercaria .
when the tadpole is ingested by an amphibian , reptile , or rodent , the mesocercariae remain in the tissues of the paratenic host .
the mesocercariae migrate through the stomach , across the diaphragm and into the lungs where the mesocercariae develops into metacercariae .
the metacercariae are able to travel up the trachea and are then swallowed , at which point they develop into adult trematodes in the small intestines . while infection with alaria species typically does not cause disease in the definitive host , there are reports of the mesocercariae causing neurologic disease due to aberrant migration through the central nervous system and of respiratory illness due to migration through the lungs in domestic dogs ( kimber and kollias , 2000 , kazacos , 2001 )
. other species of trematodes have caused disease in north american river otters , but the pathogenicity of alaria in american marten is not known ( kimber and kollias , 2000 ) .
if a pregnant female rodent or carnivore becomes infected with alaria , the mesocercariae can migrate to the mammary glands and can be transmitted to nursing young ( bowman et al . ,
not yet weaned , in the nlp , histopathology revealed infection with alaria species in the duodenum , confirming the potential for lactogenic transmission of mesocercariae in this mustelid . in the report by veine - smith et al .
( 2011 ) , faeces from the large intestines of 140 american marten carcasses from the up were examined for flukes using a sedimentation technique and found a prevalence of 39% ( n = 54 ) . the difference in prevalence between that report and ours ( 55.6% , n = 10 ) may be due to differences in sample size , methodology or a true difference ( veine - smith et al . , 2011 ) .
we recommend the use of both faecal sedimentation and zinc sulfate flotation for identification of trematode eggs in faecal samples obtained from live animals .
euryhelmis squamula has been reported in raccoons ( procyon lotor ) , mink , and american marten in washington and uses amphibian intermediate hosts in this region ( hoberg et al .
american marten from washington were documented with 6% prevalence from the southern cascades , confirming that marten are ingesting anuran prey in this area ( hoberg et al . ,
e. squamula has been reported in mink in north america and is a common parasite of the polecat in europe ( ameel , 1938 , miller and harkema , 1964 ) . a related parasite ,
the mink is the natural host for euparyphium beaveri in michigan , while euparyphium inerme has been reported to infect river otters in the pacific northwest ( miller and harkema , 1964 , hoberg et al . , 1997 ) .
hymenolepis nana is a zoonotic cestode of rodents , carnivores and humans found worldwide , but other species of hymenolepis infect galliformes , including potential american marten prey species .
the parasite may use intermediate hosts or paratenic hosts , including dung beetles , stable flies and fleas ( drew , 2003 , joslin , 2003 , loomis , 2003 , sainsbury , 2003 ) . without knowing the species of hymenolepis found from the american marten in the nlp
, it is not known whether this may have been a pass - through finding or was truly an infection of the american marten ; the zoonotic potential is not known .
taenia species are cestodes ( commonly known as tapeworms ) that maintain a completely sylvatic life cycle . the definitive hosts for taenia mustelae and taenia martis americana are primarily mustelids .
adult parasites live in the small intestine and eggs are passed in the faeces of the host .
the ingested larvae form cysticercus in skeletal muscle and viscera , and the life cycle is completed when a carnivore consumes the infected intermediate host .
while the definitive host typically shows no signs of disease , the intermediate host may suffer morbidity or mortality , including liver damage , as a result of infection ( jones and pybus , 2001 ) .
t. mustelae has a wide distribution across the northern hemisphere and has been reported in north american mustelids , including american marten , short - tailed weasel ( mustela erminea ) , mink ( neovison vison ) and least weasel ( m. nivalis ) .
the cestode has been found in 2 sciurid definitive hosts , marmota broweri and m. caligata ( jones and pybus , 2001 ) .
taenia martis americana infects mustelids , including american marten , fisher ( martes pennanti ) and the ringtail ( bassariscus astutus , family : procyonidae ) , as definitive hosts .
rodents in north america reported with the larval stage of infection include lemmus sibiricus , microtus xanthognathus , mus musculus and ondatra zibethicus ( jones and pybus , 2001 ) .
two american marten from the southern cascades were co - infected with both t. mustelae and t. martis americana ( hoberg et al .
adult parasites are found in the small intestine of definitive hosts , which include canids , felids and mustelids ( bowman et al . ,
larval or adult parasites can also infect birds , reptiles and other mammals ( wardle and mcleod , 1952 ) .
eggs , or gravid proglottids , are suspected to be ingested by a first intermediate host , a coprophagic insect or a mite ( chowdhury and aguirre , 2001 , bowman et al . ,
the insect is consumed by a second intermediate host , which may include birds , mammals , reptiles and amphibians .
lastly , the definitive host becomes infected by ingesting the second intermediate host ( bowman et al .
definitive hosts may have clinical signs of infection including anorexia , low serum albumin and vomiting ( chowdhury and aguirre , 2001 ) .
humans can be incidental definitive hosts and become infected by consuming undercooked game ( chowdhury and aguirre , 2001 , fuentes et al .
mesocestoides lineatus was identified in the small intestine of 33% ( n = 14 ) of american marten from eastern washington with significantly higher rates in juveniles than in adults .
current taxonomy , however , suggests that this species may have actually been m. variabilis , which occurs in north america , while m. lineatus is an old world species ( fuentes et al . , 2003 ) .
coinfections with m. lineatus and capillaria putorii occurred in 35% of parasitized american marten , and juveniles had statistically higher rates of coinfection than adults ( foreyt and langerquist , 1993 ) .
hookworms are zoonotic nematode parasites infecting carnivores ( taylor et al . , 2007 ) .
carnivores may become infected via ingestion of larvae or eggs , paratenic hosts , or via percutaneous or lactogenic transmission .
larvae migrate to the lungs , moult , and are coughed up and swallowed to lay eggs in the small intestine . ingested larvae may bypass pulmonary migration or migrate out of the lungs into the muscle and remain in an infected female mammal until pregnancy occurs , at which point the larvae migrate to the mammary gland leading to lactogenic transmission ( taylor et al . , 2007 ) .
hookworm infection in dogs and foxes can result in bloody diarrhoea , anaemia , poor hair coat , poor growth in puppies and respiratory signs ( taylor et al . , 2007 ) .
we found that the odds of having anaemia ( haematocrit < 42% as described by nieminen et al .
, 2007 ) were 8.75 higher for american marten infected with hookworms than in uninfected american marten in this study . while there was a significant association between presence of hookworm infection and presence of anaemia in this report , other clinical signs related to hookworm infection
in addition , molecular techniques could be used to determine the species of parasite infecting american marten in michigan . because of the potential for anaemia to affect fitness of an individual american marten or the growth of kits infected via transmammary transmission , treatment of hookworm - infected american marten destined for relocation may be warranted .
( previously known as capillaria putorii ) infects the gastrointestinal tract of mustelids and other species , while other capillarids infect the respiratory tract , bladder or liver of their respective definitive hosts ( bowman et al .
a. putorii can have a direct or indirect life cycle in which adult parasites shed eggs in the gastrointestinal tract of the mammalian host .
the eggs are capable of infecting other susceptible hosts directly or using an earthworm as an intermediate host ( segovia et al . , 2007 , taylor et al . , 2007 )
a. putorii was found in 77.8% ( n = 14 ) of american marten from the up in the current report , which was significantly higher than the 29.0% ( n = 9 ) prevalence in the nlp ( p < 0.05 ) .
however , during the initial stage of this parasite survey , some capillaria ova were not identified to species , and these are represented as
if the 42.0% ( n = 13 ) prevalence of unidentified capillaria found in the nlp were in fact a. putorii , then there would not be a significant difference in prevalence of a. putorii between the up and nlp .
the prevalence of a. putorii from the up in this report is higher than the 47% ( n = 66 ) previously reported from american marten carcasses from the up ( veine - smith et al . , 2011 ) .
a. putorii has also been reported in the stomach of ferret , mink , short - tailed weasel , raccoon , fisher and striped skunk ( mephitis mephitis ) and in the small intestine of bobcats , bears , raccoons , swine , hedgehogs and the domestic cat ( foreyt and langerquist , 1993 , bowman et al . , 2009 ) .
in northeastern washington , c. putorii was found in 86% ( n = 13 ) of american marten carcasses , mostly in the stomach and less frequently in the large or small intestine of american marten .
using faecal flotation alone , capillaria species ova were found in 64% ( n = 21 ) of samples examined from the same population ( foreyt and langerquist , 1993 ) .
additional future comparisons between carcass and faecal examinations to determine prevalence of capillaria and other parasites is warranted .
eucoleus aerophilus , previously known as capillaria aerophila , infects the respiratory tract of hosts including american marten and other carnivores , such as fisher , red fox ( vulpes vulpes ) , raccoon , coyote ( canis latrans ) , striped skunk and badger ( taxidea taxus ) ( bowman et al . , 2009 ) .
e. aerophilus rarely infects humans ( laloevi et al . , 2013 ) .
the life cycle of e. aerophilus may be direct or indirect with earthworms serving as intermediate hosts ( bowman et al .
aerophilus can lead to respiratory disease and clinical signs include coughing , wheezing , failure to thrive , pneumonia and even death .
cats and dogs have also been infected but typically do not suffer the same degree of clinical signs as foxes since their infections are not as intense ( bowman et al . , 2009 ) .
e. aerophilus was identified in 4% of the respiratory tracts of american marten from ontario , but it is unknown whether infection resulted in disease or increased risk of being trapped ( seville and addison , 1995 ) .
eucoleus boehmi has been reported to infect the respiratory tract of foxes and dogs ( bowman et al . , 2009 ) .
its ova can be differentiated from the similar e. aerophilus by its pitted surface ( bowman et al . , 2009 ) .
the relative contribution of earthworms to the american marten diet in michigan is not known but may be significant given the high overall prevalence of capillarid parasites seen there .
physaloptera species are found in the stomach of infected carnivores , including mink , striped skunk , raccoons , dogs , and cats , and eggs are shed intermittently ( chowdhury and aguirre , 2001 , veine - smith et al . , 2011 ) .
crickets , beetles or other invertebrates act as intermediate hosts , while rodents and reptiles may be paratenic hosts ( chowdhury and aguirre , 2001 ) .
while most infections do not cause disease in the host , severe ulcerative gastritis has been reported in the bandicoot , a marsupial ( perameles species ) ( holz , 2003 ) .
crenosoma species is a lungworm found within the respiratory tract of carnivores and insectivores ( craig and anderson , 1972 ) .
adult parasites lay eggs in the lungs ; larvae are coughed up , swallowed , and passed in host faeces .
heavy infection with crenosoma species can cause clinical signs such as coughing , sneezing , nasal discharge and difficulty breathing ( chowdhury and aguirre , 2001 ) .
the red fox is the typical host for crenosoma vulpis and is sympatric with american marten in both the up and nlp . given the global distribution of c. vulpis , it is likely that this parasite exists in the nlp red fox population although the parasite was not identified in american marten from the nlp in this study .
crenosoma petrowi has been reported from free - ranging russian sable , a captive fisher in the united states and a badger from canada ( craig and anderson , 1972 ) .
a single american marten from ontario was found to be infected with crenosoma petrowi ( < 1% prevalence ) , but the reported prevalence in fisher from the same region was 15% ( seville and addison , 1995 ) .
olsen ( 1952 ) examined 62 carcasses of martes caurina from colorado and found 18 ( 29% ) to be infected with crenosoma , which he designated crenosoma coloradoensis .
crenosoma species was found in the lungs of 2% ( n = 3 ) of american marten from the up of michigan ( veine - smith et al . , 2011 ) .
strongyloides martis and s. lutrae have been reported in river otters ( hoberg et al . , 1997 ) .
parasites of this genus are generally species or host - specific and undergo both a direct life cycle and a free - living stage .
infective larvae or eggs in the soil are consumed by the host ; larvae of some species can also enter the host through the skin ( morris and shima , 2003 ) .
pathogenicity of strongyloides species in mustelids is not known , but disease could result from migration of the parasite through the lung ( kimber and kollias , 2000 ) .
dioctophyme renale , commonly known as the giant kidney worm , is one of the largest roundworms infecting wild and domestic species worldwide including wolves , bears , foxes and mink , as well as domestic dogs , cattle , horses and pigs .
humans have also been reported with d. renale ( chowdhury and aguirre , 2001 ) .
the adult worm is typically found in the right kidney because the infective larvae exits the intestinal tract on the right side near the stomach .
when the parasite dies , the kidney is essentially destroyed , and the host becomes reliant on the remaining left kidney . occasionally , both kidneys are infected or the parasite is found elsewhere in the abdomen .
an adult female worm lays eggs within the kidney , and the eggs are shed in the urine of the host mammal .
it takes about 6 months for the egg to become infective , at which point it may be swallowed by the intermediate host , lumbriculus variegatus , an aquatic annelid commonly known as blackworm .
if the annelid is eaten by an american marten , or other mammalian host , the life cycle is completed when the larvae finds its way to a kidney .
fish , frogs and crayfish may act as paratenic hosts by consuming the infected annelid ( cheng , 1986 , chowdhury and aguirre , 2001 ) .
a single american marten from the up was reported with nephritis due to suspected prior infection with d. renale ( spriggs , unpublished data ) . in an examination of 405 american marten from ontario ,
d. renale was found in only 2% of american marten and only from districts with previous reports of infected mink . in 4 of the 5 infected american marten
, there was evidence only of past infection such as the entire right kidney missing or merely a fibrous capsule remaining , while in 1 american marten the actual parasite was identified ( seville and addison , 1995 ) .
filaroides martis is a helminth parasite found in the trachea , bronchi and lungs and has been reported to infect mustelids , including mink and american marten , as well as canids ( chowdhury and aguirre , 2001 ) .
the larvae moults in the stomach mucosa of the definitive host and migrates to the thoracic cavity over the next month .
larvae increase in size over 100-fold during this timeframe ( ko and anderson , 1972 ) .
infection with f. martis has been reported to cause pneumonia in other species , but its effect on american marten is not known ( chowdhury and aguirre , 2001 ) .
of 405 american marten examined from ontario , 8% had lung or aortic nodules associated with the parasite . because yearlings had a significantly higher prevalence of infection than other ages ,
the authors suggest that infection could have an effect on survival of yearlings or that american marten are able to recover from infection at older age groups ( seville and addison , 1995 ) .
f. martis was found in the lungs of 4% ( n = 5 ) of american marten carcasses from the up of michigan ( veine - smith et al . ,
histopathology revealed lesions consistent with verminous pneumonia in 60% ( n = 9 ) of american marten carcasses from michigan ( spriggs , unpublished data ) . while these were incidental findings and the causative parasite was not identified
, it is possible that pneumonia may have resulted in mild respiratory compromise in affected american marten .
pearsonema plica , previously known as capillaria plica , was identified in the urinary bladder of 6% of american marten from ontario ( seville and addison , 1995 ) .
p. plica has been reported in the urinary tract of the domestic cat , dog , raccoon , red fox , coyote , wolf , striped skunk and fisher , as well as american marten ( butterworth and beverley - burton , 1980 ) .
the definitive host begins to shed eggs of c. plica in the urine about 8 weeks after consuming an earthworm , the paratenic host ( bowman et al . , 2009 ) .
infection usually does not cause disease for the host , but there is a suggestion that p. plica resulted in poor growth in fox kits ( bowman et al . ,
baylisascaris devosi is a nematode reported in both american marten and fisher ( kazacos , 2001 ) .
eggs shed from the definitive host become infective after 1114 days , at which point small mammals such as rodents and squirrels become infected by ingesting the eggs while foraging .
the larvae migrate throughout the tissues of these paratenic hosts and typically localize to the muscle of the forelimbs and thorax .
neural larval migrans , a neurologic disease , is rare or non - existent with b. devosi ( kazacos , 2001 ) .
in contrast , the larvae of the related baylisascaris procyonis , or raccoon roundworm , frequently migrate to the central nervous system of non - adapted hosts and cause neural larval migrans .
humans are susceptible to neural larval migrans caused by baylisascaris species , but b. devosi is less likely to cause disease in humans than b. procyonis ( kazacos , 2001 ) .
adult b. devosi inhabits the small intestine ; the definitive host does not typically show signs , but intestinal blockage is possible with a severe infection ( chowdhury and aguirre , 2001 ) . in 42 m. caurina carcasses examined from idaho ,
one was infected with b. devosi ( erickson , 1946 , poole et al . , 1983 ) .
soboliphyme baturini , commonly known as stomach worm , is a nematode parasite distributed from central siberia across beringia to the pacific northwest of the united states ( koehler et al . , 2009 ) .
other mustelids reported with the parasite include ermine ( m. erminea ) , mink and ferrets ( mustelo putorius furo ) ( levine , 1968 , swartz , 1968 , koehler et al . , 2009 ) .
s. baturini has been reported in other carnivores , including the fox and domestic cat ( levine , 1968 ) .
mature female worms are found in the stomach or small intestine of the mustelid host , and eggs are passed in the faeces of the host .
earthworms are the intermediate host , while shrews become paratenic hosts when they ingest the earthworm .
american marten may be infected by consuming either infected shrews or earthworms ( koehler et al . , 2009 ) .
( 2009 ) used genetic molecular data of s. baturini to shed light on the expansion of the ancestral american marten across beringia into north america , its speciation during isolation in glacial refugia , and re - colonization in alaska and reinfection with s. baturini .
clinical manifestation of infection in sable includes anaemia and gastric ulceration ( thomas et al . , 2008 ) .
there was a 55% ( n = 155 ) prevalence of infection with s. baturini in american marten from prince of wales island in alaska ( table 3 ) .
there was no correlation between intensity of s. baturini infection and fat deposits which measured to assess nutritional condition ( thomas et al .
american marten carcasses were collected over an 8-year time period from 3 locations in alaska and stomachs were examined for s. baturini ( table 3 ) .
none of the american marten in that study had any sign of negative health impact from the parasite infection ( zarnke et al . , 2004 ) .
a study conducted in idaho examined 42 m. caurina carcasses and found one with s. baturini ( erickson , 1946 ) .
it is found worldwide except in antarctica and australasia ( chowdhury and aguirre , 2001 ) .
adult t. spiralis are found in the small intestine , and the females give birth to larvae which migrate through the body to become encysted in skeletal muscle .
an american marten may become infected by consuming a rodent or other mammal with the encysted parasite .
once consumed , the larvae are freed and migrate to the small intestine of the host to complete their life cycle .
humans can acquire trichinellosis by eating undercooked meat ( taylor et al . , 2007 ) .
the first report of t. spiralis in american marten in north america was from manitoba , canada , where a single yearling was found infected of 139 american marten examined ( poole et al . , 1983 ) .
prevalence of trichinella in american marten and other species may vary from year to year ( dick et al . , 1986 ) .
american marten from the northern and southern cascades of washington were found to have trichinella encysted in the diaphragm , with a prevalence of 50% and 31% , respectively ( hoberg et al . , 1990 ) .
another study examined tongue muscle from 42 american marten from northeastern washington and found only 5% prevalence , which the authors attribute to differences in technique and tissues examined ( foreyt and langerquist , 1993 ) .
a study conducted in ontario found a 3.4% prevalence ( n = 68 ) of infection with t. spiralis in american marten ( dick et al . , 1986 ) .
the prevalence in fishers in the study was slightly higher at 4.5% ( n = 83 ) , and a single mink ( of 12 tested ) was positive .
the authors suggested that american marten and fishers are key in the sylvatic transmission of trichinella in this part of canada ( dick et al . , 1986 ) .
other reports of t. spiralis in american marten are found in table 3 .
( 1986 ) in which only mustelids were found with t. spiralis , this study found a high prevalence in coyote ( 61% , n = 22 ) and confirmed infection in a variety of other mammals including carnivores and rodents ( schmitt et al . , 1976 ) .
dracunculus insignis is a parasite known to infect raccoons , dogs , mink , fishers and skunks in the united states east of the rocky mountains and in ontario , canada ( crichton and beverly - burton , 1973 , cheng , 1986 ) .
experimental infections in ferrets are used as a model for human dracunculiasis ( eberhard et al . , 1988 ) .
dracunculus lutrae infects otters in north america ( crichton and beverly - burton , 1973 ) .
both species have a similar life cycle to the related the old world parasite didelphis medinensis , commonly called the guinea worm , a zoonotic parasite .
female d. lutrae migrate to the tissues under the skin of the animal to give birth to live larvae while secreting a substance that causes a blister to form .
when the blister ruptures and is exposed to water , the infective larvae is released into the water .
the free - living larvae can survive for several days until they are consumed by an aquatic copepod , the intermediate host .
d. insignis develops through several more stages within the copepod over the next 3 weeks .
when a definitive host ingests the copepod while drinking water , the larvae are freed in the stomach or small intestine and migrate to abdominal organs and tissues where they continue to develop into adult worms over the next 812 months ( cheng , 1986 ) .
d. insignis was found in only a single american marten of 405 examined in ontario despite the parasite being commonly found in raccoons of the same region .
the authors suggested a higher true prevalence , but detection was low due to the pelt being removed for commercial reasons in most of the examined specimens ( seville and addison , 1995 ) .
this parasite is of economic concern as it can affect pelt quality in fur - bearer species .
a single american marten from the nlp was found to be shedding sarcocystis species sporocysts .
sarcocystis species is a protozoal parasite that has infrequently been reported in carnivores such as domestic cats , dogs , raccoons , cougars , bobcats , mink , striped skunks , sea otters , fishers and pacific harbor seals ( foreyt and langerquist , 1993 , gerhold et al . , 2005 , larkin et al . , 2011 ) .
( 2005 ) reported a case of meningoencephalitis in a fisher caused by sarcocystis neurona in maryland , usa . the virginia opossum ( didelphis virginiana ) is the definitive host for the parasite in north america , but the natural intermediate host for s. neurona has not been discovered ( gerhold et al . , 2005 ) .
sarcocystis species was found in the tongues of 10% ( n = 4 ) of american marten examined from northeastern washington ( foreyt and langerquist , 1993 ) .
antibodies to toxoplasma gondii were detected in 10.8% ( n = 15 ) of american marten in ontario .
american marten in michigan were found to have a 58% seroprevalence ( n = 47 ) , and there was no significant difference between the up and nlp ( spriggs , unpublished data ) .
t. gondii has been reported to cause mortality in the related black - footed ferret ( mustela nigripes ) , captive - raised american mink ( neovison vison ) and southern sea otters ( enhydra lutris ) ( dubey et al . ,
2003 , burns et al . , 2003 , jones et al . , 2006
coccidian parasites have been only rarely reported in american marten , but this is likely because few studies have used faecal flotation and/or histopathology to detect parasites .
all coccidian parasites are obligate , intracellular parasites and undergo stages of asexual and sexual reproduction in the life cycle , which may be direct or indirect .
coccidian oocysts from 5 american marten in this report were sporulated to allow identification to the genus level .
eimeria species were identified in 2 of the 5 sporulated samples from american marten in the current study , and cystoisospora species in the remaining 3 samples .
it is unknown whether coccidian oocysts seen in faecal samples were pass - through from prey species .
taxonomy of coccidian parasites has been controversial ; some have considered cystoisospora species to be synonymous with isospora species , while others believe it to be a distinct genus based on molecular and morphological characteristics ( yi - fan et al . , 2012 ) .
cystoisospora species were identified in american marten from michigan in this report based on morphological features of the sporulated oocysts .
( 2012 ) reported cystoisospora species in steppe polecats in china and suggested that previously reported isospora species in various other mustelids , including that of sable , should be reassigned to cystoisospora species .
faeces from 33 american marten from eastern washington were examined , and 6% were found with coccidian oocysts ( foreyt and langerquist , 1993 ) .
cryptosporidium and giardia species cysts were detected in the faeces of american marten from the up of michigan , both having a prevalence of 4% ( n = 5 ) ( veine - smith et al . , 2011 ) .
north american marten are infected with a wide variety of endoparasites , but information regarding the pathologic effects of parasitism remains limited .
american marten infected with hookworms in the current study were found to be at risk for anaemia , and this association warrants further investigation .
some parasites are infrequently reported in only a single location , while those reported in multiple locations typically have a varying prevalence .
multiple ecological factors , including habitat , prey availability , sympatric carnivore community , and host adaptation , are likely involved in the variation of prevalence at different geographic locations . a number of parasites known to infect american marten have zoonotic potential . as american marten
are frequently trapped as a furbearer species , this information could guide prevention of disease transmission to trappers and researchers working with the species .
parasite infections could cause illness under conditions of stress and presumed immunosuppression associated with reintroduction programs ( kimber and kollias , 2000 ) .
the majority of previous studies examined american marten carcasses for parasitism , which allows characterization and speciation of the adult parasite and information about intensity of infection .
however , a non - invasive method of detecting endoparasitism is desired for reintroduction programs and our results provide baseline information about parasites detected by faecal examination from american marten in michigan .
multiple faecal exams from individuals destined for relocation , including sedimentation and molecular techniques when available , are warranted to identify novel parasites and parasites with pathologic or zoonotic potential and to make appropriate treatment or management decisions .
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the american marten ( martes americana ) was reintroduced to both the upper ( up ) and northern lower peninsula ( nlp ) of michigan during the 20th century .
this is the first report of endoparasites of american marten from the nlp .
faeces from live - trapped american marten were examined for the presence of parasitic ova , and blood samples were obtained for haematocrit evaluation .
the most prevalent parasites were capillaria and alaria species .
helminth parasites reported in american marten for the first time include eucoleus boehmi , hookworm , and hymenolepis and strongyloides species .
this is the first report of shedding of sarcocystis species sporocysts in an american marten and identification of 2 coccidian parasites , cystoisospora and eimeria species .
the pathologic and zoonotic potential of each parasite species is discussed , and previous reports of endoparasites of the american marten in north america are reviewed .
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Introduction
Materials and methods
Results
Discussion
Conclusion
|
gemcitabine is a modified
cytidine analog having two fluorine atoms
at the 2-position in the deoxyribose sugar moiety ( scheme 1 ) . for nearly 20 years , it has been widely used
to treat specifically pancreatic cancer .
it has been proposed that gemcitabine inhibits
ribonucleotide reductase ( rnr ) activity as well as acting as
a replication stop , thereby affecting dna synthesis
and elongation .
the two highly electronegative
f - atoms at c2 substantially increase the acidity of h3
through the inductive effect .
it is well established
that in the absence of oxygen , thiyl radicals
are able to abstract hydrogen ( h ) atoms to form neutral c - centered
radicals .
for example , h - atom abstraction by thiyl radicals has been
shown to induce isomerization of cis-2,5-dimethyltetrahydrofuran
to the trans-2,5-dimethyltetrahydrofuran . on this basis , stubbe et al .
, in their enzymatic and electron
spin resonance ( esr ) studies with gemcitabine , have proposed that
inhibition of the rnr activity by gemcitabine should occur via radical
formation at the c3 site by direct h - atom abstraction to produce
a c3 via a enzymatic thiyl radical ( reaction 1 ) .
subsequently , the c3 intermediate
has been proposed to form 3-keto c2 via hf
loss .
this c2 is stabilized by an oxy radical resonance
contribution ( reaction 2 ) .
c2
and its immediate precursor c3 ( reactions 1 and 2 ) play a key role in
the rnr inactivation.12 the h - atom abstraction reaction ( reaction 1 ) is not only important in rnr activation but also
plays a very key
role toward stable product formation in other biologically damage
processes in dna , such as oxidative intrastrand cross - link formation and in the formation of sugar radicals that
are strand break precursors .
it is noteworthy that pulse radiolysis
experiments with a 1,4-anhydro-5-deoxy-6-thio - d - ribo - hexofuranitol
detected the formation of ribosyl - based carbon - centered radical(s )
after h - atom abstraction by thiyl radicals .
these studies are supportive of reactions 1 and 2 but unequivocal , and direct detection
of c3 and c2 employing esr or pulse
radiolysis during rnr - catalyzed deoxygenation of the natural substrates or during inactivation by gemcitabine still remains elusive . in this work , we report the formation of c2 from
a likely c3 in a gemcitabine analog which mimics the
mechanism proposed above . from the structural formula of gemcitabine
( scheme 1 )
, it is expected that the negative
inductive effect ( i ) of two highly electronegative f - atoms
at c2 should increase the acidity of h3. from our
previous work on nucleoside cation radicals , the gemcitabine cation radical formed upon one - electron oxidation
is expected to produce c3 after deprotonation of the
acidic proton h3. in this work , esr spectroscopy has been
employed to investigate one - electron oxidation of gemcitabine and
other 2-modified derivatives , for example , 2-deoxy-2-fluoro-2-c - methylcytidine ( mefdc ( psi-6130 ) ; scheme 2 ) and 2-fluoro-2-deoxycytidine ( 2-fdc , scheme 2 ) , in order to test the influence of 2-
substituent on radical site formation .
it is noteworthy that mefdc
is a well - known clinically efficacious inhibitor of hepatitis c virus .
the esr results clearly identify the c3 formation
in one - electron oxidized gemcitabine and the production of c2
in one - electron oxidized mefdc .
these calculations show
that in the case of one - electron oxidized mefdc , the lowest energy
path is the rapid formation of c2 from c3
via f loss .
this f loss is a
barrierless reaction between the 2-f - atom and the proximate
h3o which was formed via deprotonation of h3
in the cation radical .
gemcitabine ( scheme 1 ) and 2-fdc
( scheme 2 ) were obtained
from carbosynth ltd .
mefdc ( scheme 2 ) was prepared as described or
purchased from adooq bioscience ( irvine , ca ) .
lithium chloride ( licl ) ( ultra
dry , 99.995% ( metals basis ) ) was
obtained from alfa aesar ( ward hill , ma , usa ) .
2-deoxycytidine
( 2-dc ) was obtained from sigma chemical company ( st louis ,
mo , usa ) .
deuterium oxide ( d2o ) ( 99.9 atom % d ) was purchased
from aldrich chemical co. inc .
( paris , ky , usa ) . cytidine-5,6-d2 ( [ 5,6-d , d]-cyd , 99 atom % d ) was purchased from cdn isotopes ( quebec ,
canada ) .
homogeneous solutions
of gemcitabine were prepared by dissolving 210 mg / ml either
in 7.5 m licl in d2o or in h2o .
solutions of
other compounds ( 2-dc , 2-f - dc , mefdc , and [ 5,6-d , d]-cyd )
were prepared by dissolving ca .
k2s2o8 ( 616
mg / ml ) was added as an electron scavenger so that only the formation
of the one - electron oxidized species and its subsequent reactions
can be followed by employing esr spectroscopy .
the above - mentioned
procedure for preparation of solutions is according to our ongoing
studies on various model systems of dna and rna .
the ph of gemcitabine in 7.5 m licl / d2o was adjusted to the range of ca .
the ph of gemcitabine in 7.5 m licl / h2o
and the ph of other compounds ( 2-dc ,
2-f - dc , mefdc ,
and [ 5,6-d , d]-cyd ) in 7.5 m licl / d2o was adjusted at ph
ca . 10 .
these ph adjustments were performed by adding l amounts
1 m naoh as per our previous efforts .
these solutions have high ionic strength ( 7.5 m licl ) ; therefore ,
the ph meters would not provide accurate ph measurements of these
solutions . instead , as per our previous works ,
ph values reported in this work were obtained using ph papers and
are approximate measurements . as
per our previous works , these ph - adjusted homogeneous solutions were thoroughly bubbled
with nitrogen to remove the dissolved oxygen . immediately , these solutions
were drawn into 4 mm suprasil quartz tubes ( catalog no .
buena , nj , usa ) and were rapidly cooled in
liquid nitrogen ( 77 k ) .
the rapid cooling of these homogeneous liquid
solutions at 77 k leads to the formation of transparent homogeneous
glassy solutions .
these glassy solutions were later used for the irradiation
and subsequent progressive annealing experiments .
all glassy samples
were stored at 77 k in teflon containers in the dark .
all samples
were ( co)-irradiated ( absorbed
dose = 1.4 kgy ) at 77 k and stored at 77 k in teflon containers in
dark following our previous efforts .
a variable - temperature
assembly was employed which passed liquid nitrogen cooled nitrogen
gas past a thermister and over the sample as described in our earlier
studies .
the glassy samples have been
annealed anywhere from ( 140170 ) k for 15 min .
annealing leads
to one - electron oxidation of the solute by the matrix radical cl2 thus , forming only the cation
radical of the solute , e.g. , gemcitabine .
following our earlier studies , immediately after -irradiation of the glassy sample at 77
k , the esr spectrum was recorded at 77 k. also , immediately after
each annealing step , the sample was cooled to 77 k by immersing in
liquid nitrogen ( 77 k ) , and the esr spectrum was recorded at 77 k
which maximizes signal height and allows for comparison of signal
intensities . a varian century series x - band ( 9.3 ghz ) esr spectrometer
with an e-4531 dual cavity , 9 in .
magnet , and a 200 mw klystron was
used , and fremy s salt ( gcenter = 2.0056 , a(n ) = 13.09 g ) was employed for the
field calibration .
all esr spectra have been recorded at 77 k and
at 40 db ( 20 w ) .
anisotropic simulations of esr spectra
have been performed using the win - epr and simfonia programs of bruker
as per our previous works .
the simulated spectra thus obtained
were compared to experimental spectra , and esr parameters were adjusted
for the best fit ( also supporting
information figure s3 ) .
gaussview and jmol programs were used to plot the spin densities
and molecular structures .
the geometries of all the radicals considered
in the present study were fully optimized using the b97x functional and 6 - 31g(d ) basis set .
we note here that b97x
functional was developed by the group of head gordon and found
to be very successful for the calculations of various properties of
molecules in their different spin states .
the hyperfine coupling constants ( hfccs ) of the radicals were calculated
using the same method and basis set , i.e. , b97x/6 - 31g(d ) in
the gas phase . in order to treat the effect of solvent on hf loss
from the c3 in gemcitabine and in mefdc , we employ
the integral equation formalism polarized continuum model ( ief - pcm ) as implemented in gaussian 09 . in
addition to pcm , for c3 in both systems
a h3o is placed in the vicinity of the c3-oh bond
for c3 in gemcitabine and for c3 in
mefdc and have optimized the structures .
the electronic energy profile
of f dissociation from c2 site of c3
in mefdc as well as the electronic energy profile of f dissociation for each of the two f - atoms from c2 site of
c3 in gemcitabine were obtained in the presence of
a single water molecule at the same level of theory ( supporting information figure s4c , d ) .
furthermore , employing
the wb97x/6 - 31++g(d , p ) method along with the ief - pcm model for the
solvent effect , the pka of the c3-oh
group for the c3 of gemcitabine and also of the c3-oh
group for the c3 of 2-dc was calculated .
in figure 1a , we show the experimentally recorded ( 77 k ) esr spectrum
( green ) of one - electron oxidized gemcitabine at ph ( pd ) ca . 7 in a
homogeneous glassy 7.5 m licl / d2o solution .
the one - electron
oxidation of gemcitabine was induced by cl2 attack after annealing at 155 k in the dark .
912 showed identical spectra after
one - electron oxidation of gemcitabine by cl2 on annealing at 150155 k. thus , only the spectrum
obtained from the gemcitabine sample at pd ca .
10 is presented in
figure 1b along with the simulated spectrum
in blue .
it is evident from figure 1a , b , the
line shape , total hyperfine splitting , and the center of the simulated
spectra match with those of the experimentally recorded spectra quite
well .
each of the spectra in figure 1a , b show
two anisotropic -f - atom hyperfine couplings and a -h - atom
hyperfine coupling .
the -h - atom hyperfine coupling creates
the doublet splitting in the line components in figure 1a , b .
figure 1a is best matched
with a simulation
employing the two different anisotropic -f - atom ( nuclear spin
= 1/2 ) hfcc values of ( 15.0 , 15.0 , 105 ) g and ( 15.0 , 15.0 , 69.0 ) g ,
one -h hfcc as ( 15.0 , 15.0 , 24.0 ) g , gxx , gyy , gzz ( 2.0080 , 2.0050 , 2.0020 ) along with a mixed ( lorentzian / gaussian
( 1:1 ) ) line - width of 14 g. the simulated spectrum in blue is superimposed
on the experimentally recorded spectrum in figure 1a . on the other hand , the best fit for figure 1b
is obtained employing the two identical anisotropic -f - atom
( nuclear spin = 1/2 ) hfcc as ( 17.0 , 17.0 , 86.0 ) g , one -h hfcc
as ( 15.0 , 15.0 , 24.0 ) g , gxx , gyy , gzz ( 2.0060 , 2.0050 ,
2.0020 ) along with a mixed ( lorentzian / gaussian ( 1:1 ) ) line - width
of 10 g. following our work on the radicals produced in monomers
of dna
and rna , the a ( i.e. ,
the azz ) component of each of the two
anisotropic -f - atoms ( see table 1 ) as
well as the a of the -h are directly measured
from the width of the experimentally recorded spectra with an uncertainty
of 2 g ( see supporting information figure s3 ) . on the other hand ,
the theoretically obtained values
of axx and ayy components of each of the two anisotropic -f - atoms and of the
-h - atom in table 1 were adjusted to
fit the experimentally recorded spectra with estimated uncertainty
of 4 g ( see supporting information figure s3 ) .
7 show two nonequivalent anisotropic -f - atom hfccs , whereas ,
the one - electron oxidized gemcitabine spectrum at ph ca .
the -h - atom
hfcc does not show any observable change in the one - electron oxidized
gemcitabine spectrum throughout the ph range ca .
the coupling to two -f - atoms ( c2 ) and one -h - atom
( c4 ) is clear evidence for the generation of c3
after one electron oxidation of gemcitabine at 150155 k. the
electron - withdrawing effect of the two electronegative f - atoms at
c2 increases the acidity of h3 , which leads to deprotonation
( see supporting information table t2 ) and
prevents observation of the initially formed cytosine base -cation
radical ( c ) in gemcitabine as indicated in reaction 3 .
therefore , the mechanism of c3
formation due to one - electron oxidation of gemcitabine is proposed
as follows : one - electron oxidation of gemcitabine results in the formation
of metastable c , which is unstable even at ca .
155 k. the metastable c quickly deprotonates at
c3 in the sugar moiety producing c3 ( reaction 3 ) via a proton - coupled electron - transfer ( pcet ) mechanism.3 esr
spectra obtained from matched gemcitabine samples [ concentration
of gemcitabine in each sample = 2 mg / ml in 7.5 m licl / d2o ] in the presence of the electron scavenger k2s2o8 ( 8 mg / ml in each sample ) .
each sample has been -irradiated
( absorbed dose = 1.4 kgy at 77 k ) , subsequently annealed to 155 k
for 15 min in the dark at various phs ( a ) ph ca .
7 ( green ) and ( b )
ph ranging ca . 912 ( pink ) . here
the blue spectra that are superimposed on the experimentally
recorded spectra are the simulated spectra of c3.
see text for the details of simulation .
all esr spectra are recorded
at 77 k. the three reference markers ( open triangles ) in this figure
and in the subsequent figures show the position of fremy s
salt resonance with the central marker at g = 2.0056 .
the spacing
separating the markers is 13.09 g. as is evident from the hfcc
values of the spectra shown in figure 1 and
also in table 1 , the ph of the solution clearly
affects the individual -f - atom anisotropic hfcc but not the
sum of the two -f - atom anisotropic hfcc along with the c4
-proton hfcc ( see section above ) . at ph ca .
912 , the two -f - atom anisotropic hfccs
are equivalent ; whereas , at ph ca . 7 ,
the sum of the two -f - atom
anisotropic hfccs remain the same , but they individually differ .
the presence of two 2-f - atoms in gemcitabine will lower the
pka value of both h3 and c3-oh
hydrogens .
for example , the oh in 2,2-difluoroethanol has its pka lowered by 3.5 units in comparison with ethanol .
further , radical formation has also been shown
to lower the pka of the alcoholic oh group
( e.g. , pka ( ch3)2choh = 17.1 , pka ( ch3)2coh = 12.03 ) . based on
these factors , the pka value of the c3-oh
group for c3 in gemcitabine is estimated to be in
the range of 79 .
employing dft/b97x/6 - 31++g(d , p ) method
along with the ief - pcm model for the solvent effect , the pka value of the c3-oh group for c3
in 2-dc has been calculated as 14.2 .
the same level of calculation
for the c3-oh group of c3 in gemcitabine which
replaces each of the two hydrogens at c2 with fluorine predicts
a pka of 6.8 ( see supporting information pages s8 , s9 ) .
therefore , the two 2-f - atoms
are predicted to lower the pka of the
c3-oh group in c3 by 7 full units .
considering
the sensitivity of the calculations for predicting pka to the small changes in free energy , these results are very reasonable .
furthermore , the theoretically
calculated pka value 6.8 of the c3-oh
group for c3 in gemcitabine is in good agreement with
its experimentally estimated value ( 79 ) . the spectrum
in figure 1a ( ph ca .
7 ) should
therefore be for c3 in gemcitabine with a c3-oh
group and the spectrum in figure 1b ( ph ca .
912 ) should be for c3 with the deprotonated
group , i.e. , c3-o ( reaction 4 ) .
thus , we attribute the variation of the two -f - atom
anisotropic hfcc to the deprotonation of the c3-oh group at
higher phs.4 esr spectral studies
of one - electron oxidation of gemcitabine in h2o glasses
( 7.5 m licl / h2o ) were performed and compared with the results
found in d2o glasses ( 7.5 m licl / d2o ) .
no observable difference in spectra other than a small
line broadening was observed on formation of c3 in
h2o glasses versus in d2o glasses .
since c3
only has the c3-oh as an exchangeable proton at ph 7 and this
does not contribute to a significant hyperfine coupling , the esr spectrum
found on formation of c3 is not altered by a change
of the solvent from d2o to h2o .
similar experiments to those performed for gemcitabine
were carried out for the methyl / fluoro analog mefdc ( scheme 2 ) .
using mefdc , we investigated whether the formation
of c3 observed in gemcitabine bearing geminal difluoro unit at c2 ( figure 1 ) is
affected by the substitution of one of the f - atoms ( i ) with
a methyl ( me ) group ( + i ) .
( a ) esr spectrum ( black ) obtained from mefdc [ concentration
= 2
mg / ml in 7.5 m licl / d2o ] in the presence of the electron
scavenger k2s2o8 ( 8 mg / ml ) , ph ca .
10 , -irradiated to a dose of 1.4 kgy at 77 k and subsequently
annealed to 155 k for 15 min .
( c )
spectrum ( black ) obtained after subtraction of 60% of spectrum ( b )
from spectrum ( a ) .
for comparison , the c spectrum
( blue ) in 2-dc ( supporting information figure s2 and pp s3s5 ) is superimposed .
( d ) spectrum ( black )
assigned to c2 is obtained after subtraction of 50%
of spectrum ( c ) from spectrum ( a ) .
the simulated c2
spectrum ( for simulation parameters see figure 2 and text ) is superimposed
on the experimentally isolated spectrum for comparison .
all the spectra
are recorded at 77 k. shown in figure 2a is the esr spectrum
( black )
of a matched sample of mefdc that has been -irradiated ( absorbed
dose = 1.4 kgy ) , subsequently annealed to 155 k for 15 min in the
dark , and recorded at 77 k. figure 2b
was obtained by annealing this
sample for 15 min to 170 k. comparison of spectrum 2a with spectrum
2b shows clearly that a central doublet decreases along with an increase
of the other line components upon annealing .
therefore , the central
doublet ( black ) shown in figure 2c is isolated
by subtraction of 60% of the spectrum 2b from
spectrum 2a .
the doublet due to c spectrum ( blue ) in 2-dc ( see supporting information figure s2 and its discussion ( pp s3s5 ) )
is superimposed on it for comparison . from the spectral similarities
of both doublets , the doublet in black shown in figure 2c
subtraction
of 50% c spectrum 2c ( black ) from
spectrum 2a results in the black spectrum shown in figure 2d .
this overall quintet spectrum arises from 4 isotropic
-proton couplings : three methyl -protons ( ca .
25.5 g due to a -proton
assigned to the c1-h ( vide infra ) .
the experimental ( black )
spectrum is simulated using the above - mentioned hfcc values along
with a 10 g line - width and g - value = 2.0033 ( this g - value is typical for c - centered sugar radicals ) . the simulated spectrum ( red ) in figure 2d
matches the overall line components of the experimental spectrum well .
since the c2 ( reaction 5 ) is
the only likely radical structure that would explain the large hyperfine
coupling to a methyl group and the additional -proton hyperfine
coupling ( assigned to c1 ) , the experimental spectrum in figure 2d has been assigned to c2 ( reaction 5 ) . the spectra 2a and 2b are a composite of c ( black ,
figure 2c ) and c2 ( black , figure 2d ) in different amounts . under the same constant
gain and constant microwave power and upon gradual and stepwise annealing
of the sample from 155 k ( spectrum 2a ) to 170 k ( spectrum 2b ) ,
no
loss of spectral intensity was observed , and our analyses show an
additional ( ca .
20% ) conversion of the c to c2. consideration of the results presented in figures 1 and 2 suggest that for mefdc , c is produced first and on annealing converts to a transient
c3 , which is not observed .
in contrast , the line shape ,
line width , and the overall hyperfine splitting of the c3
spectrum in gemcitabine do not change upon annealing to ca .
thus , unlike
the rapid conversion of c3 to c2 found
in one - electron oxidized mefdc , a similar conversion of c3
to c2 is not observed for one - electron oxidized gemcitabine
at these low temperatures .
the lack of observation of the transient
c3 in one - electron oxidized mefdc and the low temperatures
employed in these experiments implies a very low activation barrier
for the conversion of c3 to c2 ; whereas ,
the activation barrier for the conversion of c3 to
c2 for one - electron oxidized gemcitabine should be
4 kcal / mol .
calculations suggest that the proximity of the
lost h3 proton as h3o to the 2-f - atom
quite likely provides the driving force for this rapid unimolecular
reaction ( see figure 3 ) .
therefore , we propose
that c3 in one - electron oxidized mefdc readily converts
to c2 via a barrierless f loss
( see reaction 5 , figure 3 , and supporting information s5 ) . the b97x/6 - 31g(d )
calculated hfccs of c3 and c2 found
in gemcitabine and in mefdc along with experimental hfccs ( in gauss )
are presented in table 1 .
it is evident from
table 1 that experimental and theoretically
calculated hfccs are in reasonably good agreement .
estimated errors are of 2 g for
azz and 4 g for axx and also for ayy .
calculated in the presence
of one
water molecule only isotropic
hfcc values have
been considered . to explore the reaction mechanism of c2
formation from c3 in one - electron oxidized mefdc and
gemcitabine , using the dft b97x/6 - 31g(d ) method
, we considered
three possible reaction paths : ( i ) hf loss due to deprotonation of
3-hydroxyl group , ( ii ) hf loss due to fluorine dissociation , and additionally , ( iii ) hf loss in the presence of a hydronium ion
( h3o ) .
the electronic energy profile
of hf loss from c3 in mefdc and also from c3
in gemcitabine in the presence of a water molecule is shown in figure
s4 in the supporting information . from supporting information figure s4a ,
it is evident
that for c3 of mefdc , the formation of c2
via hf loss with deprotonation of 3-oh has a significant barrier
of ca . 18 kcal / mol .
for c3 in gemcitabine , the hf
loss associated with deprotonation of 3-hydroxyl group was
calculated to be ca .
we have also considered the dissociation of
fluorine in c3
of mefdc and in c3 of gemcitabine and found that stretching
the c2-f bond up to 1.8 needs ca .
this shows that dissociation of fluorine for c3 in
mefdc occurs at lower energy than dissociation of fluorine for c3
in gemcitabine ( supporting information figure
s4 ) .
alternatively , we consider the fact that deprotonation
of h3
will form h3o initially in close proximity
of the c2-c3 bond which may then induce hf loss ( reaction 5 ) . employing the b97x/6 - 31g(d ) method and
considering the full solvent effect through the polarized continuum
model ( pcm ) , this mechanism have been modeled by placing a h3o in the vicinity of the c3-oh bond for c3
in gemcitabine and for c3 in mefdc and have optimized
the structures . from our calculations , we have observed that for c3
in gemcitabine , a minimum structure exists in the electronic energy
profile in which the h3o stabilizes the f-atom
through forming a hydrogen bond ( 1.57 ) ( see figure 3a and supporting information figure s4c ) .
a second minimum structure was also found in the electronic
energy profile in which the h3o stabilizes
the f-atom through forming a hydrogen bond of identical length ,
1.57 ( see supporting information figure s4d ) .
a barrier of 5 kcal / mol and the overall reaction energy
of 7 to 8 kcal / mol were found for hf formation in
both cases ( see figure 3a and supporting information figures s4c , d ) .
however , for c3
in mefdc , the h3o reacts with the 2-f - atom
without a barrier and forms hf and c2 ( see figure 3b ) .
the bond distances of c3-o3
and o3-h bonds for c3 in gemcitabine are calculated
as 1.34 ( primarily c o single bond character ) and 0.97 , respectively .
the values of
the corresponding bond distances of c3-o3 and o3-h
bonds for c3 in mefdc are obtained as 1.28
( mainly double bond character and 1.0
respectively ) .
thus , these calculations show that the
hf loss from c3
in gemcitabine has a ca .
5 kcal / mol barrier ( supporting
information figure s4c , d ) while for c3 in mefdc ,
the loss of hf is barrierless , and the c2 production
is exothermic in nature as shown in figure 3 .
these findings support our experimental observations that in mefdc ,
c3 is too unstable to be observed , and only c2
is found .
in contrast , in gemcitabine only c3 formation
is observed without any conversion to c2 in the same
temperature range .
pcm-b97x/6 - 31g(d ) calculated structures of c3
in ( a ) gemcitabine and in ( b ) mefdc .
as indicated in ( a ) the c3
in gemcitabine involves a barrier of 5 kcal / mol ( detailed electronic
energy profiles are provided in supporting information figure s4c , d ) to hf loss in the presence of h3o . in ( b ) , the c3 in mefdc is unstable in the presence
of h3o and reacts without a barrier to form
c2 via hf loss . the animations ( movies ) of the optimization steps for reaction involving h3o for c3 in both gemcitabine ( a )
and mefdc ( b ) are provided in the supporting information ( s5 ) .
our
work has the following two salient findings : ( i ) one electron
oxidation leads to cytosine base -cation radical ( c ) in 2-dc and 2-f - dc but to c3
in gemcitabine . as expected from the one - electron redox potentials
of the bases and the backbone , one - electron oxidation of 2-dc and 2-f - dc leads
to c formation as evidenced by the ca .
16 g doublet
that is characteristic of c. however , gemcitabine
( scheme 1 ) leads to the formation of c3
on one - electron oxidation .
the two highly electronegative f - atoms
at 2-position , through their negative inductive effect , lead
to a substantial increase in the acidity of h3. therefore ,
c in gemcitabine is highly unstable toward the
loss of h3 as deprotonation at 150155 k. this is evidenced
by the free energy changes of the cation radical for the loss of h3
as deprotonation to the surrounding solvent ( see supporting information table t2 ) ; this deprotonation shifts
the unpaired spin from the cytosine base of metastable c in gemcitabine to sugar at c3 via a pcet
process .
( ii ) c2 formation does not occur in
gemcitabine
but does in its analog mefdc .
it has been proposed in the literature
that in gemcitabine both c3 and c2
( reactions 1 and 2 ) play
an important role in the rnr inactivation .
conversion of
c3 to c2 takes place via an irreversible
f loss from c2 during rnr inactivation
by gemcitabine .
however , experimental and theoretical results shown
in this work have clearly demonstrated that in our system ( supercooled
homogeneous glassy solutions ) , c3 in gemcitabine does
not convert to c2 on annealing up to 170 k owing to
theoretically predicted barrier of greater than 5 kcal / mol .
theoretically ,
dft calculations support the mechanism involving a h3o induced barrierless conversion of c3 to c2
in one - electron oxidized mefdc .
experimentally , c2
is observed in one - electron oxidized mefdc upon annealing to ca .
160170
k. thus , our study in one - electron oxidized mefdc provides the first
evidence of formation of c2 ( via the unstable intermediate
c3 ( reaction 5 ) ) in a nonenzymatic
system even at low temperature.5
|
gemcitabine
is a modified cytidine analog having two fluorine atoms
at the 2-position of the ribose ring .
it has been proposed
that gemcitabine inhibits rnr activity by producing a c3
intermediate via direct h3-atom abstraction followed by loss
of hf to yield a c2 with 3-keto moiety .
direct
detection of c3 and c2 during rnr
inactivation by gemcitabine still remains elusive . to test the influence
of 2- substitution on radical site formation ,
electron spin
resonance ( esr ) studies are carried out on one - electron oxidized gemcitabine
and other 2-modified analogs , i.e. , 2-deoxy-2-fluoro-2-c - methylcytidine ( mefdc ) and 2-fluoro-2-deoxycytidine
( 2-fdc ) .
esr line components from two anisotropic -2-f - atom
hyperfine couplings identify the c3 formation in one - electron
oxidized gemcitabine , but no further reaction to c2
is found .
one - electron oxidized 2-fdc is unreactive toward
c3 or c2 formation . in one - electron
oxidized mefdc , esr studies show c2 production presumably
from a very unstable c3 precursor .
the experimentally
observed hyperfine couplings for c2 and c3
match well with the theoretically predicted ones .
c3
to c2 conversion in one - electron oxidized gemcitabine
and mefdc has theoretically been modeled by first considering the
c3 and h3o+ formation via h3-proton
deprotonation and the subsequent c2 formation via
hf loss induced by this proximate h3o+ .
theoretical
calculations show that in gemcitabine , c3 to c2
conversion in the presence of a proximate h3o+ has a barrier in agreement with the experimentally observed
lack of c3 to c2 conversion .
in contrast ,
in mefdc , the loss of hf from c3 in the presence of
a proximate h3o+ is barrierless resulting in
c2 formation which agrees with the experimentally
observed rapid c2 formation .
|
Introduction
Materials and Methods
Results and Discussion
Conclusion
|
vascular diseases are among the most common causes of morbidity and mortality , and both number and severity of morbid vascular conditions increase with age . regulations of angiogenesis , coagulation , and inflammation are very important issues in vascular biology , both in normal physiology and pathology .
it is now well established that disruption of endothelial integrity represents a crucial event in the initiation and development of cardiovascular ( cv ) diseases .
numerous studies have reported that microparticles ( mps ) play an important role in endothelial dysfunction .
endothelial dysfunction occurs when a perturbed homeostatic endothelium disrupts vascular competency resulting in reduced vasodilatation and increased proinflammatory and prothrombotic properties of the vascular network .
recently , mps originating from various cells have been found to be associated with several vascular related diseases .
moreover , exposed procoagulant phospholipids and specific receptors at the surface of mps act as biomessengers linking inflammation , coagulation , and angiogenesis [ 35 ] . although mps were first described as cellular debris that are believed to have no biological significance , recent studies documented that mps of endothelial and other origins are biological effectors in inflammation , vascular injury , angiogenesis , and thrombosis [ 68 ] .
mps isolated from granulation tissue are derived from endothelial cells , monocytes , platelets , erythrocytes [ 913 ] , and myofibroblasts .
they exchange biological signals and information intercellularly and each kind of mp carries the antigens and receptors of the cells they originated . mps may transfer part of their components and content to the selected target cells , thus mediating cell activation , phenotypic modification , and reprogramming of cell function .
although 70% to 90% of all circulating mps in the peripheral blood of healthy individuals are derived from platelets , marked elevations of all kinds of mps have been observed in many vascular diseases .
specifically , endothelium - derived microparticles ( emps ) represent a relatively small ( 515% ) but very important subset of all circulating microparticles [ 1618 ] .
this number may vary in different cardiovascular and inflammatory diseases [ 18 , 19 ] .
new insights into endothelial dysfunction and alterations in angiogenesis are emerging from studies of vascular microparticles , particularly endothelial microparticles in elderly populations .
vascular aging with impairment of endothelial cell function leads to altered angiogenesis , a key factor in the etiology of various cardiovascular disorders .
73% of individuals aged 6079 have a cv disease , including stroke , hypertension , or heart failure , and at > 79 years of age prevalence of these diseases increased to 86% in females and 82% in males ( 2012 nhlbi fact book ) .
recently published data have shown that these diseases are the leading cause of death for individuals aged > 65 and morbidity increased from 32% for individuals aged 66 to 48% for individuals aged 85 . an important factor which significantly decreases the incidence of coronary heart diseases in postmenopausal women is estrogen [ 2224 ] . in women already having coronary artery disease or ischemic stroke , the therapeutic benefit of estrogen is not clear [ 25 , 26 ] although it has been reported that estrogen induces rapid vasodilation , exerts anti - inflammatory activity , and regulates vascular cell growth , migration , and protection of cardiomyocytes from injury , all of which prevent atherosclerotic deterioration in vessels .
this review focuses on the role of emps in angiogenesis , coagulation , and inflammation during age - related vascular diseases and the contribution of estrogen to these diseases .
emps are small vesicles that are released from endothelial cells and can be found circulating in the blood . defined by their small size ( 0.1 to 1.0 m ) , they are a heterogeneous population of vesicles which are shed from plasma membranes in response to cell activation , injury , angiogenesis / neovascularization , and/or apoptosis .
emps consist of a small amount of cytosol surrounded by a plasma membrane and display negatively charged phospholipids on their surface that can initiate and accelerate coagulation .
circulating emps have been demonstrated as a marker of preeclampsia [ 29 , 30 ] , acute coronary syndromes , and severe hypertension [ 30 , 32 , 33 ] suggesting their association with pathological processes within the endothelium .
furthermore , circulating emp levels are also implicated in the progression of atherosclerotic lesions , heart failure , arrhythmias , inflammatory vascular disease , sickle anemia , and endotoxemia .
jimenez et al . demonstrated that the surface antigens of emps are distinctive depending on the type of endothelial cell injury , as in apoptosis versus activation .
high levels of the surface antigens e - selectin , intracellular adhesion molecule-1 ( icam-1 ) , and vascular cell adhesion molecule-1 ( vcam-1 ) are on emps derived from activated endothelial cells .
in contrast , the low levels of these antigens are on emps derived from apoptotic endothelial cells .
platelet cell adhesion molecule ( pecam-1 ) , endoglin , and vascular endothelial - cadherin ( ve - cadherin ) are at low levels on emps derived from activated ecs [ 3740 ] ( figure 1 ) .
additionally , emps generated from apoptotic endothelial cells have higher levels of phosphatidylserine on their surface and different phospholipid composition and oxidation status compared with emps generated from activated endothelial cells [ 41 , 42 ] .
these data suggest that there are distinct mechanisms for the formation of emps in apoptotic and activated cells and several studies suggest that these types of emps have different functions in vascular diseases [ 40 , 44 ] .
both inflammatory cytokines and coagulation factors participate in the generation of emps ( figure 2 ) .
recently , it has been shown that p38 mitogen - activated protein kinase ( mapk ) is a critical molecule in the production of proinflammatory emps and increased icam-1 production by endothelial cells , providing a paracrine loop to enhance the endothelial response to inflammation . in vitro studies
have shown that the proinflammatory agent , tnf , activates endothelial cells and induces release of emps ( figure 2 ) .
another potent stimulus for emp formation both in vivo and in vitro is angiotensin ii ( ang ii ) .
this effect is mediated by ang ii receptor type i that signals through nadph oxidase and rho kinase .
furthermore , in ang ii - infused apoliprotein e ( apoe / ) hyperlipidemic mice , a model of significant endothelial dysfunction , ang ii has been shown to increase emp formation by a redox - sensitive and blood - pressure - independent process .
another important factor pai-1 ( plasminogen activator inhibitor type 1 ) plays an important role in the formation of emps .
it has been shown by brodsky et al . that pai-1 promotes formation of emps with reduced transmembrane asymmetry of phospholipids in a dose dependent manner .
these findings could possibly link elevated levels of pai-1 with endothelial dysfunction and tendency toward thrombosis [ 4850 ] . increased levels of pai-1
might serve as an initiator of emp formation followed by increased procoagulant activity and thrombin generation .
in addition , it is also known that thrombin stimulates pai-1 synthesis suggesting constant production of these two factors .
all these data indicate that formation of emps links together inflammation , coagulation , and angiogenesis and causes the impairment of the last two phenomena ( figure 3 ) . among many signaling molecules , t - cadherin ( t - cad ) on the surface of ecs
might be upregulated and may serve as a characteristic marker of ec activation and stress .
recently , philippova et al . have demonstrated a mechanism of t - cad - dependent signaling in the vascular endothelium .
the authors identified that t - cadherin levels in plasma are increased in early atherosclerosis and correlate with endothelial dysfunction , which may lead to increased release of emps from ecs .
increasing evidence has also accumulated to implicate an impaired coagulation system in many vascular diseases .
this coagulation imbalance is the net result of activation of coagulation , impaired activity of natural coagulation inhibitors , and suppressed fibrinolysis .
activation of coagulation proteases , for example , thrombin , is one of the earliest events following tissue injury .
thrombin modulates tissue repair responses by altering vascular permeability , stimulating endothelial cell , fibroblast , and neutrophil migration , and promoting their spreading and adhesion .
it activates various cell types and induces secretion of several proimmune , profibrotic , and proinflammatory factors [ 45 , 56 , 57 ] .
recent studies by sapet et al . have shown that release of emps by endothelial cells in response to thrombin involves a group of genes that regulate angiogenesis and are linked to the cytoskeleton reorganization family . among these genes ,
rho - kinase rock - ii was transcribed at a high rate and was identified as a target of thrombin in emp generation .
the involvement of caspase-2 in rock - ii activation , independent of cell death , points out a novel signaling pathway that emphasizes the proteolytic activity of caspase in emp generation in response to cell activation ( figure 1 ) . however , further studies are needed to determine the molecular mechanisms involved in emp release .
emp release is initiated when thrombin binds to its receptor , proteolytically activated receptor-1 ( par-1 ) , which induces gene transcription that is mediated by thrombin via trail / apo2l , a cytokine belonging to the tumor necrosis factor alpha ( tnf ) superfamily .
this mechanism of emp generation depends on the nuclear factor ( nf ) b activation and involves the soluble form of trail , which is secreted by the endothelial cells under thrombin or inflammatory stimulation [ 4 , 59 ] ( figure 2 ) .
phospholipids expressed on emps bind coagulation factors leading to a prothrombotic state and an increase in procoagulant activity of tissue factor ( tf ) .
these phospholipids are exposed on the outer membrane of mp and are considered to be the main initiators of the coagulation cascade .
additionally , it has been demonstrated that sphingosine 1-phosphate ( s1p ) strongly potentiates thrombin - induced tf expression in ecs suggesting its role in blood coagulation .
s1p has also been shown to be involved in the process of angiogenesis and inflammation [ 6264 ] .
another important role of mps is their contribution to the development of platelet- and fibrin - rich thrombi at sites of vascular injury via the recruitment of cells and the accumulation of tf .
data suggest that emp - mediated coagulation has clinical significance ; for example , an association between the number of circulating mps and the risk of thrombolytic complication has been reported . because emps interact with coagulation proteins and with inflammatory or vascular cells , their role in cardiovascular diseases has been intensively studied .
it has also been observed by jy et al . that emps carry von willebrand factor ( vwf ) and factor viii that promote platelet aggregates and increase their stability ( figure 1 ) .
moreover , the authors postulated that emps released during vascular injury may arrest bleeding by rapid interaction with platelets via membrane - associated vwf multimers and adhesions to stabilized platelet aggregates in the microenvironment .
sabatier demonstrated that emps also carry tf and bind to monocytes causing further tf expression and resulting in enhanced transmigration of monocytes through endothelial junction [ 66 , 67 ] .
one of the important key genes for aging - associated cardiovascular disorders is plasminogen activator inhibitor-1 ( pai-1 ) , a main inhibitor of fibrinolysis .
the expression of pai-1 is not only elevated in the elderly but also significantly induced in a variety of pathologies associated with the process of aging .
increased levels of pai-1 and its procoagulant activity have been recognized as hallmarks of endothelial dysfunction in vascular aging ( figure 4 ) .
furthermore , elevated levels of pai-1 were found in werner syndrome , a disease characterized by premature aging [ 68 , 69 ] and atherosclerosis , which in advanced stages may lead to myocardial infarction and death .
recently , it has been shown that emps expressing both activators and inhibitors of coagulation have fibrinolytic properties that counteract their procoagulant activities , which may enable them to contribute to haemostatic balance .
it has been observed that endothelial and leukocyte microparticles generate fibrinolytic activity , whereas erythrocyte and platelet microparticles do not have this property .
additionally , different plasminogen activators were identified on leukocyte microparticles , urokinase - type plasminogen activator ( upa ) , and emps where tissue plasminogen activator ( tpa ) has been found .
the authors provide evidence that microparticles with plasminogen activators are rare in healthy populations but are observed more frequently in pathological conditions suggesting that plasmin generation on microparticles may be important in the modulation of hemostatic balance . therefore , complex functions of emps have an ambivalent role both in physiological and pathological conditions , either promoting or inhibiting coagulation , inflammation , or angiogenesis .
inflammatory mediators increase several procoagulant factors , inhibit endogenous anticoagulants , and attenuate the fibrinolytic response
. however , the interaction between these two systems is bidirectional , as coagulation is also capable of modulating inflammatory activity .
thrombin mediated inflammatory molecules such as il-8 and il-1ra and il-1 participate in emp release [ 4 , 59 ] ( figure 2 ) and are key factors involved in coagulation , inflammation , and angiogenesis .
inflammation and coagulation are linked processes in many diseases and emps may amplify the responses by activating the endothelium .
it has been reported that in addition to activation of d - dimers and c - reactive proteins in coagulation , the inflammatory cytokine il-6 is associated with mortality , declines in all measures of function , and leads to the frailty phenotype in the elderly .
recently , it has been shown that increased levels of il-6 are present in aged aortas and that aging induces a proinflammatory phenotype in vascular smooth muscle cells ( vsmc ) due in part to increased signaling of toll - like receptor 4 ( tlr4 ) and its signaling adaptor myd 88 .
these observations support the notion of a high prevalence of proinflammatory conditions in advanced age .
all of these factors lead to increased generation of emps and impaired coagulation , inflammation , and angiogenesis in cv diseases ( figures 3 and 4 ) .
the process of angiogenesis is complex and requires endothelial cells ( ecs ) to detach from pericytes and the extracellular matrix ( ecm ) , proliferate , invade the surrounding tissues , migrate , and differentiate to form capillary tubes that connect to newly developed vascular networks leading to vascular stabilization [ 7476 ] .
defective angiogenesis has been found in many vascular diseases and it has been established that emps play an important role in this process .
mps can act on angiogenesis directly through ligand / receptor interaction or indirectly by modulating production of soluble factors involved in endothelial cell differentiation , proliferation , migration , and adhesion .
brodsky et al . have shown that in low concentration , emps did not affect the endothelium . however , increased levels of circulating emps are an important factor in the pathophysiology of cv diseases , directly affecting the endothelium and other circulating cells .
brodsky et al . have demonstrated that emps directly impair vasorelaxation via diminishing production and/or bioavailability of nitric oxide .
this result was correlated with increased superoxide levels in aortic rings isolated from rats and cultured endothelial cells treated with microparticles .
other studies have demonstrated that mps of endothelial origin induce the expression of endothelial cyclooxygenase type 2 , different adhesion molecules , release of cytokines , and impaired release of nitric oxide from vascular endothelial cells .
a pathological concentration ( 10 ) of emps affects angiogenesis by diminishing the cell proliferation rate and decreasing the total capillary length of human umbilical vein endothelial cells ( huvec ) plated on a matrigel substrate .
moreover , huvecs or human microvascular endothelial cells ( hmvecs ) treated with emps demonstrated disorganized tube formation in the presence or absence of vegf .
impairments in the regulation of vascular tone , coagulation , and hemostasis contribute to damage of the vascular system and dysfunctional angiogenesis [ 8183 ] .
recently , in vitro studies published by burger et al . have proved that a long term culture of mouse aortic ecs leads to a senescent phenotype with increased rock activity and formation of mps ( figure 4 ) .
furthermore , it has been shown that mps promote premature ec senescence through the stimulation of endothelial cell ros production ( figure 4 ) .
brodsky et al . demonstrated a significant increase in the number of circulating emps in obesity - induced diabetes rats as well as premature endothelial cell senescence and vasculopathy .
this disorder was characterized by impaired vasorelaxation , nitric oxide production , and defective angiogenesis .
an increased number of circulating emps have been identified in patients with certain diseases , such as hypertension , coronary artery disease , acute coronary syndrome , and stroke . in patients with established endothelial dysfunction , levels of circulating emps
are inversely correlated with the amplitude of flow - mediated dilation , independent of blood pressure [ 11 , 8587 ] .
another study published by thomasow et al . has shown elevated levels of cd31 ( pecam-1 ) , which are suggestive of ec apoptosis , in severe and mild chronic obstructive pulmonary disease ( copd ) and emphysema .
cd31 emps were positively related to emphysema and were inversely associated with pulmonary microvascular blood flow .
in contrast , cd62e ( e - selectin ) emps indicative of endothelial activation were elevated in severe copd and hyperinflation .
previous studies have demonstrated an impairment of cell proliferation , migration , tube formation , and sprouting in older individuals ( > 65 years , male or female ) when compared to their younger counterparts ( < 65 years , male or female ) suggesting that these changes contribute to the decrease in effective blood vessel growth and repair mechanisms in the elderly .
a decrease of proangiogenic factors and loss of circulating endothelial progenitor cells ( epcs ) have also been observed with increased aging ( > 50 years , male ) , which may lead to compromised angiogenesis .
furthermore , age - related changes in epc number and function may directly correlate with the degree of senescent endothelial impairment .
furthermore , in older men ( > 60 years ) with myocardial infarction , circulating microparticles selectively impair the nitric oxide transduction pathway in endothelial cells , which contributes to the general vasomotor dysfunction observed after myocardial infarction . additionally , an environmental alteration such as a decrease in ecm proteins particularly fibrillar collagen production and small leucine rich proteoglycans ( slrps ) and changes in the expression of matrix metalloproteinases ( mmps ) have also been observed with increased aging .
emps are clearly implicated in the impairment of angiogenesis in vascular diseases associated with aging however , the specific pathways through which emps augment this process are unknown .
moreover , emps may be one of the major factors leading to reduced effectiveness of therapies for treating impaired angiogenesis in humans and should be further explored .
furthermore , estrogen levels have been connected to thrombin generation , a central molecule in the coagulation cascade in postmenopausal women .
in particular , estradiol increases migration , proliferation , and formation of capillary - like networks of huvecs by the classic estrogen receptor ( er ) pathway [ 9698 ] .
most reports have concentrated on the role of ers in mediating big vessel relaxation and contraction . in a rat model of acute myocardial infarction
it has been shown that estradiol promotes myocardial angiogenesis by increasing microvascular density through estrogen receptors .
furthermore , in ovariectomized rats it has been demonstrated that an increased number of genes in the aged heart , including tnf and map kinase - activating death domain protein ( madd ) , play a role in the release of emps [ 100102 ] ( figure 4 ) .
another in vivo study has shown that ovariectomy in female rats is associated with reduced pai-1 expression , while estrogen replacement counteracts this change promoting emp formation . in vitro studies
other mechanisms have also been revealed to be involved in the proangiogenic effect of estrogen including increased expression of both vegf and its receptors [ 104 , 105 ] and bfgf , as well as expression of vascular adhesion molecules .
moreover , estrogen is known to enhance nitric oxide production and release by endothelial cells .
studies published by reed and edelberg have suggested that a physiological decrease in the concentration of steroid hormones ( e.g. , estrogen and testosterone ) as a result of menopause and chronological aging may contribute both directly and indirectly to subsequent deficits in the synthesis and function of the angiogenic growth factor tgf- . in humans endogenous
estrogen contributes to the anticoagulant , anti - inflammatory , and antithrombotic properties of the endothelium .
the numbers of endothelium , platelet , and monocyte - derived microparticles have been found to be elevated in low - estrogen menopausal women .
the authors implied that increased numbers of procoagulant microparticles provide a resource to study mechanisms for cardiovascular risk development in newly menopausal women . on the other hand , studies demonstrated by rank et al .
had shown that hormone replacement therapy in postmenopausal women increased the concentration of mps derived from platelets .
the emp levels were unchanged excluding their primary role in the initiation of a thromboembolic event in these women .
another study has shown that emp concentration was diminished in older patients ( > 80 years , male or female ) but mp procoagulant activity was preserved in comparison to younger patients .
the authors suggested that a decreased emp level was associated with age and any effect of gender was ruled out by multivariate analysis .
the study performed by mateos - caceres had shown an increased level of circulating emps in elderly ( > 66 years ) male and female patients with acute stroke and that tnf activated emps were the major player in stroke induction .
furthermore , simak et al . demonstrated that circulating emp phenotypes may be associated with the severity , lesion volume , and outcome of acute ischemic stroke ( ais ) in male patients ( > 78 years ) . on the other hand , studies published by williams et al .
have shown that in elderly patients of both genders ( > 66 years ) , emp levels were similar in ais and stroke mimic patients . moreover
, they demonstrated that emps were generated via activation and not by apoptosis / necrosis of endothelial cells .
this suggested that emps may not be an appropriate marker for ais , given the incapability to distinguish between ais and stroke mimic .
the tree - city cohort studies have shown that increased thrombin generation is an independent predictor of ais in elderly women suggesting that hypercoagulability may play an important role in the pathogenesis of ais .
indicated that elevated plasma pai-1 levels are a strong risk factor for stroke at old age in people of both genders ; however , the 4g/5 g polymorphism variant of pai-1 is associated with reduced incidence of stroke .
it has been shown that the single polymorphism pai-1 4g/5 g genotype is associated with higher central systolic , diastolic , and mean arterial blood pressure in women ( > 70 years ) , while no association was found in men suggesting gender specific biology of pai-1 in addition to an advanced age specific factor . moreover ,
elevated levels of circulating mps have been reported in patients ( > 58 years , male or female ) with acute myocardial infarction and coronary artery disease .
studies published by sinning et al . have shown that circulating emps , but not mps of other cellular origin , are a strong predictor of cardiovascular mortality and major cardiovascular events in patients ( > 66 years , male or female ) with coronary artery disease and pulmonary hypertension .
all these data may indicate that estrogen probably does not exert its protective effects on cv diseases through the emp axis .
however , more analyses are needed in order to confirm if a direct connection occurs between estrogen and emps in age - related vascular diseases ( figure 4 ) .
endothelial microparticles , as pleiotropic factors , play a role in both physiological and pathological conditions and thus may contribute to regulation of vascular homeostasis .
emps not only reflect the stage of disease but also play a causative role in the development of various vascular diseases .
they can modulate coagulation , and their elevated levels have been observed in many conditions associated with inflammation and angiogenesis .
the prothrombotic properties and proinflammatory effects of microparticles on endothelial cells affect vascular aging and lead to structural changes in the heart and other organs .
thrombin and pai-1 seem to be key factors involved in emp generation in age - related vascular disease .
furthermore , in age - related vascular diseases , steroid hormones are among the factors that have been shown to have an influence on vascular homeostasis .
specifically , estrogen plays a regulatory function on vessel inflammation , injury , and repair .
lack of estrogen has been suggested to be directly involved in the endothelial cell injury with emp release that is observed in ischemic diseases .
however , the direct link between emp generation , emp release , and estrogen is understudied and the further investigation of cellular and molecular mechanisms of these correlations is imperative for understanding and providing a basis for new translational investigations .
furthermore , circulating emps show great promise not only as biomarkers in the diagnostics of vascular diseases but also as a target for the treatment of these disorders , especially in elderly patients .
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endothelial microparticles ( emps ) are complex vesicular structures that originate from plasma membranes of activated or apoptotic endothelial cells .
emps play a significant role in vascular function by altering the processes of inflammation , coagulation , and angiogenesis , and they are key players in the pathogenesis of several vascular diseases .
circulating emps are increased in many age - related vascular diseases such as coronary artery disease , peripheral vascular disease , cerebral ischemia , and congestive heart failure .
their elevation in plasma has been considered as both a biomarker and bioactive effector of vascular damage and a target for vascular diseases .
this review focuses on the pleiotropic roles of emps and the mechanisms that trigger their formation , particularly the involvement of decreased estrogen levels , thrombin , and pai-1 as major factors that induce emps in age - related vascular diseases .
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1. Introduction
2. Endothelial Microparticles and Factors That Stimulate Their Formation and Release
3. Endothelial Microparticles in Coagulation and Vascular Aging
4. Endothelial Microparticles in Inflammation
5. Endothelial Microparticles and Their Effect on Vascular Function in the Elderly
6. Endothelial Microparticles and Estrogen in Age-Related Vascular Diseases
7. Conclusions
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one of them , adiponectin , also known as acrp30 , adipoq , gbp28 , and apm1 , was discovered by four independent research teams in 1995/1996 , [ 25 ] as the most abundant product of the adipose tissue .
the hormone is a 244-amino acid protein with 30 kda molecular weight that circulates in the serum in different multimeric forms .
actions of adiponectin are mediated by two types of receptors : adiponectin receptor 1 ( adipor1 ) and 2 ( adipor2 ) .
the receptors are highly related and share 67% sequence identity in mice , but they differ in their tissue distribution and the ability to bind various forms of adiponectin .
adipor1 shows high affinity for the globular form of adiponectin , whereas adipor2 is characterized by intermediate binding affinity for both globular and full - length adiponectin .
the highest levels of adipor1 expression are observed in skeletal muscles , whereas adipor2 is most highly expressed in the liver [ 7 , 8 ] . due to the extensive distribution of adiponectin receptors in peripheral tissues and organs , adiponectin exerts pleiotropic effects on metabolism , including regulation of homeostasis by fatty acid oxidation , stimulation of glucose uptake and gluconeogenesis inhibition , which leads to intensified thermogenesis and weight loss .
consequently , adiponectin level correlates negatively with body fat and positively with insulin sensitivity .
the existing evidence points to the presence of a common endocrine system comprising several hormones , including ghrelin , leptin , and orexin that controls metabolism and reproductive functions [ 1215 ] .
adiponectin mrna and protein were found in the ovaries of several species , including humans , rats , and cows [ 1619 ] .
adiponectin mrna and protein were also detected in the ovaries of prepubertal but not sexually mature gilts .
the influence of the hormonal status of swine related to the stage of the oestrous cycle on adiponectin expression in corpora lutea , granulosa , and theca interna cells remains unknown .
studies indicating higher concentrations of adiponectin in female than in male blood suggest that ovarian steroids may regulate adiponectin secretion [ 20 , 21 ] .
similar conclusions can be drawn from varied plasma levels of adiponectin during the oestrous cycle in pigs . despite the above ,
the possible influence of adiponectin on the production of steroid hormones by porcine ovarian cells remains unexplored .
therefore , the aim of this study was to investigate the expression of the adiponectin gene by real - time pcr , to determine the concentrations of adiponectin protein in the porcine ovary ( corpora lutea , granulosa and theca interna cells ) and to compare gene and protein expression levels during different stages of the oestrous cycle in pigs . additionally , we sought to determine the in vitro effect of adiponectin on the secretion of steroid hormones ( progesterone , oestradiol , testosterone , and androstenedione ) by ovarian luteal , granulosa , and theca interna cells of sexually mature swine .
all experiments were carried out in observance of the ethical standards of the animal ethics committee at the university of warmia and mazury in olsztyn . the experimental material comprised mature gilts ( large white and polish landrace ) from a private breeding farm , aged 7 - 8 months and weighing 130140 kg .
twenty gilts were divided into four experimental groups as follows : days 2 - 3 , 1012 , 1416 , and 1719 of the oestrous cycle .
the day of the second oestrus was designated as day 0 of the oestrous cycle .
the phase of the oestrous cycle was also determined based on ovarian morphology . additionally , to fully confirm correctness of the evaluation of the oestrous cycle phase , the level of progesterone was determined .
dissected corpora lutea ( cls ) from different stages of the oestrous cycle ( days 2 - 3-corpora haemorrhagica , 1012-mature cls , and 1416-regressing cls ) were either immediately frozen in liquid nitrogen and stored at 80c until rna and protein analysis or , similarly to the ovaries from days 1719 of the cycle , placed in cold pbs buffer and transported to the laboratory where luteal , follicular granulosa , and theca interna cells were isolated .
granulosa and theca interna cells are precursor cells of large and small luteal cells , respectively .
dissected corpora lutea from ovaries on days 2 - 3 , 1012 , and 1416 were minced into small fragments and dispersed in f-12 medium containing bovine serum albumin fraction v ( bsa ; 1% ) and antibiotics .
corpora lutea were enzymatically dissociated in 0.125% trypsin solution ( 46 times ) at 37c , centrifuged ( 300 g , 10 min , 4c ) , and washed three times .
isolated luteal cells were filtered through nylon mesh ( 40 m in diameter ) and resuspended in fresh f-12 medium .
the cells were counted using a haemocytometer , and their viability ( ~90% ) was determined by 0.4% trypan blue dye exclusion .
granulosa and theca interna cells were isolated from large follicles ( diameter > 6 mm ) without signs of atresia .
granulosa cells were aspirated with a syringe and additionally washed out with a strong stream of media directed to the internal wall of the follicle .
the theca interna layer was scraped off from granulosa cells and enzymatically dispersed in 0.25% trypsin solution .
dispersed cells were centrifuged ( 800 g for 10 min ) and washed two and three times to isolate granulosa and theca interna cells , respectively .
the cells were filtered through nylon mesh ( 40 m in diameter ) and resuspended in eagle 's medium enriched with bsa ( 5% ) and antibiotics .
the cells were counted using a haemocytometer , and their viability ( ~98% ) was determined by 0.4% trypan blue dye exclusion .
some of granulosa and theca interna cells ( 5 10 viable cells within each experiment ) were immediately resuspended in trizol reagent ( invitrogen , usa ) and stored at 80c until processing for rna analysis .
total rna was extracted from luteal tissues from days 2 - 3 , 1012 , and 1416 of the oestrous cycle using the absolutely rna miniprep kit ( stratagene , usa ) . in the case of granulosa and theca cells
approximately 1 g of rna was reverse - transcribed into cdna in a total volume of 20 l with 0.5 g oligo ( dt)15 primer ( roche , germany ) using the omniscript rt kit ( qiagen , usa ) at 37c for one hour and was terminated by incubation at 93c for 5 min .
quantitative real - time pcr analysis was performed using a pcr system 7300 ( applied biosystems , usa ) .
sense and antisense primers ( adiponectin , cyclophilin a ) were chosen according to lord et al .
the chosen primers were as follows : adiponectin forward : 5atgatgtcaccactggcaaattc-3 , reverse : 5-gaccgtgacgtggaaggaga-3 ; cyclophilin a , forward : 5-gcactggtggcaagtccat-3 , reverse : 5-aggacccgtatgcttcagga-3 ; gapdh forward : 5-ccttcattgacctccactacatggt-3 , reverse : 5-ccacaacatacgtagcaccagcatc-3. adiponectin primers ( access no : ay135647 ) were complementary to positions 514536 ( f ) and 565584 ( r ) of pig adiponectin gene sequence ; cyclophilin a primers ( access no : ay266299 ) were complementary to positions 219237 ( f ) and 269299 ( r ) of pig cyclophilin a gene sequence , gapdh primers ( access no : u48832 ) were complementary to positions 6185 ( f ) and 219243 ( r ) of pig gapdh . a constitutively
expressed genes , cyclophilin a and gapdh , were used as the internal control to verify the quantitative real - time pcr . to ensure that cyclophilin
a and gapdh were the suitable reference genes for this study , we revealed that there were no statistically significant differences in ct values between the examined ovarian structures throughout all investigated stages of the oestrous cycle .
both of the housekeeping genes were also indicated as suitable reference genes ( internal controls ) in the independent studies .
the pcr reaction included 20 ng cdna , 900 nm ( adiponectin forward ) , 300 nm ( adiponectin reverse , cyclophilin a forward and reverse ) and 60 nm ( gapdh forward and reverse ) primers , 12.5 l sybr green pcr master mix ( applied biosystems , usa ) , and rnase free water in a final volume of 25 l .
quantitative real - time pcr cycling conditions were as follows : initial denaturation and enzyme activation at 95c for 10 min , followed by 40 cycles of denaturation at 95c for 15 s and annealing at 59 for 1 min .
negative controls were performed in which water was substituted for cdna , or reverse transcription was not performed prior to pcr .
the specificity of amplification was tested at the end of the pcr by melting - curve analysis .
calculation of relative expression levels of adiponectin was conducted based on the comparative cycle threshold method ( ct ) and normalized using the geometrical mean of reference genes expression levels : gapdh and cyclophilin a. western blotting analysis was performed as described by smolinska et al . .
briefly , equal amounts of porcine corpora lutea as well as granulosa and theca cells lysats ( 10 g ) were resolved by sds - page on 12.5% polyacrylamide gel and then transferred to a nitrocellulose membranes ( whatman , usa ) .
blots were blocked for 5 h at 4c in 1 tbst containing 5% skimmed milk powder and then incubated overnight at 4c with the rabbit polyclonal adiponectin antibodies at the dilution of 1 : 150 ( santa cruz biotechnology , usa ) or rabbit polyclonal actin antibodies ( sigma , usa ) diluted 1 : 200 , which were used as an internal control for equal loading and to quantify porcine adiponectin protein . to identify immunoreactive bands ,
membranes were incubated for 1.5 h at room temperature with mouse anti - rabbit igg for adiponectin ( sigma , usa ; diluted 1 : 2000 ) , goat anti - rabbit igg for actin ( diluted 1 : 5000 ) conjugated with alkaline phosphatase ( santa cruz biotechnology , usa ) .
nonspecific foetal calf serum ( mp biomedicals , usa ) was used instead of primary antibodies to produce negative control blots .
the immunocomplexes were visualized using 4-nitroblue tetrazolium chloride ( nbt ) and 5-bromo-4-chloro-3-indolyl phosphate ( bcip ) , according to the manufacture 's protocol ( promega , usa ) .
the results of western blotting were quantified by densitometric scanning of immunoblots with gelscan for windows ver . 1.45 software ( kucharczyk , poland ) .
data were expressed as a ratio of adiponectin protein relative to actin protein in arbitrary optical density units .
luteal cells ( 250000/1 ml medium ) were resuspended in f-12 medium enriched with foetal calf serum ( fcs ; 20% ) , bsa ( 1% ) and antibiotics and preincubated for 48 hours in a humidified incubator with 95% air and 5% co2 atmosphere .
the serum - containing medium was discarded , and the cells were washed using serum - free f-12 medium .
after washing , luteal cells were cultured for 24 hours in f-12 medium with bsa ( 1% ) and antibiotics , with or without treatment agents .
granulosa and theca interna cells ( 250000/1 ml medium ) were resuspended in eagle 's medium supplemented with 10% fcs , 5% bsa , and antibiotics .
after 24 hours of preincubation , the medium was discarded , and the cells were washed and cultured for 24 hours in eagle 's medium with 5% bsa and antibiotics , with or without treatment reagents .
the cultured cells were treated with 1 or 10 g / ml of recombinant human adiponectin ( biovendor , usa ) alone or in combination with insulin ( granulosa and theca interna cells ; 10 ng / ml ; sigma , usa ) , fsh ( granulosa cells ; 10 ng / ml ; nhpp , usa ) and lh ( theca interna cells ; 10 ng / ml ; nhpp , usa ) , and combination of those treatments ( insulin + fsh for granulosa cells and insulin + lh for theca interna cells ) .
adiponectin doses used in the experiment were close to physiological concentrations of this hormone in the porcine blood .
those cells are thought to be generally more autonomic than follicular cells which is a reason of luteal cells ' hard response to stimulators [ 3133 ] .
the alamar blue test revealed that none of the treatments affected the viability of the cultured cells .
following incubation , the media were harvested , centrifuged ( 800 g for 10 min ) , and the supernatants were collected and stored at 20c until analyses of progesterone ( cultures of luteal , theca interna , and granulosa cells ) , oestradiol ( cultures of theca interna and granulosa cells ) , androstenedione , and testosterone ( cultures of theca interna cells ) . progesterone ( p4 )
was analysed according to the method described by ciereszko et al . , oestradiol ( e2 ) was determined according to the method of hotchkiss et al . modified by kotwica , androstenedione ( a4 ) as described by dziadkowiec et al . , and testosterone ( t ) according to kotwica and williams .
the specificity of the antibodies against a4 , t , and e2 has been reported by ciereszko et al . and szafranska et al . .
the sensitivities of the assays for p4 and a4 were 1 pg / ml and for e2 and t were 0.5 pg / ml .
intra- and interassay coefficients of variation of the p4 , e2 , a4 , and t assays were 0.91% , 0.7% , 0.81% , 0.97% and 8.5% , 10.1% , 14.2% , and 7.8% , respectively .
data from real - time analysis and western blot were analysed by one - way anova and least significant difference ( lsd ) post hoc test and are reported as the means sem from five independent observations .
all data concerning in vitro cell cultures were analysed by anova for repeated measurements and least significant difference ( lsd ) post hoc test and are reported as the means sem from five ( for all types of cells ) independent observations .
each experiment was performed on different day using separate pool of luteal or follicular cells .
the expression of adiponectin mrna was significantly higher in corpora lutea during all investigated days of the luteal phase than in theca interna cells isolated on days 1719 of the cycle ( p < 0.05 ) .
no significant differences were noted in adiponectin gene expression in granulosa cells compared with theca interna cells and cls ( figure 1(a ) ) . in general ,
adiponectin protein concentration in the porcine ovary was higher in the luteal phase than in the follicular phase .
in particular , the adiponectin protein content was the greatest in cls from days 2 - 3 in comparison with cls and follicular cells from the remaining stages of the oestrous cycle ( p < 0.01 ) . within
the luteal phase adiponectin concentration was the lowest in cls during days 1012 ( p < 0.01 ) .
there was no significant differences in protein expression between granulosa and theca interna cells ( figure 1(b ) ) .
treatment of luteal cells from days 1012 of the oestrous cycle with adiponectin at both doses ( 1 g / ml ; 10 g / ml ) resulted in significant ( p < 0.05 ) decrease in progesterone secretion ( figure 2 ) .
adiponectin at any dose did not affect p4 secretion by luteal cells collected on days 2 - 3 and 1416 ( data not shown ) .
adiponectin at the higher dose ( 10 g / ml ) in combination with insulin provoked the increase in p4 secretion in comparison with the cells stimulated by insulin alone ( p < 0.05 ) .
in contrast , there was no effect of adiponectin ( both doses ) on fsh- and fsh + insulin - induced production of p4 in porcine granulosa cells ( figure 3 ) .
basal secretion of p4 was unaffected independently on adiponectin dose ( data not shown ) .
adiponectin at the higher dose ( 10 g / ml ) increased basal secretion of e2 ( p < 0.05 ) ( figure 4 ) .
adiponectin did not change secretion of e2 by granulosa cells cultured with insulin and/or fsh ( data not shown ) .
adiponectin at both doses reduced basal secretion of t by porcine theca interna cells ( p < 0.05 ) ( figure 5 ) .
the effect of adiponectin on basal p4 , e2 , and a4 release was negligible ( data not shown ) .
adiponectin ( 10 g / ml ) decreased lh + insulin - stimulated secretion of p4 by theca interna cells ( p < 0.05 ) .
there was no effect of adiponectin on lh- or insulin - induced production of p4 by these cells ( figure 6(a ) ) .
adiponectin did not influence lh- or insulin - stimulated secretion of e2 by theca interna cells .
however , lh + insulin - induced secretion of e2 by the cells was increased in response to the higher dose ( 10 g / ml ) of adiponectin ( p < 0.05 ) ( figure 6(b ) ) .
secretion of a4 by theca interna cells was inhibited by combination of adiponectin ( 10 g / ml ) with insulin compared to insulin alone ( p < 0.05 ) .
adiponectin at the lower dose ( 1 g / ml ) increased lh + insulin - induced a4 secretion by the cells ( p < 0.05 ) ( figure 6(c ) ) .
induced secretion of t was not changed under adiponectin influence ( data not shown ) .
this is the first ever study to demonstrate the changes in adiponectin gene and protein expression in the porcine ovary throughout the oestrous cycle .
our results indicate that adiponectin is more highly expressed in luteal cells derived from ovaries in all examined stages of the luteal phase than in follicular granulosa and theca interna cells .
adiponectin was also found to affect basal and stimulated secretion of steroid hormones by porcine ovarian cells .
in addition to porcine ovarian structures , adiponectin gene expression and the presence of the hormone protein were also found in woman theca interna cells and in theca interna cells , granulosa cells , corpora lutea , and oocytes of rats and cows [ 18 , 19 ] .
singh and krishna localized the adiponectin protein in bat ovarian structures that showed positive immunostaining in theca interna cells , oocytes of growing follicles , and the corpus luteum .
the expression of adiponectin gene and protein was also determined in porcine granulosa cells of prepubertal gilts .
the levels of adiponectin expression and also its receptors seem to vary subject to cell type and the stage of follicle and corpora lutea development . in the bovine ovary ,
the expression of adiponectin and its both receptors was lower in granulosa cells than in theca interna cells and oocytes .
contrary results were presented by ortega et al . who observed the highest levels of adiponectin expression in granulosa cells of sheep ovaries .
throughout follicular development , adiponectin mrna increased in granulosa cells and decreased in theca interna cells of cows . in the corpora lutea ,
bovine theca interna cells derived from large follicles revealed higher expression of both receptors than cells isolated from medium - sized and small follicles , indicating that follicular growth influences the transcript levels of adiponectin receptors .
the synthesis of ovarian adiponectin and the expression of adipor1 and adipor2 are probably hormonally controlled , as suggested by an increase in adiponectin concentrations in ovarian follicular fluid of women administrated lh in the in vitro fertilization procedure .
following pmsg pretreatment , an injection of hcg also significantly increased the expression of adiponectin and adipor1 ( but not adipor2 ) genes in rat ovaries .
in bovine theca interna cells , lh increased the concentrations of adipor2 mrnas , whereas igf - i suppressed the expression of the above gene .
the results of the present study seem to confirm the hypothesis that adiponectin production is regulated hormonally .
higher levels of adiponectin gene and protein expression during the luteal phase and lower expression levels during the follicular phase could point to the stimulatory effect of progesterone and the inhibitory influence of oestradiol on ovarian adiponectin production . in our previous study ,
adiponectin serum concentrations were higher during the luteal phase than the follicular phase , which suggests that ovarian steroids influence plasma adiponectin levels . in this study
the results of our in vitro studies and the presence of both adiponectin receptors in porcine ovaries indicate that adiponectin may directly affect ovarian steroidogenesis .
in fact , progesterone secretion decreased due to adiponectin 's influence on luteal cells in mid - luteal phase ( days 1012 ) .
greater variations in adiponectin action were observed in porcine follicular cells . induced progesterone production increased in granulosa cells and decreased in theca interna cells .
basal testosterone output and insulin - induced androstenedione secretion were inhibited , while lh + insulin - stimulated release of androstenedione was enhanced by adiponectin .
the effect of adiponectin on ovarian steroidogenesis was also suggested in studies of cows , rats , and humans .
adiponectin was found to inhibit insulin - induced secretion of progesterone and oestradiol by bovine granulosa cells . in a study of theca interna cells of bovine ovaries ,
lagaly et al . observed that adiponectin decreased lh + insulin - induced production of progesterone .
the above authors also noted that adiponectin inhibited the mrna expression of lh receptor in granulosa cells .
granulosa cells of rats and women treated with igf - i responded to adiponectin by increasing progesterone and oestradiol secretion [ 16 , 17 ] .
in the cited studies , adiponectin did not influence fsh - induced production of progesterone and oestradiol in human and rat granulosa cells which is consistent with the response of porcine cells noted in this study .
it seems that adiponectin affects ovarian functions by binding adipor1 and adipor2 , in two ways .
an experiment where rnai was used to block adipor1 and adipor2 expression in the kgn cell line derived from human granulosa cells contributed to new information about adiponectin 's role in the ovaries .
the absence of adipor1 in the analysed cells enhanced apoptosis , whereas the elimination of adipor2 reduced fsh- and igf - i - induced the production of progesterone and oestradiol and inhibited mitogen - activated kinase activity , relative to control , in response to adiponectin or fsh treatment .
the above results suggest that adipor1 is more involved in the survival of granulosa cells , whereas adipor2 regulates steroidogenesis through mapk activation .
the influence of adiponectin on the ovaries of sexually mature pigs has not been studied to date , but the response of granulosa cells derived from prepubertal gilts to the analysed hormone was described by ledoux et al . .
the above authors observed higher levels of cyclooxygenase-2 , prostaglandin e synthase , and vegf gene expression in cells primed with adiponectin , an increase in the expression of the star gene and a decrease in the expression of the p450 aromatase gene .
adiponectin treatment contributed to an increase in progesterone levels and a decrease in androstenedione and oestradiol plasma concentrations in comparison with control bats . in the same study , adiponectin treatment increased p450scc and 3-hsd enzyme levels but decreased aromatase , star and lh - r levels in comparison with controls .
an in vitro study by caminos et al . demonstrated that adiponectin significantly inhibited basal and hcg - stimulated testosterone secretion by testicular explants .
in another study , adiponectin decreased corticosterone secretion by freshly isolated rat adrenocortical cells but did not affect aldosterone production .
adiponectin treatment enhanced cortisol secretion , which was accompanied by increased expression of steroidogenic pathway genes , including star protein expression , via erk1/2 and ampk - dependent pathways .
in addition to its direct effects on steroidogenesis in the ovary , adiponectin could also indirectly affect gonadal functions by controlling the secretory activity of the hypothalamus and pituitary .
a study by wen et al . demonstrated the inhibition of gnrh release from gt1 - 7 hypothalamic gnrh neurons after the administration of adiponectin . in in vivo study by cheng et al .
adiponectin also lowered gnrh secretion by activating the ampk and inhibiting the erk pathways . in the lower branch of the hpg axis ,
the release of lh from rat pituitary cells and the murine lt2 cell line was decreased after the administration of adiponectin alone and in combination with gnrh [ 53 , 54 ] . in vivo observations also demonstrated an inhibitory effect of adiponectin on lh secretion : the intravenous administration of the adenovirus expressing the adiponectin gene to male mice decreased plasma lh levels without changes in fsh levels .
the inhibitory influence of adiponectin on lh secretion could be attributed to a decrease in gnrh receptor gene expression in the pituitary .
the presence of adiponectin mrna and protein in porcine ovaries observed in this study as well as the presence of adiponectin receptors 1 and 2 in the gonads noted in our previous work could provide evidence for auto / paracrine actions of the analysed hormone .
the variations in adiponectin gene and protein expression during the oestrous cycle indicate that adiponectin expression is determined by the hormonal status of pigs .
the effect of adiponectin on ovarian steroidogenesis suggests that this adipokine influences reproductive functions in pigs . yet for definitive conclusions to be drawn , especially concerning precise localization of adiponectin mrna and protein in different ovarian structures , intracellular mechanisms of adiponectin influence on the gonadal steroidogenesis and its possible effect on other functions of the ovaries , further studies are required to determine the role of adiponectin in ovarian physiology .
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adiponectin is an adipose - secreted hormone that regulates energy homeostasis and is also involved in the control of the reproductive system .
the goal of the present study was to investigate changes in adiponectin gene and protein expression in porcine ovarian structures during the oestrous cycle and to examine the effects of in vitro administration of adiponectin on basal and gonadotrophin- and/or insulin - induced secretion of ovarian steroid hormones .
both gene and protein expression of adiponectin were enhanced during the luteal phase of the cycle .
adiponectin affected basal secretion of progesterone by luteal cells , oestradiol by granulosa cells , and testosterone by theca interna cells .
the gonadotrophin / insulin - induced release of progesterone from granulosa and theca interna cells and the release of oestradiol and androstenedione from theca cells was also modified by adiponectin . in conclusion , the presence of adiponectin mrna and protein in the porcine ovary coupled with our previous results indicating adiponectin receptors expression suggest that adiponectin may locally affect ovarian functions .
the changes in adiponectin expression throughout the oestrous cycle seem to be dependent on the hormonal status of pigs related to the stage of the oestrous cycle .
the effect of adiponectin on ovarian steroidogenesis suggests that this adipokine influences reproductive functions in pigs .
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1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusions
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the knowledge of mechanical materials behaviour is of great importance particularly for some innovative engineering applications , which involve a single material , bimaterials , or multiple materials .
some bimaterials are obtained by means of a coating process and consist of coupled layers that may or may not contain filler elements , such as a foaming operation , or may be developed as welded substrates . usually , at the end of the process , the material is identified as a new single material .
the coating procedure developed and studied in this work refers to the hot - dip galvanizing treatment , considered as a better treatment to fight against corrosion .
the role of a hot - dip galvanizing treatment consists in the deposition of a protective external layer of metallic zinc obtained by immersing the steel in a zinc bath at a temperature of around 460c .
when the material ( i.e. , steel ) is introduced into the zinc bath and then removed , several changes in the chemical composition and in the mechanical structure can occur .
these changes produce a new structural arrangement on zinc substrate and are usually revealed by the generation of cracks in the zinc layer .
the behaviour of materials may change as a result of various events such as fatigue , fracture , wear , fretting fatigue , creep , hydrogen embrittlement , thermal shock processes , or atmospheric attacks .
an important role in these failure processes is played by any discontinuity in the material that can appear during the manufacturing process or during working period , or as a result of inappropriate use . in our case
it appears as a consequence of the hot - dip galvanizing treatment applied as described above in association with the action of the mechanical loading .
recently , krishnan and xu focused on failure mechanics of adhesive joints ( i.e. , considered as bimaterials ) using a fringe pattern concentrations technique to describe the bimaterials interface .
as analytical model , they used the fracture mechanics approach , while to compute the stress singularity in the bimaterial layer they took into account the stress intensity factor in mode i. the formula used to express this stress intensity factor is
( 1)ki = rekai ,
where k is the stress intensity factor in the case of the bimaterial component :
( 2)k = ytaaiei ,
where t = p(3s / w ) and y , are the calibrating factors that depend on a / w , b / w , respectively , dundurs ' parameters .
then a , b , and w represent the length of the possible crack , the thickness , and the length of the specimen .
is a function of dundurs ' parameters , , and is presented as
( 3)=12ln11+ ,
where , dundurs ' parameters material , is expressed by
( 4)=112221211122+2121
in which 1 and 2 denote the material , while and are the shear modulus and poisson 's ratio , respectively .
the elastic fracture theory is a suitable tool to compute the mechanical behaviour in case of coatings , composites , and welded structures .
the bimaterial produced by the reactions that occur during the hot - dip galvanized steel process must have the same conditions on the contact surface as the linear spring - like model .
the discontinuity of displacement across the interface is assumed to be linearly proportional to the displacement at the interface of the constituent where the stress source is located .
the present authors agree that the two materials that form the bimaterial should be considered as a functionally autonomous subsystem with a different young 's modulus and poisson 's coefficients .
this implies a strain incompatibility between the two solids and the formation of a periodic distribution of tensile and compressive stress in checkerboard patterns under the uniaxial tensile test .
a robust approach was proposed by yu et al . in order to formulate analytical solutions through the use of linear spring - like model . according to this theory we can define the type of contact between the surfaces by the fraction of the adherent area , applying the following formula :
( 5)=aacac ,
where is used to quantify the extent of bonding at the interface , a is the total area of the interface , and ac is the area covered by paper ( see example on figure 2 from ) . according to the study made by panin et al .
, the interface of solid materials covers a sinusoidal surface layer due to a rotation of successive constraints in tensile and compression stress in the structure .
this effect is described by the following equation :
( 6)=aysinxlxt2 ,
where
t is the thickness of the coating , x is the distance of crack propagation , y is the stress coefficient .
this paper addresses two goals : first of all , to experimentally characterize the layer of zinc applied during the hot - dip galvanization process , in order to obtain necessary information about the uniformity of the zinc layer and the number of cracks and their length , and finally to estimate the behaviour of the cracks located in zinc layer when the mechanical loading is imposed .
secondly , we proposed to corroborate the experimental results with analytical and numerical computations , ascertaining where the crack discontinuities spread while considering the three main possibilities of crack development : arrest at the bimaterial interface , crossing into the second material , in our case steel , and finally creating a deflection between the two materials . in the literature
k. m. mrz and z. mrz predicted the behaviour of bi- and multimaterial interfaces according to a simplified approach using the mk - criterion based on the linear elastic fracture mechanics ( lefm ) . in this criterion
it is assumed that the crack growth follows the direction of minimum distortion energy density at a distance corresponding to a specified value of dilatation energy .
figure 2 shows a specific case in which the crack is considered to propagate perpendicularly to the interlayer and make a bifurcation at the interface .
the decohesion phase may present as one of the four possible scenarios whereby the crack will propagate as follows : ( i ) the plastically weaker material ( wm ) to the plastically stronger material ( sm ) , wm - sm , ( ii ) the plastically weaker material ( wm ) to the plastically stronger interlayer ( si ) , wm - si , ( iii ) the plastically stronger material ( sm ) to the plastically weaker material ( wm ) , sm - wm , and ( iv ) the plastically stronger material ( sm ) to the plastically weaker interlayer ( wi ) , sm - wi . in simple terms , the criterion for crack initiation and propagation along the interface could be considered , where the maximum stress , max , is expressed as
( 7)max=x+y2+x+y22+xy . to solve the problem of deflection at the interface of two materials , hutchinson proposed the following condition :
( 8)gcgc1<gdgp1 ,
where g = gc represents release energy rate of the interface bimaterial .
studies , related to the problem of cracks running perpendicular to a bimaterial , yields a different expression for the stress intensity factor that is connected to the stress tensor as follows :
( 9)ij = kr1fij. to determine the propagation energy of the crack running perpendicular to the interface , also called energy release rate , the following equation studied by madani et al .
can be applied :
( 10)gd=(11)/1+(11)/24cosh2k12+k22gp=1222kp2 .
in ( 8) , ( 10 )
gd and gp are the strain energy release rate for deflection and penetration ; k1 and k2 are the stress intensity factors for the interface crack ; kp is the value of stress intensity factor , for a particular case , when the crack from material 1 penetrates into material 2 ( see figure 2 ) .
analytical techniques and the green function can be employed to describe the behaviour of bimaterial compounds by calculating numerical solutions of singular integral equations , as was applied by erdogan et al . and used by pruncu et al . and azari et al . , which yield the following expression :
( 11)ki(a)=211+k1a0g(1)kib=211+k1a0g1 . a more general form of such approach could be structured using an algorithm that describes the numerical procedure for obtaining the different values of the function g(ti ) by the following .
( 2 ) for k ranging from 1 to n , we need a computing route for xk and f(xk ) .
( 3 ) for every xk and for i variants from 1 to ( n 1 ) , we have to calculate the first ti that is a weight function of the jacobi polynomials described as follows :
( 12)1ng(ti)1tixk+k(xk , ti)=0 withti = cos2i12n , i=1, ,nxk = coskn , k=1, ,n1 .
( 4 ) the computation then yields a matrix relationship expressed as follows:(13)1t1x1+k(x1,t1)1t2x1+k(x1,t2) .... 1tnx1+k(x1,tn) .............................. 1t1xk+k(xk , t1)1t2xk+k(xk , t2) .... 1tnxk+k(xk , tn)gt1gt2 .... gtn = fx1fx2 .... fxk .
g are obtained by multiplying the transposed of the matrix by the vector f(xi ) .
one has the following : a0 is half - length of the crack ; 1 , 2 are shear modulus for materials 1 and 2 ; c is the distance from the middle of the crack at the interface plane ; r , are polar coordinates , k = 3 4 for the plane strain case and k = ( 3)/(1 + ) for the plane stress case , is poisson coefficient , and g is the parameter that expresses the magnitude of the applied load . another analytical technique , which was employed by chen et al . using complex potential values ,
was obtained in the following manner :
( 14)ki(b)=limr02rx=22k2 + 1a0m=11mmki(a)=22k2 + 1a0m=1m ,
where a0 is half - length of the crack , m = m/b , m = 0,1 , 2,3 , , b is the distance between the interface and the crack front , x is the distribution of stress in front of the crack , k = 3 4 for the plane strain case and k = ( 3 4)(1 + ) for the plane stress case , and 2 is shear modulus for the material in which the crack is located .
because of its multiple properties , steel is one of the most versatile materials used in industrial engineering applications .
the european standard ( en ) reveals different types of steel employed in fields such as the automotive industry and aeronautical design . in this work we considered two steel alloys : he360dr and s420mc .
their mechanical characteristics are summarized in table 1 . during the lifetime of service , in contact with the environment
this metal may develop problems that could be prevented by applying the above - described process of hot - dip galvanization .
the specimens obtained after hot - dip galvanization were submitted in laboratory to fatigue tensile test in order to detect their behaviour under fatigue conditions conforming to real life and to observe the changes that occurred in the metallic zinc layer .
the fatigue tests were performed on a servohydraulic testing machine capable of applying axial loads up to 100 kn , using a sinusoidal load wave , with a frequency of 30 hz and a strength ratio r = 0.1 .
after the fatigue test the specimens were experimentally analysed by optical microscopy and scanning electron microscopy ( sem ) . during observation with the optical microscope ,
a sample of length 5328 m was considered . to simplify the management of these optical microscope observations , we divided the sample size into 16 units with a length of about 333 m .
the main purpose of this was to analyse ( i ) the number of cracks per unit of length ; ( ii ) whether the evolution of the number of cracks was constant over all layers applied ; ( iii ) which material was prone to develop longer cracks , and finally if the zinc layer was constant over the whole steel surface .
the first result was the average number of cracks per unit of length , as summarized in table 2 .
the letters b and h in table 2 indicate the time of immersion in the zinc bath , b being 3 minutes of immersion and h 7 minutes .
the numbers 9 , 8 , 7 , and 2 indicate the specimen number analysed . from table 2
we can observe that the average number of cracks per unit of length is about 6/11 for he360dr and 7/8 for s420mc .
a first remark proves that the number of cracks increases with the time of immersion .
the results are plotted in figure 3 and highlight the number of cracks for all 16 units of length .
figure 3 shows that the number of cracks on the entire length of zinc layer that encloses each unit is overall scattered ; however , on the local case of two / three consecutive units , the number of cracks per unit seems to be almost uniformly distributed . a key element for determination of the efficiency of the coating process
may be deduced from the evolution of cracks lengths before and after the fatigue tests . according to the observations shown in figure 4
, there was a significant change of crack length as a consequence of fatigue test .
a gap of about 25 m in crack length before and after cyclic load was found due to the weakened structure of material .
this observation was done in a useful sample area ( ua ) and then in the area where the test had less influence on the behaviour of the piece , marked as the nonuseful area ( nua ) .
the uniformity of the zinc layer was considered as another issue that can stimulate the crack behaviour .
, the zinc layer deposited over the steel alloy is uniformly distributed , having a value of 5080 m .
the particularity of the size of substrate of he360dr h8 materials , that is , the thin thickness , may be explained as a consequence of immersion time .
so , because the time of immersion within the bath of zinc was less than the time imposed by our methodology , the thickness of substrate was lower ; this means that instead of 7 minutes we found that the real time of immersion was about 5 minutes .
the optical measurements confirmed that the thickness deposited over the steel pieces was uniform and show that the zinc layer is composed of several multilayers .
the substrates are denominated by gamma ( ) , delta ( ) , zeta ( ) , and zinc eta ( ) , as shown in figure 6 .
figure 6 proves that most of the cracks are located in substrate and may occur along the zinc grain boundaries . indeed , from our observations these cracks arise in this substrate and then spread toward the steel - zinc interface .
however , the analysis indicates a decrease of the crack magnitude near the interface , which means the crack growth is interrupted before it reaches the interface .
observations from scanning electron microscopy ( sem ) exhibit even more clearly that , during the hot - dip galvanized process ( hdg ) , the quality of the bonded layers may influence the direction of the cracks , if the cracks touch the steel - zinc interface .
this troublesome problem produces a sort of debonding area which forms a path for surface deflection of the cracks .
the debonding area in steel - zinc adhesive layers is illustrated in figure 7 , created by the propagation of cracks at the interface between the zinc and steel and into the zinc substrates zeta ( ) and eta ( ) . for bimaterials , the interface crack problems could be due to the nonhomogeneity of the materials , which develop in the direction parallel to the crack tip . from these preliminary findings
we could confirm that during the hdg process the zinc layer is uniformly deposited and the average number of cracks is significant on this layer .
it seems that during the fatigue process the cracks get longer and spread toward the bonding interface , arresting at the bimaterial interface . in the worst scenario ,
numerical computational technique was implemented by abaqus software , in order to corroborate the experimental data with analytical and numerical simulations and to highlight the effects of cracks that develop during the hot - dip galvanizing process . in order to implement the experimental data in the numerical model ,
besides , this value corresponds with the thickness measured under the s420mc h2 materials ( see figure 5 ) , and it allows making the assumption that the crack / cracks imposed in our models will spread only toward the interface steel / zinc . if we consider a smaller thickness , for example , the size of layer measured under he360dr b9 material , and we adopt in the numerical model the critical size of crack detected after the loading test ( see figure 4 ) , it will be obvious that the crack will cross all the coating substrate .
but this issue is far from our experimental observation , since we had observed that the crack growth is only toward interface steel / zinc .
experimental data were introduced in our numerical model as the thickness size of zinc layer ( figure 5 ) , while the contour of zinc layer on the entire material surface is considered homogenous and uniform ( figure 5 ) .
an initial length of crack and the maximum length of crack ( see figure 4 ) were imposed as constraint in the numerical program .
the principles of linear fracture mechanics ( lfm ) were implemented in the numerical model in order to explain behaviour of crack at the interface steel / zinc that is assuming that all the materials contain a minimum flow that will grow during the mechanical loading .
so , considering the crack located in zinc substrate ( figure 5 ) that will develop perpendicular to the applied load , it is possible to denote this state by mode i fracture .
it should be noted that in the numerical model a mechanical loading of 500 mpa corresponding to an average of ultimate tensile strength of galvanized materials ( see table 1 ) was imposed .
a value of about 30 m was selected as the initial length of the crack / cracks before propagation , in agreement with experimental data ( figure 4 ) .
however , we know that the crack is bounded by two fronts , a and b. in this model , we assumed that the front of crack b was static and so it could not propagate , whereas the only front of crack a would spread .
a basic model composed of two materials , m1 and m2 , as shown in figure 8(a ) , which forms the bimaterial body , was implemented .
m2 represents the steel and m1 the zinc layer , delimited by the following values : h1 = 0.08 mm , h2 = 19.84 mm , c = 0.045 mm , w = 15 mm , 2a0 = 0.030 mm , and = 0.5 m and submitted to a mechanical load , = 500 mpa .
the applied load in this system will have direct consequence to the crack behaviour and define the presence of stress singularity on both fronts of the crack , tips a and b. the configuration for the crack fronts is shown in figure 8(b ) .
settlement of the crack propagates performance was evaluated using the stress intensity factor ( sif ) parameter , derived from the singularity 1/r advanced in front of the crack tip .
since the value of the stress intensity factor ( sif ) is obtained , we proceed to conclude if the crack crosses into the second material , ends at the bonded interface , or deflects between these two bodies .
( a ) model with one crack in the bimaterial : in this case one single crack of an initial size of 30 m was considered . then the length was increased by 3 m in each simulation up to the maximum of 60 m ( i.e. , close to the experimental analysis shown in figure 4 ) .
figure 9 shows how the stress distribution increases with the length of crack , assuming that the initial crack had the same stress value at both fronts a and b. two - dimensional finite - element meshes are virtually symmetrical near this bottom ( crack fronts ) and the symmetry showed almost the same value as the stress distribution , for each of the crack fronts , during the simulation .
the shape of the stress - strain curve as a function of the crack length is illustrated in figure 10 .
the shape of the curve grows wide with the increasing of crack length in front of crack tip a. the effect of stress distribution in front of crack can be converted into basic parameters that show the impact of crack behaviour , that is , the stress concentration factor ( scf ) . obviously , the stress concentration factor is the ratio of the maximum stress over the nominal stress , denominated kt and expressed as
( 15)kt=maxnom. while the front of crack b does change , the main role in this research was played only by the front of crack a located near the interface of the bimaterial .
thus , figure 11 shows the effect of scf in the front of crack a. these results were corroborated with the theoretical stress concentration factor ( scf ) obtained with the online software .
the results presented in figure 11 show a good agreement and underline that when the crack reaches the interface , there is a sudden increase in the stress distribution value .
this sharp rise in values may result in the creation of a surface called a debonding area , as shown in figures 9(c ) and 9(d ) .
then , by assessing the crack propagation with lfm using ( crack ) stress intensity factors ( sif ) , k , it is possible to establish where the crack stops . to achieve even better accurate results , a comparison between an analytical model reported by erdogan et al . and
the difference between the numerical and analytical results is due to the use of a small number of constants in the analytical model .
the results are shown in figure 12 , demonstrating the trend of the ( crack ) stress intensity factors ( sif ) during applied stress , in front of cracks a and b. from the curves drawn below , it can be seen that the value of the sif increases with the crack length , and the crack propagates up to a length of about 50 m . then stabilization and even a decline of the sif value
the value of sif will increase sharply only if the flow area denoted as well as
since the value of the ( crack ) stress intensity factors ( sif ) is low for the front of both cracks a and b , it seems that the crack can not cross into the second material .
( b ) model with two or more cracks : in the second assumption , a multiple cracks model , namely , with 3 cracks , was implemented using almost the correspondent algorithm but specifying 3 initial cracks with an initial size of 30 m for each crack , and then each crack would increase by 3 m , up to the maximum size of 60 m .
thus , the maximum cracks value is assigned when the cracks reach the steel - zinc interface . in this model with 3 cracks the applied load ( 500 mpa ) was considered as statement from the case of the model with a single crack .
the motivation to implement a model with three cracks aims to detect whether the situation changed due to the increased ( crack ) stress intensity factor ( sif ) values , as a result of the cumulative energy developed by these 3 cracks .
related work has been reported by song et al . who declared that the interface crack will propagate if the principal maximum stress at the crack tip exceeds a critical value .
thus , confirming our initial supposition , the results presented in figure 14 show an increase for maximum stress value within front of the cracks . at the same time , the surface denominated debonding area appears more pronounced .
the tendency was confirmed from the development of the ( crack ) stress intensity factors ( sif ) , k values , where the results are reported in figure 15 .
the shapes of the curves in figure 15 show a similar trend obtained by the one in the case with one crack except for the fact that crack 2 shows the highest influence of propagation , derived from the cumulated extension efforts from another two cracks .
another thing to note is the gap difference of sif for crack lengths 57 m and 60 m .
this difference could be explained by the appearance of the previously mentioned debonding areas
( i.e. , imposed length for debonding area considered in this research was a maximum of two initial crack lengths , because after this size the peeling processes occur ) .
although the difference is evident , it does not exceed the critical value of mode i fracture toughness ; for example , the value cited by ashby for steel is about 80170 mpa m. consequently , the crack will not cross into the steel material and in the worst case the crack / cracks will form a large bifurcation at the interface between these two materials .
this research paper highlights by means of experimental , numerical , and analytical tool the behaviour of a bimaterial zinc / steel interface submitted to mechanical loading . in particular , the case of crack / cracks at the bimaterials interface was considered .
the main objectives of this paper were to assess the behaviour of the crack / cracks generated at the end of the hot - dip process and to reveal that the numerical computations are in a good agreement with the experimental outcomes .
optical microscopy and scanning electron microscopy ( sem ) techniques were involved to evaluate the experimental performance of the bimaterials compound .
postmortem analysis of the fracture surfaces emphasized the uniformity of the zinc layer over the steel surface and provided accurate information related to the average of cracks length ( figure 4 ) .
in addition , this technique allows quantifying the number of cracks ( figure 3 ) that forms in the zinc layers . in the meantime
, this assessment provides patterns about the evolution of crack from incipient phase , where the cracks arise , and how far they spread . in numerical computations , simulations of the model considered
were run in two different situations , namely , when the bimaterial contains just one crack or else 3 cracks , in order to prove the agreement between computation and experimental outcomes .
besides , to get even accurate results , the analytical model used by erdogan et al . was also confronted .
the sif were calculated to obtain value of the stress singularity that characterizes the crack evolution in the bonding area of bimaterial , for both crack tip values , that is , the fronts of cracks a and b. the analytical , numerical , and experimental assessments prove that the crack / cracks that arise during the hot - dip galvanized steel process will propagate during the mechanical fatigue test .
it was also established that the crack / cracks will stop near the steel - zinc interface at a distance of about 3 m and only in particular cases yield a deflection at the bimaterial interface . to summarize
, the results may confirm that cracks reduce the life span of the bimaterial but are not responsible for a direct degradation of the steel .
this method is therefore useful for employment in damage models that include the safety factor condition .
finally , a nondestructive method should be employed to detect the behaviour of bimaterials for a better calibration .
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the behaviour of materials is governed by the surrounding environment .
the contact area between the material and the surrounding environment is the likely spot where different forms of degradation , particularly rust , may be generated .
a rust prevention treatment , like bluing , inhibitors , humidity control , coatings , and galvanization , will be necessary .
the galvanization process aims to protect the surface of the material by depositing a layer of metallic zinc by either hot - dip galvanizing or electroplating . in the hot - dip galvanizing process
, a metallic bond between steel and metallic zinc is obtained by immersing the steel in a zinc bath at a temperature of around 460c .
although the hot - dip galvanizing procedure is recognized to be one of the most effective techniques to combat corrosion , cracks can arise in the intermetallic layer .
these cracks can affect the life of the coated material and decrease the lifetime service of the entire structure .
in the present paper the mechanical response of hot - dip galvanized steel submitted to mechanical loading condition is investigated .
experimental tests were performed and corroborative numerical and analytical methods were then applied in order to describe both the mechanical behaviour and the processes of crack / cracks propagation in a bimaterial as zinc - coated material .
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1. Introduction
2. Objectives
3. Material and Experimental Procedures
4. Simulation Procedure
5. Numerical Results and Discussion
6. Conclusion
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epilepsy is a common and serious chronic neurological disease that affects over 50 million people worldwide .
anti - epileptic drugs ( aeds ) are effective at controlling seizures in many patients , acting through a range of neuronal targets , including ion channels , neurotransmitter release , and receptor mechanisms.1 , 2 however , a third of patients with epilepsy fail to achieve seizure freedom , and current aeds do not show disease - modifying properties .
future treatments for epilepsy need to differentiate from currently marketed drugs , for example , by having a novel mechanism(s ) of action or providing disease - modifying effects.1 , 2 acquired epilepsy is thought to result from disturbances to the expression and function of neurotransmitters and their receptors , ion channels , and signaling components , as well as altered metabolism , neuronal and glial microstructure , neuroinflammation , and other mechanisms.3 , 4 , 5 it is likely that disease - modifying treatments will need to influence multiple genes within a given pathway or in various independent pathways .
potential gene network control points were recently identified and include master regulators of transcription and neuroinflammation , epigenetic factors , and noncoding rnas .
micrornas ( mirnas ) are a conserved family of endogenous small noncoding rnas that regulate protein levels in cells by post - transcriptional control of mrna stability and translation.9 , 10 this is achieved by sequence - specific binding of the mirna to complementary bases in the mrna target .
since this generally requires only a 7- to 8-nucleotide match , individual mirnas can potentially target large numbers of mrnas .
this provides the ability to regulate entire gene networks or multiple biological processes and has been demonstrated in the brain .
microrna-134 ( mir-134 ) was identified as a brain - enriched , neuronally expressed mirna that controls dendrite spine morphogenesis , via repression of lim domain kinase 1 and other targets.14 , 15 this is potentially important for disorders of hyperexcitability , since dendritic spines are contact points for excitatory communication and there is evidence that reorganization of dendrites is a feature of experimental and human epilepsy.16 , 17 we identified mir-134 among upregulated mirnas profiled in areas of hippocampal damage following status epilepticus induced by intra - amygdala kainic acid ( ka ) in mice .
expression of mir-134 is also upregulated after neuronal stimulation and seizures in various other in vitro and in vivo models,15 , 19 , 20 , 21 , 22 and mir-134 is overexpressed in the resected human neocortex from patients with intractable temporal lobe epilepsy ( tle ) . silencing mir-134 by intracerebroventricular ( i.c.v . )
injection of locked nucleic acid ( lna ) , cholesterol - tagged antagomirs ( ant-134 ) reduced levels of mir-134 for at least a month and rendered mice refractory to the convulsant effects of both ka and pilocarpine.19 , 22 consistent with known mir-134 targets , lim domain kinase 1 and levels of other mir-134 targets were higher in ant-134-treated mice after status epilepticus and the dendritic spine volume was increased.19 , 22 notably , injection of ant-134 after status epilepticus suppressed the later occurrence of spontaneous recurrent seizures in the intra - amygdala ka model in mice .
there is significant interest in developing mirna - based therapies.23 , 24 there are also barriers to antisense - based approaches and there has been some disengagement of industry from pursuing such efforts .
this is likely to be a problem for any new therapy for epilepsy , particularly one that is not based on traditional small molecule chemistry.1 , 2 pre - clinical development of an mir-134-based treatment for epilepsy might be more attractive if additional important questions are answered .
principally , work to date has only tested ant-134 in mouse models of status epilepticus .
proof of seizure - suppressive effects of ant-134 in a non - status epilepticus model or in another species would bridge the gap in evidence and facilitate pre - clinical development of ant-134-based treatment for epilepsy.26 , 27 here , we evaluated mir-134 expression in the resected hippocampus from patients with pharmacoresistant tle and we tested ant-134 in three different models .
we used the pentylenetetrazol ( ptz ) model in mice , a popular method for screening putative anticonvulsive effects that triggers seizures via -amino butyric acid ( gaba ) blockade.26 , 28 second , the perforant pathway stimulation ( pps ) model , a toxin - free model of acquired epilepsy based on nonconvulsive status epilepticus that avoids the confounding influence of exposing the brain to chemoconvulsants , was used in rats .
finally , we used a high - potassium model of epileptiform activity , using ex vivo brain slices from rats pre - treated with ant-134 .
our results establish that targeting mir-134 offers broad anticonvulsant and possibly disease - modifying effects in experimental epilepsy , which may encourage pre - clinical development of ant-134 as a treatment for epilepsy .
for this , we analyzed levels of mir-134 in the surgically obtained hippocampus from adults with pharmacoresistant tle and we compared findings to autopsy hippocampal samples from people who died of causes unrelated to neurological disease .
these samples were used previously to analyze expression of other genes , and clinical data for both groups are briefly summarized in tables s1 and s2 .
there were no between - group differences in patient age , and each group included males and females .
we detected higher levels ( p = 0.037 ) of mature mir-134 in the tle specimens compared to autopsy samples ( figure 1a ) .
this difference is unlikely to be an artifact of post mortem delay , since mir-134 levels did not decrease in simulated autopsy experiments lasting up to 12 hr . we began by establishing dosing parameters in the mouse ptz model .
generalized tonic - clonic seizures were first reliably elicited at a dose of 60 mg / kg in c57bl/6j mice ( figure 1b ) .
a dose of 80 mg / kg elicited generalized tonic - clonic seizures in all mice and with a more consistent and shorter latency than with the 60 mg / kg dose ( figure 1b ) .
a dose of 100 mg / kg also produced rapid - onset tonic - clonic seizures in all mice but was associated with high mortality ( figure 1b and data not shown ) .
ordinal logistic regression analysis was used to explore the dose dependence of the ptz - induced convulsions .
ptz dose was significantly associated with racine scores ( figure 1c ) . a dose of 80 mg / kg was selected for further experiments .
to obtain molecular evidence for ptz - induced seizure activity in the model , we measured expression of activity - regulated genes in the hippocampus .
transcript levels of fbj osteosarcoma oncogene ( fos ) , a calcium - dependent marker of neuronal activity and activity regulated cytoskeletal - associated protein ( arc ) , another immediate early gene responsive to intense neuronal activity , were strongly increased in the hippocampus 30 min , but not at 4 hr , after ptz - induced seizures ( figures 1d and 1e ) .
there was no evidence of irreversible neuronal injury in tissue sections from mice after ptz , as assessed by fluoro - jade b ( fjb ) and neun ( rna binding protein , fox-1 homolog [ c. elegans ] 3 ) staining ( data not shown ) .
expression of mir-134 is known to increase in various in vitro and in vivo seizure models.15 , 19 , 20 , 21 , 22 we therefore investigated whether ptz - induced convulsions alter mir-134 levels in mice .
taqman mirna assays showed that levels of mature mir-134 were significantly increased in the hippocampus , but not in the cortex , 30 min after ptz - induced generalized tonic - clonic seizures in mice ( figure 1f and data not shown ) .
injection of antagomirs targeting mir-134 ( ant-134 ) in mice.19 , 22 previous work has established a dose of these antagomirs that reduces hippocampal mir-134 levels by over 90% by 24 hr after injection .
accordingly , we tested ptz responses 24 hr after injection of ant-134 and compared them to responses in mice that received a scrambled antagomir ( scr ) . in scr mice , delay to onset of first tonic - clonic seizure after ptz injection was similar to that observed before in control mice ( figure 2a , and compare to figure 1b ) .
thus , the control antagomir did not alter the normal response to ptz in the model .
the time to first ptz - induced tonic - clonic seizure was significantly longer in mice injected with ant-134 compared to scr - injected animals ( figure 2a ) .
the severity of racine - scored ptz - induced seizures was also significantly lower in ant-134- compared to scr - injected mice ( figures 2b and 2c ) .
analysis of electrographically recorded seizure activity determined that electroencephalography ( eeg ) total power was lower in ant-134 mice compared to the control group after ptz ( figure 2d ) .
we next analyzed brain tissue samples from scr- and ant-134-injected mice for evidence of mirna silencing and differences in expression of activity - regulated genes .
analysis of the hippocampus obtained after ptz - induced seizures confirmed that mir-134 expression was lower in ant-134 pre - treated mice compared to scr - treated animals ( figure 3a ) .
ant-134 had no effect on levels of an unrelated mirna ( figure 3b ) .
next , we assessed expression of arc and c - fos as molecular markers of recent neuronal activity to support the clinically scored behavior and eeg differences in ant-134 mice . quantitative real - time pcr analysis revealed increased c - fos expression in the hippocampus of scr - injected mice after ptz - induced seizures ( figure 3c ) .
in contrast , c - fos levels were lower in ant-134-injected mice , consistent with reduced seizure severity ( figure 3c ) .
this difference was also apparent in the neocortex from the same animals ( figure 3c ) .
expression of arc was increased in the hippocampus of scr - injected mice that received ptz .
arc levels were not significantly different in ant-134 mice compared to scr - injected animals in the hippocampus and neocortex after ptz - induced seizures ( figure 3d ) .
we turned next to the pps model in rats , first investigating effects on mir-134 levels .
taqman mirna assays showed that levels of mature mir-134 were not significantly modified in the hippocampus of rats at two different time points ( figure 4a ) .
next , we assessed the effect of silencing mir-134 before stimulation on seizure severity during status epilepticus .
rats were pre - treated with ant-134 or scr ( i.c.v . ) 24 hr before the 8-hr stimulation protocol .
analysis of electrographically recorded seizure activity determined that scr - injected rats presented a mean ( sem ) increase of 1,304.3% 94.9% in eeg total power relative to baseline .
this was similar to animals that were stimulated but not receiving the oligonucleotide ( data not shown ) . in rats
pre - treated with ant-134 , the severity of evoked seizures in the pps model was similar : eeg total power during seizures was a mean ( s.e.m ) of 1,244.6% 82.7% ( percent of baseline ) . despite not seeing an acute anticonvulsant effect of ant-134 in the pps model , we were nevertheless interested in whether silencing mir-134 after status epilepticus in the model would alter the emergence of recurrent spontaneous seizures . a key prior
finding was that silencing mir-134 after status epilepticus induced by intra - amygdala ka reduced the subsequent occurrence of spontaneous recurrent seizures by over 90% .
for these studies , rats underwent 8-hr stimulation of the perforant pathway to induce epilepsy and the i.c.v .
rats were then monitored with video - eeg until a first spontaneous seizure was recorded , typically 1025 days after pps , and they were then followed for a further 8 weeks ( figure 4b ) .
epilepsy developed in all scr - injected rats over the expected course , with the first spontaneous seizures appearing 14 weeks after status epilepticus ( figures 4b and 4c ) .
scr - injected rats averaged 20 spontaneous seizures recorded during the 8-week recording time ( figures 4d and 4e ) .
in contrast , no spontaneous seizures were detected over the 8-week period in six of seven rats injected with ant-134 after status epilepticus ( figures 4d4f ) .
epilepsy emerged in only a single rat treated with ant-134 ( figures 4d and 4f ) .
the fisher exact test determined that there was a significant difference in epilepsy development between the groups ( p = 0.005 ) . in terms of effect size , ant-134 prevented 86% of the risk of epilepsy , with a 95% confidence interval ( ci ) of 12%98% .
an analysis of the duration of spontaneous seizures ( electrographically defined ) revealed that when spontaneous seizures occurred , they were similar between groups .
that is , the average duration of a spontaneous seizure in scr - treated rats was 53.7 14.4 s ( n = 20 seizures , from five rats ) , which was similar to the duration in epileptic rats in the pps model that did not receive an i.c.v .
injection ( 49.4 15.9 , n = 20 seizures ) . in the ant-134 rat that developed epilepsy ,
the average duration of a spontaneous seizure was 52.3 16.2 s ( from n = 12 seizures ) .
having observed a disparity between the acute anti - seizure effects of ant-134 pre - treatment in the ptz and pps models , we sought to examine the effect of ant-134 on naive , non - epileptic tissue and whether the effects were due to long range networks or if local circuitry was sufficient to mediate any seizure protection .
we injected ant-134 in vivo in rats and subsequently prepared ex vivo brain slices ( figure 5a ) .
these slices were then exposed to a 9-mm k seizure challenge , to determine whether ant-134 protects against epilepsy in local circuits of the isolated slice in the absence of epileptogenesis .
onset of activity was delayed in ant-134-treated slices by 176 s relative to the control ( figures 5b and 5c ) .
this effect was statistically significant ( mann - whitney u test , p = 0.002 , = 0.025 , power = 0.87 ) .
we also compared the power of epileptiform activity and duration of seizure - like events in these data ; these effects were not statistically significant ( power : t test , p = 0.70 ; duration : mann - whitney u test , p = 0.067 ) .
this finding suggests that ant-134 can confer seizure resistance in naive tissue and thus mediate its effect even in the absence of epileptogenesis .
in addition , preservation of the seizure resistance in isolated slices indicates that at least some of the effects of ant-134 are due to local changes in activity .
the present study provides important additional evidence of the anticonvulsant and disease - modifying effects of lna - based antagomirs targeting mir-134 in animal models of epilepsy .
our study shows that ant-134 is an anticonvulsant in an acute chemoconvulsant model ( ptz in mice ) and ant-134 had anti - epileptogenic activity in the perforant pathway stimulation model of epilepsy in rats .
ant-134 also confers resistance to epileptic activity in naive tissue , reflected as a delay to activity onset following a seizure challenge in ex vivo brain slices .
we also report that mir-134 levels are elevated in the hippocampus of patients with pharmacoresistant tle .
the therapeutic potential of targeting mir-134 in established epilepsy will require additional studies , but our findings may encourage further research or pre - clinical development of this mirna - based treatment for epilepsy . despite the recent introduction of two additional aeds ( perampanel and brivaracetam ) , new drug development in epilepsy
faces significant challenges.1 , 2 , 32 new targets are required that can treat pharmacoresistant patients or have disease - modifying effects that are not served by current therapies .
mirnas have emerged as potential targets of the future via their influence on neuronal microstructure , gliosis , neuroinflammation , and ion channels.8 , 33 silencing mir-134 has produced the most convincing anti - seizure effects to date , reducing status epilepticus in pre - treatment paradigms in the intra - amygdala ka and pilocarpine models and exerting potent long - term effects on spontaneous seizure development.19 , 22 this mirna is also an attractive target , since studies show increased levels of mir-134 within the temporal lobe of adult and pediatric patients with intractable epilepsy.19 , 20 we report here that mir-134 is also elevated in the hippocampus of adults with pharmacoresistant tle , although there are potential confounders with comparing resected material from patients to autopsy control samples . the present study has answered key questions that could facilitate pre - clinical development by showing that ant-134 has anticonvulsant effects in non - status epilepticus models and is disease modifying in another species .
the first important finding was that pre - treatment with ant-134 strongly suppressed seizures in the mouse ptz model .
effects on both clinical behavior , including delaying the time to first convulsive seizure , and electrographic seizure activity were seen .
thus , lowering brain levels of mir-134 can exert anti - excitability effects , and this implies that mir-134 has a role in setting excitability thresholds in the brain .
this is thought to be due to regulation of proteins that control dendritic morphology,14 , 15 although other targets are known .
ant-134 did not fully prevent seizures in the ptz model ; rather , it delayed and minimized their severity , a finding most closely matching the effect of ant-134 in the pilocarpine model of status epilepticus
. it may not be possible to fully prevent all seizures using ant-134 in chemoconvulsant models , but further adjustments to antagomir dose or delivery may yield optimal seizure - suppressive effects .
ptz triggers seizures via gabaergic blockade ; this differs from the mechanism by which status epilepticus is induced by ka and pilocarpine , which work by promoting glutamatergic and cholinergic transmission , respectively .
this suggests that ant-134 may be effective for epilepsies of various etiologies , which , in patients , can vary considerably between trauma , stroke , infection , genetic , and other causes .
our studies also show that ptz - induced convulsions increase brain levels of mir-134 , transcription of which is probably mediated by myocyte enhancer factor 2c .
we did not observe an increase in mir-134 after perforant pathway stimulation in the rat .
this was surprising , although changes to mir-134 levels are not always detected after chemoconvulsive stimuli , and we can not rule out a technical explanation ( e.g. , the time points selected or a subfield - specific effect that was masked by the use of the whole hippocampus ) .
our study included an analysis of the short - term effects of ant-134 on neuronal activity - regulated genes in the ptz model .
expression of genes such as c - fos and arc is a well - established surrogate marker of recent seizure activity , the protein products of which are involved in coordinating synaptic plasticity and adaptation to excessive neuronal stimulation.38 , 39 unexpectedly , only levels of c - fos , and not arc , showed the expected reductions in ant-134 mice after ptz seizures .
it is known that c - fos induction is calcium dependent , whereas arc expression can be induced via calcium - independent pathways.38 , 39 therefore , ant-134 treatment may have reduced seizure severity sufficient to prevent c - fos induction , such as by minimizing n - methyl - d - aspartate ( nmda ) receptor opening , but enough residual neuronal excitation remained to upregulate arc .
these findings provide additional evidence of the scale and nature of seizure suppression by ant-134 and suggest molecular markers that can distinguish effects of mirna manipulations on different components of seizure responses . in the present study
, we observed that pre - treatment of rats with ant-134 did not protect against perforant pathway stimulation - induced status epilepticus .
while there are examples of anticonvulsants producing effects in some but not all models , these findings suggest that ant-134 may have the most clinical value as an anti - epileptogenic treatment .
previous work showed that injection of ant-134 after status epilepticus in mice suppressed the later occurrence of spontaneous seizures by 90% , evidence of a disease - modifying effect in the intra - amygdala ka model . here
, we found nearly identical anti - epileptogenic effects of ant-134 in a different model in rats .
this is the only demonstration , to our knowledge , of any disease - modifying treatment in the pps model in rats .
the pps technique induces hippocampal sclerosis and recurrent spontaneous seizures,29 , 40 and it provides a means to trigger epilepsy without the bias or other issues associated with chemoconvulsants .
ant-134 was injected shortly after triggering status epilepticus in order to model a reasonably realistic clinical scenario , such as a patient being treated shortly after an initial precipitating injury .
ant-134 had dramatic and potent effects , with the majority of ant-134 rats ( six of seven , 86% ) never developing spontaneous recurrent seizures .
notably , spontaneous seizures in the study with the intra - amygdala ka model did occur in all ant-134 mice , albeit with dramatically lower seizure frequency .
this difference may relate to the extent of hippocampal injury or another aspect of the epileptic phenotype , such as the frequency of spontaneous seizures .
thus , presently we see a possible additional therapeutic action of ant-134 , whereby post - treatment is capable of fully preventing epilepsy at least in some models .
we can not , of course , exclude that epilepsy might have developed in some of the ant-134 rats at some point in the future . however , we recorded for 8 weeks beyond the standard latent period .
we further corroborated the anti - seizure effect of ant-134 by using an acute seizure challenge with high potassium in otherwise healthy ( ex vivo ) tissue .
inhibitory neurotransmission is retained in this model ( in contrast to ptz ) and there is no hippocampal sclerosis ( unlike pps ) , further suggesting that ant-134 can be therapeutic in multiple epileptic syndromes with varying mechanisms .
we observed a delay in the onset of epileptiform bursts in brain slices that had been pre - treated with ant-134 .
this finding suggests that epileptogenesis is not required for ant-134 to mediate its effects on seizure onset .
we did not see any significant changes in the power or duration of epileptiform activity ; however , these parameters are highly variable in brain slices , which can obscure potential effects . in addition
, we propose that ant-134 can mediate seizure protection in the hippocampus via effects on local circuitry , because long range connections would likely be severed during the slicing process .
the anti - epileptogenic actions of ant-134 may be independent of the etiology of the initial precipitating injury , although this will require further testing ( e.g. , in models of trauma or infection - induced epilepsy ) .
in addition , upregulation of mir-134 is not essential for ant-134 to produce anti - epileptogenic effects , since mir-134 levels were not altered after pps - induced status epilepticus .
thus , epilepsy can be dramatically suppressed in two different in vivo models , and one in vitro , in two different species .
this provides strong support for and is an argument for further development of ant-134 for epilepsy treatment or prevention .
antagomirs targeting a liver - expressed mirna have been given to humans and were found to be well tolerated and effective against hepatitis c in clinical trials .
there are concerns , obviously , that because ant-134 can alter dendritic spines , it could have effects on cognition . studies to date have not identified such problems , with natural exploratory behavior and performance in a hippocampus - dependent task found to be normal in mice a few days after i.c.v .
injection of ant-134.19 , 22 however , more demanding tests of cognition or learning will need to be performed .
another factor that argues for general safety is that mice and rats have been recorded for at least 2 months after brain injections of ant-134 .
nevertheless , pre - clinical development will necessitate an extensive battery of toxicity and safety testing .
work to date on ant-134 satisfies certain recommendations for testing and evaluating novel therapies for epilepsy , with seizure - suppressive effects demonstrated in five different models and two different species.1 , 2 , 43 , 44 there are , however , additional seizure models that could be used during any future pre - clinical development .
this includes kindling , a popular model that has been successful in identifying aeds with novel mechanisms of action .
while this is not an essential step before pre - clinical development , sex differences in mirna inhibitor responses may be worth investigating in future studies and sexual dimorphism in mirna expression has been reported , although not for mir-134.46 , 47 it will also be of value to understand how ant-134 produces its potent seizure - suppressive effects .
the most likely mechanism is upregulation of one or more proteins due to de - repression of a mir-134 target .
previous work has shown that mir-134 is uploaded into the rna - induced silencing complex after seizures , which facilitates targeting of mrnas , and in vitro studies suggest that the neuroprotective effects of ant-134 are due to de - repression of lim domain kinase 1 .
ant-134 treatment also increases dendritic spine volume and changes spine number in the hippocampus,19 , 22 which could influence excitability and would also be consistent with a mechanism involving protection of mir-134 targets such as lim domain kinase 1 or pumilio rna - binding family member 2
. altered spine volume and number is consistent with our observation of a robust local effect of suppressing mir-134 on delaying epileptiform activity , although additional effects on long range connections can not be ruled out .
we must also consider that the acute seizure - suppressive effects and long - term anti - epileptogenesis effects may arise from different mechanisms .
ultimately , in vivo studies will be necessary to determine the anti - seizure mechanism of ant-134 ( e.g. , by analyzing gene expression networks , quantifying the proteome , and applying techniques to validate antagomir - mirna targeting such as polysome shift assays ) .
what are the next steps for translation or pre - clinical development ? an obvious question is whether and how antagomirs could be given to patients .
their large size precludes their passing an intact blood - brain barrier ( bbb ) .
recent work has explored methods to circumvent the bbb , including conjugating antagomirs with brain - penetrating peptides or administering antagomirs via intranasal injection.19 , 51 the bbb may also be open after certain epilepsy - precipitating injuries or it can be physically breached , such as through use of ultrasound . another question is whether ant-134 injection into already epileptic animals could alter the frequency or duration of spontaneous seizures or attendant hippocampal pathology .
success with that approach could provide novel treatment opportunities for the population of drug - resistant patients for whom surgery or other alternatives to aeds are either not possible or not effective currently . in summary ,
the present study provides important additional validation of the potent seizure - suppressive and possibly disease - modifying effects of ant-134 in two different animal models and one brain slice model .
the effectiveness of ant-134 and the potency of seizure suppression suggest a target for drug development in epilepsy that could provide additional effects beyond those provided by any currently marketed drug for epilepsy .
this study was approved by the ethics ( medical research ) committee of beaumont hospital of dublin ( no . 05/18 ) and written informed consent was obtained from all patients .
the control ( autopsy ) hippocampus ( n = 12 ) was obtained from 12 individuals .
patients ( n = 12 ) were referred for surgical resection of the temporal lobe by an epileptologist ( n.d . ) following neurological assessment , video - eeg recording , and mri / neuroimaging . each patient was determined to have medically intractable tle with a history of recurring seizures ( table s2 ) .
the hippocampus was obtained and a portion of each specimen was frozen in liquid nitrogen and stored at 70c until use .
a pathologist ( m.a.f . ) assessed the hippocampus as part of the pathologic evaluation , addressed the degree of hippocampal sclerosis , and described other pathologic changes .
all animal experiments were performed in accordance with the european communities council directive ( 2010/63/eu ) .
procedures in mice were approved by the research ethics committee of the royal college of surgeons in ireland ( rec-842 ) , under license from the ireland health products regulatory authority ( ae19127/001 ) .
procedures in rats were performed under authorization of the philipps university bioethics committee and were approved by the local regulation authority ( regierungsprsidium gieen ) , or they were performed according to the uk animals ( scientific procedures ) 1986 act ( ppl 70 - 7684 ) and approved by local ethical review ( university college london ) .
male adult c57bl/6j mice ( 2025 g ) and male sprague - dawley rats ( 325350 g or 200300 g ; all from harlan ) were used in all studies .
animals were housed in on - site barrier - controlled facilities having a 12-hr/12-hr light / dark cycle with ad libitum access to food and water .
mice were equipped for eeg recordings under surgical anesthesia ( isoflurane ; 5% induction , 1%2% maintenance ) in a mouse - adapted stereotaxic frame .
body temperature was maintained within the normal physiological range with a feedback - controlled heat pad ( harvard apparatus ) .
after a midline scalp incision , partial craniectomies were performed and animals had skull - mounted recording electrodes placed and fixed with dental cement .
a second cohort of mice had eeg electrodes and a guide cannula implanted to allow i.c.v .
this cannula was placed on the dura mater with the following coordinates from the bregma : anterior - posterior ( ap ) , + 0.3 mm ; lateral ( l ) , + 0.9 mm ; and ventral ( v ) , 2.0 mm .
after recovery from surgery , mice were connected to the lead socket of a swivel commutator , which was connected to a grass twin digital eeg system .
] ) with different doses of ptz ( 60 , 80 , or 100 mg / kg ) . lorazepam ( 8 mg / kg , i.p . ) was administered after the first tonic - clonic seizure to reduce morbidity and mortality .
mice were followed for 30 min or 4 hr after ptz administration for the appearance of seizures by electrographic and behavioral methods .
after the observation period , mice were deeply anesthetized ( pentobarbital ) and transcardially perfused , and their brains were removed for analysis of gene expression or histological assessment .
injection of 0.12 nmol/2 l 3-cholesterol - tagged locked nucleic acid oligonucleotide targeting mir-134 ( ant-134 ) or its respective control , a non - targeting scrambled version of the antagomir ( scr ; 0.12 nmol/2 l ) ( exiqon a / s ) , as previously described.19 , 22 twenty - four hours later , mice were connected to an eeg system and a baseline recording was obtained .
next , mice were injected with ptz ( 80 mg / kg , i.p . ) .
lorazepam ( 8 mg / kg , i.p . ) was administered after the first tonic - clonic seizure to reduce morbidity and mortality .
these mice were followed up for 30 min after ptz administration for the appearance of seizures .
after this period , mice were deeply anesthetized ( pentobarbital overdose ) and transcardially perfused ( pbs ) for further assessments .
under isoflurane ( 5% induction , 3% maintenance ) anesthesia , bipolar stainless - steel stimulating electrodes ( nex-200 ; rhodes medical instruments ) were positioned in the angular bundles of the perforant pathway and custom unipolar recording electrodes ( crafted from 796000 ; a - m systems ) were lowered into the dorsal dentate gyrus .
electrodes and ground screws were connected to miniature wireless transmitters ( ft20 ; data sciences international ) that were implanted subcutaneously on the animal s flank .
plastic connectors ( gs09plg ; ginder scientific ) joined the electrodes with stimulation / recording equipment .
the pps protocol utilized a paradigm designed to evoke and maintain hippocampal seizure activity throughout the stimulation , but not convulsive status epilepticus , which consisted of continuous , bilateral 2-hz paired - pulse stimuli , with a 40-ms interpulse interval , plus a 10-s train of 20 hz single - pulse stimuli delivered once per minute , generated by an s88 stimulator ( grass instruments ) .
all pulses ( 0.1-ms duration ) were delivered at 2024 v , as this voltage reliably evokes granule cell discharging without tissue - damaging hydrolysis.29 , 40 the current associated with these voltages was typically between 15 and 30 a .
no samples displayed any signs of hydrolysis ( e.g. , holes around the stimulating electrodes ) .
as described previously , stimulation for 30 min ( on 2 consecutive days ) required only isoflurane to terminate seizures .
eight - hour stimulation on the third day did not induce convulsive status epilepticus and , therefore , did not require pharmacological termination .
rats were randomly divided in three cohorts to assess the following : ( 1 ) mir-134 expression after pps ( 24 hr , 4 days , and 14 days ) ; ( 2 ) the effect of ant-134 on pps - induced status epilepticus , in which rats were pre - treated with ant-134 or scr ( 0.36 nmol/6 l , i.c.v . ; exiqon a / s ) 24 hr before the 8-hr stimulation ; and ( 3 ) the effect of ant-134 on epileptogenesis , in which rats were injected with 0.36 nmol/6 l ( i.c.v . ) of ant-134 or scr ( exiqon a / s ) immediately after 8-hr stimulation .
five rats were excluded from this study due to practical issues ( e.g. , blocked cannula ) . for statistical purposes
, we pooled samples from 24 hr and 4 days after pps as an early time point , while 14-day samples were considered as the late time point .
mouse eeg data were analyzed and quantified using labchart 8 software ( ad instruments ) .
ptz - induced seizures were defined as high - amplitude ( > 2 baseline ) high - frequency ( > 5 hz ) polyspike discharges lasting > 5 s. for statistical purposes , we assigned a cut - off time of 1,800 s for those animals that did not present seizures during the observation period . from the eeg recordings ,
we calculated the delay to first seizure , the total power , and the percentage of total power / spectral bands , as previously described.19 , 54 eeg total power was plotted as the percentage of baseline recording ( each animal s eeg power post - seizure compared to its own baseline eeg ) .
clinical behavior was scored using the following adapted racine scale : score 0 , no seizures observed ; score 1 , rhythmic mouth and facial movement ; score 2 , rhythmic head nodding ( bobbing ) ; score 3 , forelimb clonus ; score 4 , rearing and bilateral forelimb clonus ; and score 5 , rearing and falling ( stay fallen on rear side ; tonic - clonic seizures ) .
the highest score reached during every 5 min during the 30 min after ptz was recorded .
for studies in rats , eeg activity during and after pps was amplified and recorded digitally at 2 khz utilizing a powerlab 16/35 and labchart software versions 7 and 8 ( ad instruments ) .
spontaneous granule cell layer activity was recorded continuously ( 24 hr / day ) and stored digitally and automatically in 3-hr epochs . each day , we analyzed the preceding 24 hr of recordings , as well as all events with amplitudes exceeding 120% of average baseline .
continuous ( 24 hour/7 day ) video monitoring utilized edimax 7010w infrared cameras .
video files were captured at 1215 frames / sec and were time - stamped for integration with the electrophysiological data using securityspy surveillance software ( ben software ) and stored digitally . confirmed seizures were scored according to the racine scale .
rats were anesthetized with isoflurane ( 5% induction,2% maintenance as appropriate ) and mounted in a stereotaxic frame .
we injected ant-134 ( 0.12 nmol ) in 1 l te buffer ( life technologies ) at coordinates relative to the bregma : ap , 0.92 mm ; medial - lateral ( ml ) , + 1.3 mm , and dorsal - ventral ( dv ) , 3.3 mm to target the lateral ventricle . to allow the rats to fully recover from the analgesic drugs and to coincide with the maximal anti - epileptic effect seen in vivo
sodium pentobarbital and transcardially perfused with ice - cold sucrose artificial cerebrospinal fluid ( acsf ; in mm : 205 sucrose , 10 glucose , 2.5 kcl , 0.1 cacl2 , 5 mgcl2 , 26 nahco3 , and 1.2 nah2po4.h2o ; osmolarity , 295 mosm ) .
the brain was quickly dissected into ice - cold sucrose acsf and transferred to a campden 700 smz slicer ( campden instruments ) . slices ( 400 m ) were prepared in the horizontal orientation between the bregma at approximately 7.1 and 5.1 mm . for storage ,
slices were submerged in oxygenated normal acsf ( in mm : 125 nacl , 10 glucose , 3 kcl , 1 mgcl2 , 2 cacl2 , 26 nahco3 , and 1.25 nah2po4.h2o ; osmolarity , 290 mosm ) and allowed to recover at room temperature for at least 60 min prior to recordings .
slice recordings were made using a membrane chamber ( scientific systems design ) , a modified submerged chamber , which improves oxygen supply to the tissue and supports greater epileptiform bursts than conventional submerged recording chambers .
slices were continually perfused with acsf , heated to 32c , at a flow rate of 16 ml / min . for local field potential ( lfp ) recordings ,
borosilicate glass recording micropipettes ( resistance , 34 m ) were placed into hippocampal ca1b stratum pyramidale and epileptiform activity was induced by raising the perfusing kcl concentration to 9 mm .
total rna was isolated from brain samples using trizol ( invitrogen ) , as described previously.19 , 54 for mrna qpcr , 1 g total rna was used to generate cdna by reverse transcription using the superscript iii reverse transcriptase enzyme ( invitrogen ) .
the pcr amplification was performed for 50 cycles on a thermocycler ( applied biosystems ) .
quantitative real - time pcr was performed using a lightcycler 1.5 ( roche diagnostics ) in combination with quantitect sybr green pcr kit ( qiagen ) as per the manufacturer s protocol with 25 m of primer pairs used .
specific primers for each gene assayed were purchased from sigma and sequences used were as follows : actin , beta ( actb ) ( forward [ f ] , ggttggccttagggttcagg ; reverse [ r ] , gggtgtgatggtgggaatgg ) ; c - fos ( f , cattcagaccacctcgacaa ; r , ggaattaacctggtgctgga ) ; and arc ( f , agcagcagacctgacatcct ; r , gtgatgccctttccagacat ) .
data were normalized to actb and relative mrna transcript levels were quantified using the ct method . for mirna ,
250 ng rna was reverse transcribed using stem - loop specific primers for mmu - mir-134 ( applied biosystems ) and real - time quantitative pcr was carried out on a 7900ht fast real - time system ( applied biosystems ) using taqman mirna assays .
expression of rnu19 was used for normalization . a relative fold change in expression of mir-134
was determined using the ct method.19 , 56 specific mirna assays were obtained from thermofisher scientific for mir-134 ( i d 001186 ) , mir-19a ( i d 000395 ) , and rnu19 ( i d 001003 ) .
data were normalized to rnu19 and relative mirna expression levels were quantified using the ct method .
statistical analysis was performed using graphpad prism and stata release 14 software , and a p value < 0.05 was considered significant .
latencies to tonic - clonic seizures were analyzed by the kruskal - wallis test , followed by the nonparametric dunn multiple comparison test when indicated .
total time spent in seizures , mean amplitude , frequency , total power of eeg recordings , and mrna levels were analyzed by the student t test or one - way anova followed by the bonferroni test , depending on the experimental design .
ex vivo brain slice data were analyzed using the mann - whitney u test .
three comparisons were made from the same data and ( significance level ) was consequently reduced to 0.025 for these experiments .
, d.f.o . , and n.d . provided human samples and data ; c.r.r . , s.s .
wrote the paper and all co - authors contributed to data interpretation , review , and editing of the final paper .
the royal college of surgeons in ireland filed for a patent on the inhibition of microrna-134 for the treatment of seizure - related disorders and other neurologic injuries .
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current anti - epileptic drugs ( aeds ) act on a limited set of neuronal targets , are ineffective in a third of patients with epilepsy , and do not show disease - modifying properties .
micrornas are small noncoding rnas that regulate levels of proteins by post - transcriptional control of mrna stability and translation .
microrna-134 is involved in controlling neuronal microstructure and brain excitability and previous studies showed that intracerebroventricular injections of locked nucleic acid ( lna ) , cholesterol - tagged antagomirs targeting microrna-134 ( ant-134 ) reduced evoked and spontaneous seizures in mouse models of status epilepticus .
translation of these findings would benefit from evidence of efficacy in non - status epilepticus models and validation in another species . here , we report that electrographic seizures and convulsive behavior are strongly reduced in adult mice pre - treated with ant-134 in the pentylenetetrazol model .
pre - treatment with ant-134 did not affect the severity of status epilepticus induced by perforant pathway stimulation in adult rats , a toxin - free model of acquired epilepsy .
nevertheless , ant-134 post - treatment reduced the number of rats developing spontaneous seizures by 86% in the perforant pathway stimulation model and ant-134 delayed epileptiform activity in a rat ex vivo hippocampal slice model .
the potent anticonvulsant effects of ant-134 in multiple models may encourage pre - clinical development of this approach to epilepsy therapy .
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Introduction
Results
Discussion
Materials and Methods
Author Contributions
Conflicts of Interest
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microglia account for approximately 10% of the adult brain cell population and represent the first and main form of immune defense in the central nervous system ( cns ; lawson et al .
microglia become activated and they can be identified and distinguished from their resting phenotype based on a combination of morphological and immunophenotypic changes ( dheen et al . , 2007 ; ransohoff and perry , 2009 ) .
microglia initiate immune responses by enhancing the expression of toll - like receptors ( tlr ) and a wide range of pro - inflammatory mediators such as tumor necrosis factor - alpha ( tnf ) , interleukin ( il)-1 and il-6 for the removal of the cns threat ( suzumura et al . , 1996 ;
microglia may also fulfill a neuroprotective role via the release of neurotrophic factors and promotion of neurogenesis for the restoration of normal physiology ( stadelmann et al . , 2002 ) .
hence , the acute inflammatory response is generally beneficial , as it tends to minimize injury and promotes tissue repair .
however , chronic neuroinflammation is closely related to various neurodegenerative disorders such as parkinson s disease ( pd ) , huntington s disease ( hd ) , dementias , and multiple sclerosis ( ms ) , although the consequences of sustained microglial activation in these diseases is unclear .
activated microglia upregulate expression of the 18-kda translocator protein ( tspo ; chen and guilarte , 2008 ; cosenza - nashat et al .
tspo are found in abundance throughout the body in peripheral organs ( i.e. , liver and adrenals ) , and hematogenous cells , but are present at very low levels in the normal healthy cns ( banati , 2002 ) .
functionally , tspo has several biological functions including the control of cholesterol transport and neurosteroid synthesis ( papadopoulos et al . , 2006 ) , and
may also be involved in the release of pro - inflammatory cytokines during inflammation ( choi et al . , 2002 ; wilms et al . ,
enhanced tspo expression can be detected in vivo by using positron emission tomography ( pet ) imaging with the selective tspo radioligand c - pk11195 ( benavides et al .
, 1993 ; banati et al . , 1999 ) , with evidence that increases in c - pk11195 binding potential ( bpnd ) correspond to activation of microglia ( stephenson et al . , 1995 ; conway et al .
although tspo is also expressed by reactive astrocytes , a c - pk11195 pet study of patients with hippocampal sclerosis , a condition histopathologically characterized by marked astrogliosis , did not yield results that were significantly different to healthy normal controls ( banati et al .
this is consistent with the view that reactive astrocytes , in vivo , do not significantly contribute to the c - pk11195 signal .
therefore , in the absence of invading blood borne cells or severe focal leakage of blood - brain barrier , the increased pk11195 binding is likely to indicate the transition of microglia from a resting to an activated state , and is due to an increase in the number , rather than the affinity , of tspo ( banati et al . , 2000 ) .
hence , the measurement of tspo uptake using pet provides an in vivo tool to monitor progression and severity of neuroinflammation and is a useful indicator of active cns disease .
this article aims to review the use of pet imaging to promote the understanding of activated microglia in neurodegenerative disease .
parkinson s disease is the second most common neurodegenerative disorder of the elderly and is associated with the motor symptoms of tremor , bradykinesia , and rigidity .
it is characterized by the extended loss of dopaminergic neurons in the substantia nigra pars compacta , resulting in a deficiency of dopamine in the striatum ( braak et al . , 2006 ) , and the presence of alpha - synuclein ( -synuclein)-containing lewy bodies .
pd is the most common of a group of parkinsonian movement disorders that also includes multiple system atrophy ( msa ) , corticobasal degeneration ( cbd ) , and progressive supranuclear palsy ( psp ) .
the presence of activated microglia close to dopaminergic neurons in post - mortem pd patient brains ( mcgeer et al . , 1988a ; mogi et al . , 1994 ; langston et al . , 1999 ; imamura et al . ,
2003 ) , and pd animal models ( czlonkowska et al . , 1996 ; kim et al . , 2009 ) suggests a close relationship between neurodegeneration and neuroinflammation in pd .
numerous investigations have proposed a deleterious role of microglial activation in pd based on the vulnerability of dopaminergic neurons to various microglia - derived pro - inflammatory cytokines ( ferrari et al . , 2006 ; stone et al . , 2009 ; de lella ezcurra et al . , 2010 ) , while -synuclein can directly induce activation of microglia ( zhang et al . , 2005 ) .
however , it seems that the plasticity of microglia must be considered with regards to their contribution in pd , and their role ; whether beneficial or detrimental , it may depend on the stimuli present and the stage of disease ( li et al .
, 2007 ; michelucci et al . , 2009 ; sanchez - guajardo et al . ,
further clues regarding the role of activated microglia has also come from in vivo pet imaging studies ( table 1 ) .
significant microglial activation , as reflected by an increase in c - pk11195 bpnd was reported in the midbrain and putamen of pd patients when compared to controls , and was found to correlate positively with the motor severity of parkinsonism ( ouchi et al . , 2005 ; bartels et al . , 2010
these findings suggest that activated microglia has a pathogenic importance in the disease and indicate that the early introduction of a neuroprotective drug to suppress microglial activation could be favorable in pd . additionally , pd patients exhibited significantly increased c - pk11195 bpnd in the basal ganglia , pons , and frontal and temporal cortical regions ( gerhard et al . , 2006a ) .
in this study , the increased microglial activation remained unchanged for 2 years , while the patients deteriorated clinically during this period .
hence , it is likely that microglia are activated early in pd , where they remain activated for longer periods and possibly drive progression of the disease ( gerhard et al . , 2006a ) .
bpnd , binding potential ; cbd , corticobasal degeneration ; msa , multiple system atrophy ; nc , normal control ; pd , parkinson s disease ; psp , progressive supranuclear palsy .
multiple system atrophy is a sporadic neurodegenerative disorder involving a progressive akinetic - rigid syndrome , autonomic failure , and cerebellar dysfunction .
it is associated by the appearance of abnormal glial cytoplasmic inclusions ( gci ) containing ( -synuclein aggregates and neuronal loss within the nigrostriatal and olivopontocerebellar regions ( lantos and papp , 1994 ) .
the presence of activated microglia is also a prominent feature of msa ( schwarz et al . , 1998 ) .
in an in vivo pet study of msa patients , significant c - pk11195 bpnd was observed in the putamen , pallidum , pons , substantia nigra pars compacta , and dorsolateral prefrontal cortex , reflecting the known distribution of neuropathological changes in msa ( gerhard et al . , 2003 ) . although the role of microglia in msa is inconclusive , microglial activation localization correlated significantly with the locations of gcis in specific neuroanatomical systems affected in msa ( ishizawa et al . , 2004 ) .
a correlation between extent of microglial activation and dopaminergic neurodegeneration has also been reported ( stefanova et al . , 2007 ) .
progressive supranuclear palsy is an adult - onset progressive neurodegenerative disease of unknown cause , characterized by pd - like symptoms such as postural instability and bradykinesia .
the pathological hallmark of the disease is neurofibrillary tangles consisting of hyperphosphorylated tau , accompanied by neuronal loss in the thalamus , basal ganglia , and specific brainstem regions ( hauw et al .
several early studies including immunohistochemical investigations have confirmed the possible involvement of activated microglia in psp ( kida et al .
, 1992 ; komori et al . , 1998 ; ishizawa et al . , 2000 ; ishizawa and dickson , 2001 ) .
c - pk11195 pet have also reported significant levels of activated microglia in brain regions known to be affected by the disease process such as the midbrain , cerebellum , pons , frontal lobe , and basal ganglia ( gerhard et al . , 2006b ) . although these results were unable to support a direct causal contribution to neurodegeneration in psp , they are at least suggestive of a role of microglia in the disease .
corticobasal degeneration is a neurodegenerative disorder that affects both cortical and basal ganglial regions , with considerable clinical heterogeneity between patients .
typically , cbd features an asymmetric hypokinetic - rigid syndrome , coupled with alien limb phenomenon and cortical sensory impairment that is unresponsive to dopaminergic therapy ( rebeiz et al . , 1968 ; gibb et al . , 1989
information on the association of activated microglia in cbd is limited , and mainly coming from immunohistochemical - based assessments ( armstrong et al . , 2000 ; ishizawa and dickson , 2001 ) .
however , more recent in vivo pet investigations have attempted to quantify microglial activation in cbd patients .
increased c - pk11195 bpnd was observed in regions such as the caudate nucleus , putamen , substantia nigra pars compacta , pons , and pre- and post central gyrus ( gerhard et al .
2004 ) that correspond to the expected neuropathological changes seen in cbd ( ishizawa and dickson , 2001 ; dickson et al . , 2002 ) .
huntington s disease is an autosomal , dominant inherited progressive neurodegenerative disorder associated with motor , cognitive , and psychiatric symptoms .
it is caused by an abnormal polyglutamine - repeat expansion on the it15 gene that codes huntingtin , and involves the progressive loss of medium spiny dopaminergic receptor - bearing striatal gaba - ergic neurons ( vonsattel and difiglia , 1998 ) .
although the role of chronic neuroinflammation in the hd pathogenesis is not fully understood , post - mortem assessments have reported high levels of activated microglia close to degenerating neurons ( mcgeer et al .
upregulated inflammatory cytokines have also been detected in the striatum and plasma , indicative of an inflammatory component in hd ( dalrymple et al . , 2007 ;
imaging studies using c - pk11195 pet have found increased microglial activation in both premanifest hd gene carriers and manifest hd patients when compared to healthy controls ( table 2 ; pavese et al .
, 2006 ; tai et al . , 2007 ; politis et al . , 2008 , 2011 ) . in premanifest
hd patients , significant increases in c - pk11195 bpnd in the striatum and hypothalamus was reported , which correlated inversely with neuronal dysfunction as measured by c - raclopride ; a marker of dopaminergic d2/d3 receptor availability ( tai et al . , 2007 ; politis et al . , 2008 ) .
interestingly , microglial activation in the striatum , and regions related to cognitive function has been shown to predict the 5-year disease clinical onset in premanifest hd patients ( tai et al .
these results imply that microglial activation is an early event in the hd disease course , with a possible pathogenic involvement that is associated with a subclinical progression of the disease .
bpnd , binding potential ; hd , huntington s disease ; nc , normal control . in manifest hd patients ,
significant c - pk11195 bpnd in the striatum , hypothalamus , and various cortical regions was found , that correlated with greater disease burden and higher motor disability ( pavese et al .
the cortical microglial activation is likely to indicate the involvement of cortical neurons in hd , a well - recognized phenomenon as the disease progresses .
collectively , these findings are consistent with the post - mortem studies ( messmer and reynolds , 1998 ; sapp et al . , 2001 ) and suggest a detrimental microglial contribution to the ongoing neuronal degeneration in hd .
dementias are a group of disorders that are expected to affect more than 100 million people by 2050 raising remarkable financial costs for healthcare ( wimo et al . , 2003 ) .
ad is the most common cause of dementia and is the most common neurological disorder of the elderly .
ad is characterized by the presence of amyloid plaques , neurofibrillary tangles , and activated microglia ( for review , see hardy and selkoe , 2002 ) .
there is a plethora of evidence from post - mortem human ad studies ( mcgeer et al . , 1988a ; venneti et al . , 2009 ) and animal models ( frautschy et al . , 1998 ; stalder et al
, 1999 ; leung et al . , 2011 ) reporting a high accumulation of activated microglia in close proximity with the amyloid plaques , and upregulated levels of pro - inflammatory cytokines ( akiyama et al . , 2000 ; eikelenboom et al . ,
positron emission tomography enables a broad range of functional processes to assess the ad brain in vivo ( table 3 ) .
c - pk11195 has been used to demonstrate increased levels of activated microglia in both ad animal models ( venneti et al . , 2009 ) and ad patients ( cagnin et al . , 2001 ; edison et al . , 2008 ; yokokura et al . , 2011 ) . in ad patients ,
significant c - pk11195 bpnd was consistently observed in the temporal , parietal , and occipital cortices , regions known to be affected by ad pathology ( cagnin et al . , 2001 ;
the increased activated microglia also inversely correlated with the patient mini - mental state examination ( mmse ) scores , which is compatible with a role of microglia in neuronal damage ( edison et al . , 2008 ) .
interestingly , elevated levels of activated microglia were also detected in patients with amnestic mild cognitive impairment ( mci ; okello et al . , 2009a ) , although this was not observed in another study assessing mci patients ( wiley et al .
mci could represent an early precursor stage of ad , since it was found that mci patients with increased amyloid load were significantly more likely to clinically convert to ad within 3 years ( okello et al . , 2009b ) .
therefore , microglial activation could be an early event in the ad pathogenesis that begins at the mci stage .
ad , alzheimer s disease ; bpnd , binding potential ; ftld , frontotemporal lobar degeneration ; mci , mild cognitive impairment ; mmse , mini - mental state examination ; nc , normal controls . despite the evidence suggestive of a pathogenic role of activated microglia in ad
, it is hypothesized that the accumulation of amyloid plaques is actually due to a failure in microglial clearance mechanisms that would normally remove the protein ( bornemann et al . , 2001 ;
this indicates a beneficial , rather than detrimental role of microglia in ad . notwithstanding the abundance of activated microglia close to senile plaques , they maybe inefficient in the clearance of amyloid , hence , resulting in aggregate formation ( bolmont et al . , 2008 ) .
it has been shown that in the presence of pro - inflammatory cytokines , phagocytic functions of microglia are compromised ( koenigsknecht - talboo and landreth , 2005 ) .
therefore , microglia may confer a dichotomous role in ad , where early microglial activation is possibly neuroprotective involving the removal of amyloid . however , chronic neuroinflammation may downregulate amyloid clearance mechanisms , thus , promoting aggregation and progression of disease .
frontotemporal lobar degeneration ( ftld ) which includes frontotemporal dementia is the name given to a group of pathologically , clinically , and genetically heterogeneous disorders involving focal atrophy of the frontal and temporal lobes , while unlike ad , with sparing of the parietal and occipital regions ( neary et al . , 1998 ) .
another important dissimilarity between ad and ftld pathology is the absence of amyloid plaque formation ( paulus et al . , 1993 ;
rather , the key histopathological features of ftld , depending on subtype , includes tau deposition ( including pick bodies ) and ubiquitin - positive , tau - negative inclusions ( munoz et al . , 2003 ; uchihara et al . , 2003 ) .
in vivo pet imaging of ftld patients detected enhanced microglial activation in the expected frontotemporal regions ( cagnin et al . ,
2004 ) . in the same study , significant c - pk11195 bpnd in the bilateral putamen is also consistent with previous neuropathological data showing the involvement of the basal ganglia in ftld ( mirra and hyman , 2002 ) .
these observations indicate the presence of an active microglial response that reflects progressive neuronal degeneration .
importantly , the detection of increased microglial activation in affected regions in ftld suggests that microglial responses occur independently of amyloid deposition , and that neuronal loss alone is enough to induce activation ( cagnin et al . , 2004 ) .
multiple sclerosis is a disease characterized pathologically by inflammatory demyelination and axonal transection , and is the most common cause of non - traumatic disability in young adults ( compston and coles , 2008 ) .
the involvement of activated microglia has long been proposed in ms ( benveniste , 1997 ) .
post - mortem investigations have detected activated microglia in the cortical gm of ms patients ( de groot et al .
2002 ) , while histopathological studies have implicated microglia in lesion pathogenesis ( for review , see lassmann , 2008 ) .
an observed correlation between neuronal loss and microglial activation was reported in animal experimental ms ( rasmussen et al . , 2007 ) .
significant levels of activated microglia was also found in ms patients , especially in the progressive forms of disease that are associated with neurodegeneration ( kutzelnigg et al . , 2005 ; magliozzi et al . ,
2010 ) , and selective ablation of parenchymal microglia was able to prevent demyelination and axonal damage ( heppner et al . , 2005 ) .
pathological aspects of ms such as neuroinflammation , demyelination , and neurodegeneration may be explored in vivo with pet ( for review , see kiferle et al . , 2011 ) .
pet with c - pk11195 and other tracers has demonstrated inflammatory processes with microglial involvement in ms ( figure 1 ; table 4 ) . in animal
experimental ms and human post - mortem brains it has been shown that c - pk11195 uptake corresponds to the distribution pattern of activated microglia ( banati et al . , 2000 ) .
it has also been demonstrated that there is increased c - pk11195 bpnd in areas of focal pathology identified by t1- and t2-weighted mri ( vowinckel et al . , 1997 ; banati et al . , 2000 ) and in gadolinium - enhanced t1-weighted mri ( debruyne et al . , 2003 ) .
increased c - pk11195 bpnd was observed in normal - appearing gray and white anatomical structures ( banati et al .
this is in line with the hypothesis that inflammatory processes initiated by microglia early in ms may constitute the real burden of disease , associated with invisible microglia - mediated damage that occur independently of relapses ( kesselring , 1990 ; confavreux et al . , 2000 ) .
a positive correlation has been suggested between ligand uptake and disease duration , disability , and brain atrophy ( banati et al . , 2000 ;
however , recent data from our group found a significant association between high c - pk11195 bpnd in the cortical gray matter and disability in patients with secondary progressive ms , and with higher c - pk11195 bpnd in the secondary progressive group than the relapse - remitting ms group ( politis et al .
enhanced microglial activation in ms has also been detected using the more recently developed tspo tracers c - vinpocetine and c - pbr28 ( vas et al . , 2008 ; oh et al . , 2011 ) .
positron emission tomography images showing increased c - pk11195 bpnd in a multiple sclerosis patient ( a ) when compared to a healthy normal control ( b ) . color bar represents intensity of c - pk11195 tracer binding ( bpnd ) .
bpnd , binding potential ; gm , gray matter ; mri , magnetic resonance image ; ms , multiple sclerosis ; nawm , normal - appearing white matter ; nc , normal control ; pp , primary progressive multiple sclerosis ; rr , relapse - remitting multiple sclerosis ; sp , secondary progressive multiple sclerosis . microglial activation may contribute to the mechanism of axonal injury via the release of soluble factors that may either directly or indirectly cause neuronal dysfunction ( peterson et al .
, 2001 ; barnett and prineas , 2004 ; dutta and trapp , 2006 ; zipp et al . , 2006 ; dal bianco et al . , 2008 ; lassmann , 2008 ; magliozzi et al . , 2010 ) ,
however , activated microglia may also exert protective functions with ms through the release of neurotrophic factors ( stadelmann et al .
, 2002 ; napoli and neumann , 2009 ) , and triggering of remyelination mechanisms ( li et al . , 2005 ; setzu et al . ,
c - pk11195 was the first tracer to be consistently used for the study of activated microglia and neuroinflammation in vivo .
however , limitations associated with the application of c - pk11195 include a high level of non - specific binding ( petit - tabou et al . , 1991 ) , and a poor signal to noise ratio , which complicates its quantification ( boutin et al . , 2007 ) .
this has prompted the search for novel pet tracers ( termed , second generation radioligands ) with improved capacities to quantify tspo expression .
radioligands such as c - pbr28 , c - daa1106 , f - fedaa1106 , and f - pbr111 have recently been developed to image tspo in vivo ( gulys et al . , 2002 ; ikoma et al . , 2007 ;
, 2011 ; for a review , see chauveau et al . , 2008 ) .
published data using the second generation ligands c - daa1106 ( ikoma et al . , 2007 ) and f - fedaa1106 ( fujimura et al . , 2006 ) in humans were promising , with both tracers showing significantly higher cerebral uptake than c - pk11195 .
furthermore , increased c - daa1106 binding was reported in ad patients ( yasuno et al .
, 2008 ) that were similar to the previous studies that used c - pk11195 ( cagnin et al . , 2001 ) . the only published study using the second generation radioligand c - pbr28 found areas of focal increases in radiotracer binding in the brain of ms patients ( oh et al . , 2011 )
interestingly , the increased focal c - pbr28 binding preceded the development of some gadolinium - enhancing lesions . brain parenchymal c - pbr28 binding in ms patients was positively correlated with the duration of the disease , however it was not significantly higher than that of healthy volunteers . interpretation of these results is limited by the lack of characterization of the binding affinity pattern , which might have significantly affected the comparison between subjects .
it has been recently demonstrated that there are three different affinity patterns for second generation tspo ligands in healthy volunteers as well as patients with ms , which was evident with all the ligands tested ( c - pbr28 ; c - pbr06 ; f - pbr111 ; owen et al . , 2010 ) .
this presents a methodological problem , as differences in pet signal across subjects can not be safely interpreted as differences in target density , but may reflect differences in the affinity pattern . a possible approach to solve this problem
is based on the use of peripheral binding affinity , which can be characterized to classify subjects into one of the groups , as differences in affinity status between individuals have been shown to be present on peripheral cells as well ( owen et al .
interestingly , the difference in binding patterns observed with second generation radioligands was not observed with c - pk11195 .
also , in vitro autoradiography data using c - pk11195 suggest a receptor density ( bmax ) significantly higher than that found using second generation ligands .
it could be speculated that c - pk11195 and newer ligands bind to distinct sites within the tspo molecule .
although , data obtained from first generation studies have been promising and suggested that c - pk11195 could be useful to image acute inflammatory lesions and microglial activation in ms , a conclusive demonstration of the potential of tspo imaging for the application as disease biomarker , indicative of microglial activation in ms , is still lacking .
furthermore , despite second generation ligands constituting a potential improvement relative to c - pk11195 at least from a methodological point of view , a clear advantage in their clinical application as disease biomarkers has not been demonstrated yet .
for these reasons , we aim to characterize a second generation tspo pet radioligand in vivo in humans , and to evaluate its application as a disease biomarker in ms . among second generation tspo tracers ,
f - pbr111 presents different advantages , as there is low difference in its affinity for tspo between high , medium , low affinity binders .
also , it could be potentially used in clinical applications as it is labeled with fluorine-18 .
promising preclinical data , and ongoing studies in neurological patients , suggest it could be a good choice amongst second generation tspo ligands to progress into studies in ms patients .
inflammation coupled with the presence of activated microglia seems to be a common feature of a wide range of cns diseases .
however , despite a large number of research studies , the exact role of microglia in chronic neurodegenerative diseases remains uncertain . in line with the high plasticity of microglia that allows them to perform numerous cns functions , microglia are likely to play a dichromatic role in disease , depending on signals present in their microenvironment and the duration of activation . while early microglial activation could represents a beneficial response ( i.e. , removal of cns threat , promoting tissue repair and removal of misfolded protein ) , chronic exposure could induces detrimental effects by promoting neuronal death ( i.e. , through the sustained release of neurotoxic factors ) , thus , contributing to progression of disease . pet imaging with the use of tspo radioligands
provides a valuable tool that allows us to track the progression and severity of neuroinflammation in the living brain , and is a useful indicator of active cns disease .
therefore , the early detection of microglia using pet could offer opportunities for pharmacological interventions to limit the potential disruptive effects of chronic microglial activation .
furthermore , with the development of newer tspo tracers , the potential for pet imaging research to promote our understanding of activated microglia in cns disease can only increase .
the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest .
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microglia constitute the main immune defense in the central nervous system . in response to neuronal injury ,
microglia become activated , acquire phagocytic properties , and release a wide range of pro - inflammatory mediators that are essential for the annihilation of the neuronal insult .
although the role of microglial activation in acute neuronal damage is well defined , the pathophysiological processes underlying destructive or protective role to neurons following chronic exposure to microglial activation is still a subject of debate .
it is likely that chronic exposure induces detrimental effects by promoting neuronal death through the release of neurotoxic factors .
positron emission tomography ( pet ) imaging with the use of translocator protein ( tspo ) radioligands provides an in vivo tool for tracking the progression and severity of neuroinflammation in neurodegenerative disease .
tspo expression is correlated to the extent of microglial activation and the measurement of tspo uptake in vivo with pet is a useful indicator of active disease .
although understanding of the interaction between radioligands and tspo is not completely clear , there is a wide interest in application of tspo imaging in neurodegenerative disease . in this article , we aim to review the applications of in vivo microglia imaging in neurodegenerative disorders such as parkinson s disease , huntington s disease , dementias , and multiple sclerosis .
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Introduction
Parkinsons Disease and Related Disorders
Huntingtons Disease
Dementia
Multiple Sclerosis
New TSPO Ligands
Conclusion
Conflict of Interest Statement
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rheumatoid arthritis ( ra ) is a chronic systemic inflammatory disease affecting predominantly joints , synovial membranes , articular cartilages , and subchondral bones .
disease progression is attributed to increases in reactive oxygen species ( ros ) and oxidative stress ( os ) in the lesion sites .
proinflammatory cytokines , such as tumor necrosis factor- ( tnf- ) , interleukin-1 ( il-1 ) , and il-6 , regulate the inflammatory and immune responses and play a pivotal role in the disease .
overproduction of nitric oxide ( no ) , as a result of induction of inducible nitric oxide synthase ( inos ) due to enhanced production of these cytokines , is associated with persistent inflammation and tissue destruction in experimental arthritis models , including rheumatoid arthritis [ 4 , 5 ] . a number of inflammation stimuli , including tnf- , il-1 , il-6 , or ros , can activate proinflammatory pathways involved in ra pathogenesis , concerning predominantly nuclear factor-b ( nf-b ) , mitogen activated protein kinases ( mapks ) , or janus kinases / signal transducers and activators of transcription ( jak / stat1/3 ) [ 68 ] .
this results in translocation of relevant downstream transcription factors from the cytoplasm to the nucleus , where they activate messenger rna ( mrna ) expression of target genes , including il-1 , tnf- , inos , and 12/15-lipoxygenase ( lox ) , leading to overproduction of corresponding proteins .
cytokines released into the synovium reach also the systemic circulation and act in other tissues and organs such as lungs , vascular tissue , liver , and heart . several recent investigations reported damage of vital organs with various degrees of impairment , considered to be secondary complications of ra and a major predictor of mortality in ra patients .
increasing evidence is pointing to the critical role of the liver in modulating the immune response in autoimmune and chronic inflammatory diseases including ra [ 5 , 11 , 12 ] .
the hepatic biochemical and immunological alterations are associated with and influenced by changes in the oxidative state of liver cells .
adjuvant - induced arthritis ( aa ) in rats not only is an experimental model of polyarthritis but also induces pathological changes in a variety of other tissues , including the liver and spleen .
it is a useful tool to study immunopathologic processes , autoimmune chronic inflammation , and inflammatory cachexia in rodents .
in addition , at the molecular level , mrna profiling suggests that this model is also similar to human ra , particularly in tissue gene expression and in the activation of regulatory pathways [ 11 , 14 ] .
numerous studies reported natural polyphenols as potential therapeutic agents of diseases caused by os and inflammation [ 1517 ] .
n - feruloylserotonin ( n - f-5ht , n - feruloyl-5-hydroxy - tryptamine ) is a conjugated serotonin , a member of the indole hydroxycinnamic acid amides , with serotonin ( 5-ht ) and ferulic acid ( fa ) as representative components of its structure .
hydroxycinnamic acid amides of serotonin , synthesized by serotonin n - hydroxycinnamoyltransferase , are present in several vegetables and wild - growing plants whose seeds are used in herbal medicine in eastern countries [ 1820 ] . in cell - based studies , under short - term high - glucose conditions ,
n - f-5ht exerted an inhibitory effect on overproduction of mitochondrial superoxide by acting as scavenger of superoxide .
n - f-5ht attenuated the upregulation of mrna and proteins of ros - dependent adhesion ( vascular cell adhesion protein-1 ( vcam-1 ) ) and migration factors ( monocyte chemoattractant protein-1 ( mcp-1 ) ) , crucial in early atherosclerosis lesions in human aortic endothelial cells , and inhibited the activation of transcription factor nf-b .
furthermore , n - f-5ht showed a protective effect on ros - related neuronal damage by decreasing the activity of proapoptotic caspase-3 .
n - f-5ht isomers isolated from seeds of leuzea carthamoides were shown to inhibit protein kinase c / ii activation and decrease the oxidative burst of human whole blood and isolated neutrophils in vitro .
n - f-5ht was also found to have a protective effect against ldl oxidation and atherogenesis in experimental animals and in human studies [ 2426 ] .
methotrexate ( mtx ) , used as a standard drug in our study , represents the most frequently used pharmacotherapy of ra in clinical practice .
its administration is , however , limited due to its toxic side effects [ 27 , 28 ] .
yet application of a combination therapy of mtx with other potential immunomodulators , synthetic drugs or natural substances [ 3032 ] , might elevate the therapeutic efficacy : decrease the dose of mtx and thus its side effects . in our previous study , we showed that administration of n - f-5ht to mtx - treated arthritic rats lowered the dose of mtx for the required sustained antirheumatic impact . in this study , we focused on the therapeutic impact of n - f-5ht and mtx administered in monotherapy and on details of the inflammatory state in the arthritic rat liver with the aim to elucidate the molecular mechanisms of their effect .
one of the possible clarifying approaches is to study the mrna expression of key proinflammatory markers ( il-1 , tnf- , and inos ) in the liver of treated and untreated arthritic rats .
further , it is of particular interest to expand our knowledge on the effect of n - f-5ht and mtx in the aa model , which in turn should allow extrapolations of these results to ra patients .
to this aim we evaluated also conventional arthritic parameters ( hpv , arthritic score , body weight change , and weight of the liver ) along with changes in plasmatic levels of il-1 and crp and the activity of 12/15-lox in the liver .
adult male lewis rats weighing 160180 g were obtained from charles river wiga , germany .
the experimental protocol was approved by the ethics committee of the institute of experimental pharmacology and toxicology and by the slovak state veterinary and food administration in accordance with the european convention for the protection of vertebrate animals used for experimental and other scientific purposes and was in line with slovak legislation . to induce a rat model of adjuvant arthritis ( aa ) ,
rats were intradermally injected with a suspension of heat - inactivated mycobacterium butyricum in incomplete freund 's adjuvant ( difco laboratories , detroit , mi , usa ) .
the third group comprised adjuvant arthritis rats treated with methotrexate ( methotrexat ebewe sol inj 20 mg/2.0 ml ) in oral dose of 0.4 mg / kg twice a week ( aa - mtx ) .
the fourth group comprised adjuvant arthritis rats treated with n - feruloylserotonin dissolved in suspension of methylcellulose tween 80 at a dose of 3 mg / kg / day orally ( aa - n - f-5ht ) .
drugs were administered orally by gastric gavage from day 0 ( the day of treatment ) to day 28 of the study .
blood for plasma preparation was taken by retroorbital puncture on day 14 and by cardiac puncture on day 28 under deep ketamine / xylazine anesthesia .
after the animals had been sacrificed under deep ketamine / xylazine anesthesia , tissues for liver and spleen homogenate preparation were taken at the end of the experiment ( day 28 ) .
blood in heparinized tubes for plasma preparation was centrifuged at 3000 rpm for 15 minutes at 4c .
fraction of four isomers of n - f-5ht ( table 1 ) was isolated from the seeds of leuzea carthamoides ( wild ) dc by solvent extraction .
this was then followed by column chromatography on silica gel and hplc separations under conditions previously reported [ 35 , 36 ] .
the hind paw volume ( hpv ) was recorded on days 14 , 21 , and 28 with the use of an electronic water plethysmometer ( ugo basile , comerio , varese , italy ) .
calculation of the increase in hind paw volume in ml assessed the intensity of the edema .
the arthritic score was measured as the total score of hpv ( ml , max .
points 5 ) + diameter of scab in the site of mb application , measured in parallel to the spinal column ( mm , max .
body weight change ( bwc ; g ) was measured on days 1 , 14 , 21 , and 28 .
bwc was calculated as the difference of the body mass measured on days 14 , 21 , and 28 to the body weight measured at the beginning of the experiment ( day 1 ) . for the determination of rat crp concentration in plasma ( g / ml ) , the elisa kit from immunology consultant laboratories , inc .
the reaction of secondary biotin - conjugated anti - rat crp antibody was evaluated by streptavidin - hrp .
the tetramethylbenzidine reaction with hrp bound to immune complex was measured at 450 nm ( microplate reader , labsystems multiskan rc ) .
for the determination of il-1 concentration in plasma , the elisa kit from r&d systems quantikine was used .
rat cytokine present in the samples binds to anti - rat cytokine antibodies absorbed in the microwells .
the reaction of secondary biotin - conjugated anti - rat cytokine antibody is evaluated by hrp .
the tetramethylbenzidine reaction with hrp bound to immune complex was measured at 490 nm in comparison with the reference wavelength of 620 nm ( microplate reader mrx ii ) .
concentration of proteins in liver homogenates was determined by using the bradford method and expressed in mg / ml of enzyme preparation ( cytosolic fraction from rat lung and liver tissues ) .
linoleic acid ( 99% , sigma - aldrich , usa ) was used as a substrate prepared in solubilized state as described in the concentration of 0.2143 100.7143 10 m. the assay of lox was monitored for 60 seconds as an increase in the absorbance at 234 nm , reflecting the formation of hydroperoxylinoleic acid . for the lox activity assay ,
an uv / vis spectrometer perkin - elmer lambda 35 ( usa ) was used .
the reaction medium contained a 50 mm tris - hcl buffer ( ph 7.0 ) , 2.5 l of the enzyme , and solubilized linoleic acid .
total rna was isolated from the rat liver and spleen using rnazol rt ( sigma - aldrich ) and converted into complementary dna ( cdna ) using the primescript rt reagent kit ( takara ) following the protocols of the manufacturers .
amplification and detection of cdna of reference and target genes were performed on a 7300 real - time pcr system ( applied biosystems ) using hot firepol evagreen qpcr mix plus ( rox ) ( solis biodyne ) .
relative mrna expressions of il-1 , tnf- , and inos were analyzed using the ct value method .
the sequences of the primers were designed and checked using primer3 and oligo analyzer 1.0.3 ( table 2 ) .
mean and sem values were calculated for each parameter in each group ( 810 animals in each experimental group ) .
statistically significant differences among treated , untreated , and control groups were tested using parametric analysis of variance ( anova ) .
post hoc tests ( tukey - kramer ( anova ) ) were applied in situations where differences among groups were significant at the level of significance = 0.05 .
after post hoc testing , the following significance levels were specified : extremely significant ( p < 0.001 ) , highly significant ( p < 0.01 ) , significant ( p < 0.05 ) , and not significant ( p > 0.05 ) .
antioxidant properties of polyphenols including n - f-5ht have been reported [ 21 , 40 , 41 ] .
nevertheless , the n - f-5ht impact on chronic inflammatory and os - inducing arthritis , which could widen the possibilities of the ra therapy , remains to be elucidated . in our previous study in the model of aa , n - f-5ht in the dosage of 15 mg / kg markedly potentiated the therapeutic effect of low - dose ( nontherapeutic dose ) mtx ( 0.3 mg / kg ) on arthritic ( hind paw volume and arthritic score ) and inflammatory parameters ( il-17 , mcp-1 , and crp ) , yet it resulted in insignificant effect in monotherapy . as data about the optimal n - f-5ht dose in the rat model
are scarce , we decided to study two doses of n - f-5ht : ( i ) when 15 mg / kg exceeded the physiologically acceptable concentration , we used 3 mg / kg , and ( ii ) when 15 mg / kg was too low to reach the maximal effect , we used 30 mg / kg .
unexpectedly , contrary to the lower dose of n - f-5ht , the higher dose exhibited minor effect on the parameters examined and/or these varied strongly among the animals .
for this reason , this report shows only the data evaluating the lower dose of n - f-5ht . in this study
, we used the therapeutic dose of mtx ( 0.4 mg / kg ) with the intention to compare each mechanism of action of mtx and n - f-5ht , both evaluated in monotherapy .
the significant rise in arthritic parameters , arthritic score , and hpv confirmed the arthritis in our model in rats .
the arthritic score showed an increase in the untreated arthritic group compared to the control group on all days monitored ( aa versus co , day 14 , p < 0.01 ; day 21 and day 28 , p < 0.001 ; table 3 ) . at the end of the experiment ,
the arthritic score was almost doubled in the aa group compared to controls . a trend toward reduction
was observed after administration of n - f-5ht to aa animals on day 28 , but the effect was not statistically significant .
the treatment with mtx significantly reduced the arthritic score on observation days 21 and 28 , compared to the untreated arthritic group , proving the therapeutic potential of the applied dose of mtx ( aa - mtx versus aa , day 21 , p < 0.05 ; day 28 , p < 0.01 ; table 3 ) .
similarly , the change in hpv showed an increase in the untreated arthritic group compared to the control group on days 21 and 28 ( aa versus co , day 21 , p < 0.01 ; day 28 , p < 0.05 ; table 3 ) .
the administration of n - f-5ht induced no modification of hpv of the arthritic animals on any day monitored .
mtx therapy significantly reduced the observed swelling on days 21 and 28 compared to the untreated arthritic group ( aa - mtx versus aa , day 21 and day 28 , p < 0.001 ; table 3 ) .
the muscle wasting condition due to high catabolic activity , known as rheumatoid cachexia , occurring in approximately two - thirds of all patients with ra , is mediated by tnf- and il-1 in ra .
papers published over the past years confirmed that oxidative metabolism was considerably enhanced in the liver of adjuvant - induced arthritis in rats [ 4346 ] .
rats used in this study revealed signs of cachexia ( table 3 ) . a significant decrease in body weight change ( bwc )
the bwc of the arthritic rats was 56% on day 14 , 19% on day 21 , and 27% on day 28 ( aa versus co , days 14 , 21 , and 28 , p < 0.001 ; table 3 ) of the bwc of healthy controls .
n - f-5ht treatment led to a significant increase of bwc on day 28 ( aa - n - f-5ht versus aa , p < 0.05 ; table 3 ) .
the administration of n - f-5ht in arthritic animals did not change these parameters on any of the days observed .
the liver weights were significantly lower ( aa - mtx versus aa , p < 0.05 ; table 3 ) only in the group of rats treated with mtx .
the reduced weight of the liver in mtx - treated rats was assumed to be the result of inhibition of the pathway of de novo dna synthesis by mtx [ 47 , 48 ] . in summary ,
the statistical significance of 3 mg / kg of n - f-5ht treatment was determined only for bwc . the arthritic score revealed a trend toward the positive effect increasing with time , indicating a late onset of n - f-5ht action ( table 3 ) .
as expected , significant differences were found in the arthritic score and hpv in the arthritic animals treated with the therapeutic dose of 0.4 mg / kg mtx compared to those treated with the low dose of 0.3 mg / kg mtx .
il-1 , a prototypic proinflammatory cytokine , is a major mediator of the inflammatory cascade in ra , which is involved in the mechanisms leading to progressive joint destruction . in the model of aa ,
the early phases of the disease seem to be characterized by a systemic increase of il-1 .
the plasmatic level of il-1 , a protein of multiorgan origin , was significantly increased in arthritic animals compared to the control group in the early phase of aa , on day 14 ( aa versus co , p < 0.001 ; figure 1(a ) ) , ascertaining the presence of inflammation .
administration of mtx did not lead to a significant change of plasmatic il-1 concentration ; only a trend toward reduction was observed on day 14 .
it is noteworthy that n - f-5ht treatment resulted in a significant decrease of il-1 level in plasma ( aa - n - f-5ht versus aa , p < 0.05 ; figure 1(a ) ) .
this result is interesting , as this molecule was reported to be relevant in driving the transition from the acute phase to the chronic irreversible phase of the disease and it has been suggested that it could be the target of early intervention to stop the course toward the chronic form of the disease .
the blocking il-1 protects bone and cartilage from progressive destruction in ra and its inhibition could be effective in the treatment of this disease .
activation of t and b cells , macrophages , and inflammatory mediators tnf- , il-1 , and il-6 aggravates the oxidative damage of the vital organs in rheumatoid arthritis , such as the liver .
the liver , in turn , influences the systemic inflammation via producing inflammatory cytokines and mediators such as tnf- , il-1 , il-6 , no , crp , and lox .
il-6 , il-1 , and tnf- promote the synthesis of crp in hepatocytes via stat3 [ 51 , 52 ] and nf-b pathways .
the level of the systemic inflammatory parameter crp in plasma , resulting from liver synthesis , was increased significantly in the group of arthritic animals compared with control animals in the chronic phase of the disease on experimental day 28 ( aa versus co , p < 0.001 ; figure 1(b ) ) .
administration of n - f-5ht and mtx significantly reduced the plasmatic levels of crp on day 28 compared to the untreated group of arthritic animals ( aa - n - f-5ht versus aa , p < 0.05 ; aa - mtx versus aa , p < 0.05 ; figure 1(b ) ) .
interaction of crp with fc - gamma receptors ( fcr ) fcri and fcriia is known to promote the production of proinflammatory cytokines , resulting in the amplification loop of inflammatory reaction .
these processes are initiated through the induction of the receptor activator of nuclear factor-b ligand ( rankl ) protein and direct stimulation of osteoclastogenesis , causing a loop between inflammation and bone destruction in ra .
crp enhances both the proinflammatory response and bone destruction . in the treatment of ra , a lowered crp level thus not only is a significant parameter in terms of disease progression elimination but also has a direct impact on decreasing the degree of bone destruction .
alterations in the oxidative state lead to the activation of nf-b and nf-b - dependent genes , such as lox .
the enzyme 5-lox catalyzes the conversion of arachidonic acid to leukotrienes , whose production has been associated with inflammation in arthritis .
suppression of 5-lox expression ameliorates clinical parameters in ra and aa [ 56 , 57 ] .
increased levels of nf-b in the lung and liver as well as increased activity of lox in the lung highlight the importance of extra - articular manifestations of aa . in our experiment ,
liver 12/15 lox activity increased in arthritic animals in comparison to healthy animals ( aa versus co , p < 0.001 ; figure 1(c ) ) .
the effect of n - f-5ht on the activity of 12/15-lox in liver homogenate was comparable with that of mtx .
after administration of mtx or n - f-5ht , a significant decrease to control levels was assessed in the liver of the aa group ( aa - n - f-5ht versus aa , p < 0.001 ; aa - mtx versus aa , p < 0.001 ; figure 1(c ) ) .
thus the anti - inflammatory effect of n - f-5ht in aa was supported by the ability of the molecule to inhibit 12/15-lox activity . similar to this result , recent observations also reported that several other flavonoids may act as lox inhibitors . in aa ,
the gene expression levels of tnf- and inos produced in the liver were reported to increase [ 60 , 61 ] .
also , in our study , the levels of tnf- and inos mrna expressions were significantly increased in arthritic animals ( both p < 0.001 , aa versus co ; figures 2(a ) and 2(b ) )
. it was proposed that these modifications in the liver of arthritic rats not only were a consequence of the metabolic alterations caused by the disease , especially the increased oxidative metabolism , but also depended on increased inflammatory parameters in the liver .
the same agents that increase oxidative metabolism , tnf- , il-1 , il-6 , and others , are responsible for increasing the activity of inos in several tissues .
an increase of inos activity as a consequence of elevated inos mrna expression was considered to play a dominant role in the pathogenesis of ra .
no generation by inos induced in chondrocytes in the initial stage of aa may play a key role in triggering the subsequent events in arthritis . in general , the use of nos inhibitors has been shown to exert beneficial effects in experimentally induced arthritis . however ,
which types of cells expressing inos are associated with the induction or progression of adjuvant - induced arthritis via no generation remains uncertain .
mrna expression of inos in rat liver was reduced following mtx ( aa - mtx versus aa , p < 0.001 ; figure 2(a ) ) and n - f-5ht treatment ( aa - n - f-5ht versus aa , p < 0.01 ; figure 2(a ) ) .
the effect of mtx treatment on tnf- protein and mrna expression differs among studies , depending on the conditions of the given study , concerning gender of patients , type of cell line , duration of treatment , mtx dose , and so forth . in our study in the rat aa model , administration of mtx
attenuated significantly the mrna expression of tnf- ( aa - mtx versus aa , p < 0.01 ; figure 2(b ) ) . in many patients , however , mtx treatment does not result in lower tnf- plasma concentration .
when mtx fails to produce an adequate response , newer therapies are used in combination with mtx .
blocking tnf- with anti - tnf- monoclonal antibodies significantly decreased the signs and symptoms of ra compared to placebo in ra patients with active disease receiving mtx [ 65 , 66 ] .
thus , the n - f-5ht - driven significant reduction of tnf- mrna expression ( a - n - f-5ht versus aa , p < 0.01 ; figure 2(b ) ) suggests an intriguing effect on ra treatment , calling for deeper investigation .
increase of mrna expression was observed for il-1 in the liver of arthritic animals ( aa versus co , p < 0.001 ; figure 3(a ) ) as expected .
administration of mtx did not lead to significant attenuation of il-1 transcription in the liver .
this is in concert with previous studies of mtx function in different types of cells ( e.g. , human peripheral blood mononuclear cells and murine peritoneal and splenic cells ) [ 67 , 68 ] . on the other hand
, mtx exhibits another mechanism of il-1 function inhibition , which involves blocking the binding of il-1 to il-1 receptor in the membrane of peripheral blood cells ( monocytes , lymphocytes , and granulocytes ) .
contrary to mtx , treatment with n - f-5ht led to a substantial inhibition of il-1 gene expression ( aa - n - f-5ht versus aa , p < 0.01 ; figure 3(a ) ) .
further , we examined il-1 mrna expression in the main immunocompetent organ , in the rat arthritic spleen , which has not been studied previously in terms of the aa model , related to il-1 expression .
we observed il-1 mrna expression activation comparable to that in the liver ( aa versus co , p < 0.001 ; figure 3(b ) ) .
interestingly , both mtx and n - f-5ht exhibited a significant and remarkably stronger inhibition of il-1 mrna expression in comparison to that in the liver ( aa - mtx versus aa , p < 0.01 ; aa - n - f-5ht versus aa , p < 0.001 ; figure 3(b ) ) .
in the spleen of n - f-5ht treated rats , the relative mrna expression decreased even to control level .
besides other events , mtx treatment leads to suppression of nf-b , a heterodimer consisting of two subunits p65 and p50 , one of the most prominent inflammatory transcription factors activated in ra .
this was confirmed in our previous work , along with the finding that also n - f-5ht ( 15 mg / kg ) suppressed the activation of nf-b ( p65 ) in the arthritic rat liver [ 21 , 33 ] .
interestingly , combination therapy ( mtx + n - f-5ht ) potentiated the effect of a single drug , suggesting different mechanisms leading to nf-b inhibition .
mtx driven reduction of cytokine transcription was attributed to abrogation of ib kinase activation and thereby suppression of ib ( nf-b inhibitor ) phosphorylation and degradation , resulting in retaining the inactive nf-b form in cytoplasm .
however , the contribution of n - f-5ht to nf-b pathway suppression needs to be further investigated .
studies of the proposed pathways involved in the transcription of tnf- , il-1 , and inos in ra could help evaluate the mechanism of action of these drugs [ 68 , 71 , 72 ] .
the gene expression of inos is mostly under the control of synergistically activating nf-b ( il-1 and tnf- stimulated ) and stat1 ( ifn- stimulated ) key proinflammatory signals in the liver .
in contrast to inos , tnf- does not contain the stat binding element in its promoter region .
the inhibition of tnf- and inos transcription observed in our study might be mostly attributed to the suppressed nf-b pathway for both mtx and n - f-5ht [ 33 , 64 , 70 ] .
however , the contribution of ap-1 to tnf- and stat1 for inos can not be excluded .
mtx - dependent suppression of nf-b was reported [ 33 , 70 , 73 , 74 ] , but in other cases mtx was not found to be effective in the attenuation of arthritic - increased mrna expression of il-1 [ 68 , 75 ] . taking into account our results , where mtx treatment did not lead to inhibition of il-1 mrna expression in the arthritic liver in contrast to the significant n - f-5ht impact , yet treatment of both mtx and n - f-5ht decreased the presumably nf-b - dependent lox activity and inos and tnf- transcription to a similar extent , the involvement of n - f-5ht in another pathway for transcription regulation of this cytokine in the arthritic liver should be considered .
after analysis of the reported pathways involved in the regulation of il-1 mrna expression , we hypothesized that tnf--driven ap-1 transcription factor activation or jak / stat3 pathway activated via il-6 or ifn- might play a role ( [ 7 , 8 , 71 , 72 , 76 , 77 ] , figure s1 in supplementary material available online at http://dx.doi.org/10.1155/2016/7509653 ) .
papers reporting involvement of other polyphenols in anti - inflammatory regulation , for example , resveratrol , claim that these compounds exhibit their anti - inflammatory effect through suppression of nf-b and jak / stat signaling pathways [ 78 , 79 ] .
the enhanced influence of mtx and n - f-5ht on il-1 transcription in the spleen in comparison to the liver may be the consequence of different predominance of inflammatory pathways in this organ , presumably with a stronger nf-b contribution .
details about the relevance of these pathways and the role of n - f-5ht in the transcription regulation of il-1 , inos , and tnf- in the liver and other organs in ra are to be further elucidated .
the present study contributed additional evidence about the beneficial effect and mechanism of action of n - f-5ht and of mtx on a systemic inflammatory process in the liver and its association with the pathogenesis of adjuvant arthritis .
n - f-5ht treatment led to amelioration of inflammatory parameters tested ( plasmatic crp and il-1 protein levels , liver lox activity , and liver and spleen cytokine expression ) .
however , this did not result in a significant change of hpv , although a trend of improvement of the arthritic score was observed after 28 days .
a synergistic effect of tnf- and il-1 was shown to influence the balance between protein degradation and protein synthesis causing among others an increase in resting energy expenditure and net efflux of amino acids from muscle to liver .
the significant increase of bwc in n - f-5ht treated rats , probably sign of the partial improvement of rheumatoid cachexia , might be the result of lowered mrna expression of tnf- and il-1 determined in the arthritic liver . moreover , taking into account the reported association of weight loss with the il-1 production by splenic cells , the n - f-5ht mediated attenuation of increased il-1 mrna expression in the arthritic spleen might contribute to this complex process
. the contribution of the affected expression of tnf- and il-1 originating from other organs can not be excluded and is to be further elucidated .
unexpectedly , chronic daily treatment with a high concentration of n - f-5ht ( 30 mg / kg ) exhibited either a minor effect on the parameters examined and/or a strong variation among the animals ( not shown ) and that in contrast to a much lower concentration ( 3 mg / kg ) . since n - f-5ht possesses a serotonin ( 5-hydroxytryptamine , 5-ht ) moiety , the question if there might be some interplay between effects of these two molecules on ra pathogenesis is to be raised . since n - f-5ht inhibited the increase of cytosolic free ca concentration in rat vascular smooth muscle cells induced by serotonin mediated by 5-ht2 receptors , it was hypothesized that at a sufficient concentration n - f-5ht may act as a competitive antagonist , which displaces serotonin from its binding site .
intake of a high concentration of a 5-ht2 receptor antagonist may lead to a variety of effects : it may influence the receptor density , even enhance the effect of serotonin , or lead to desensitization and with time to receptor resistance ( through inhibitory feedback due to binding - induced enhanced production of serotonin ) .
interestingly , serotonin is known not only as a neurotransmitter . increasing but contradictory reports associate serotonin with immunoinflammatory pathways in the periphery .
serotonin , via its 5-ht2a , 5-ht2b , and 5-ht3 receptors , has been implicated to have both proinflammatory and anti - inflammatory roles in a number of studies of rheumatoid arthritis [ 8488 ] .
the reported effects of 5-ht receptor antagonist on macrophage - like synovial cells encourage the interest to study the effect of n - f-5ht from this point of view . to confirm this hypothesis ,
a precise characterization of interaction between n - f-5ht and 5-ht receptors is to be done . on comparing the effects of the two drugs , administration of mtx ( 0.4
mg / kg ) or n - f-5ht ( 3 mg / kg ) was found to lead to a decrease of the main plasma marker of systemic inflammation crp , the liver origin protein , and to inhibition of proinflammatory lox in the liver .
the impact of mtx and n - f-5ht on mrna expression of tnf- , il-1 , and inos in the liver and on the level of crp in plasma was mentioned at the conference .
mtx and n - f-5ht reduced the arthritis - increased transcription of tnf- and inos in the liver to a comparable extent . we suppose that the inhibition of tnf- and inos transcription might be mostly attributed to the suppressed nf-b pathway for the two drugs [ 21 , 33 , 70 ] .
as previously reported [ 67 , 68 ] and also proven by our study , mtx was not able to diminish the arthritic - induced il-1 mrna transcription in the liver .
this handicap might be compensated by coadministration of n - f-5ht , since this drug was shown to lower the level of proinflammatory cytokine il-1 in plasma in the acute phase of aa and to attenuate significantly the elevation of il-1 mrna expression in the arthritic rat liver and spleen in the chronic phase .
detailed studies are required to confirm the hypothesis that n - f-5ht might function through potentially different mechanisms of inhibition of the inflammatory pathway nf-b and not through mtx , as well as the possibility of an additional pathway influencing il-1 transcription under control of n - f-5ht but not mtx .
the confirmation would support n - f-5ht as a promising agent for the treatment of ra in combination therapy with mtx .
the positive effect was shown in our previous study , where n - f-5ht markedly potentiated the therapeutic effect of low - dose mtx .
as the therapeutic dose of mtx was used in this study and the purpose of combination study is to lower the mtx dose to decrease the side effects of this drug , the effect of combination therapy was not included
. oral daily intake of n - f-5ht could overcome the inconvenient administration and high costs of biological therapy using il-1 monoclonal antibody , which was shown in clinical trials to be superior to placebo in combination with mtx in reducing signs , symptoms , and radiographic progression in patients with advanced ra [ 91 , 92 ] .
future studies of n - f-5ht mechanisms of action should shed more light on the immunomodulatory function of this natural polyphenol .
it is to be expected that n - f-5ht is able to positively affect the activity of other markers of inflammation and oxidative stress not only in the liver and spleen but also in other organs ( lung , brain , etc . ) , a hypothesis to be tested by future work .
however , to establish the optimal dosing in light of the effects achieved is of primary importance .
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rheumatoid arthritis ( ra ) is a chronic inflammatory disease , leading to progressive destruction of joints and extra - articular tissues , including organs such as liver and spleen .
the purpose of this study was to compare the effects of a potential immunomodulator , natural polyphenol n - feruloylserotonin ( n - f-5ht ) , with methotrexate ( mtx ) , the standard in ra therapy , in the chronic phase of adjuvant - induced arthritis ( aa ) in male lewis rats .
the experiment included healthy controls ( co ) , arthritic animals ( aa ) , aa given n - f-5ht ( aa - n - f-5ht ) , and aa given mtx ( aa - mtx ) .
n - f-5ht did not affect the body weight change and clinical parameters until the 14th experimental day .
its positive effect was rising during the 28-day experiment , indicating a delayed onset of n - f-5ht action .
administration of either n - f-5ht or mtx caused reduction of inflammation measured as the level of crp in plasma and the activity of lox in the liver .
mrna transcription of tnf- and inos in the liver was significantly attenuated in both mtx and n - f-5ht treated groups of arthritic rats .
interestingly , in contrast to mtx , n - f-5ht significantly lowered the level of il-1 in plasma and il-1 mrna expression in the liver and spleen of arthritic rats .
this speaks for future investigations of n - f-5ht as an agent in the treatment of ra in combination therapy with mtx .
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1. Introduction
2. Materials and Methods
3. Results and Discussion
4. Conclusions
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this retrospective observational study was approved by the western institutional review board ( olympia , washington , usa ) .
it complied with the health insurance portability and accountability act of 1996 and followed the tenets of the declaration of helsinki .
informed consent was obtained from the subjects after explanation of the nature and possible consequences of the study .
patients with ga were identified from the tertiary practice of retinal specialists ( kbf , js , and ly ) if they exhibited plateau signatures in at least one eye ( study eye ) on oct imaging and if they also had at least 3 years ' follow - up with consecutive eye - tracked spectral - domain ( sd)-oct scans taken at least every 6 months .
eyes with macular neovascularization , other retinal pathology , or media opacity preventing adequate imaging were excluded .
medical records and multimodal imaging , including color fundus photography ( cfp ) , red - free photography ( rf ) , fundus autofluorescence ( faf ) , near - infrared reflectance scanning laser ophthalmoscopy ( nir ) , and sd - oct was performed on all the patients .
cfp and rf were performed with a trc-50ix flood - illuminated fundus camera ( topcon medical systems , oakland , nj , usa ) .
faf was performed with either the spectralis hra + oct ( heidelberg engineering , heidelberg , germany ) or the topcon trc-50ix fundus camera .
oct scans taken before the acquisition of eye - tracked spectralis scans were assessed qualitatively but not included in the quantitative analysis .
the sd - oct protocol used in all eyes comprised 20 horizontal raster line scans over the area of interest , ranging from 19 to 49 b - scans per eye , with each scan spaced 115 to 250 m apart , and automatic real - time averaging set between 4 and 23 . to study the origin and progression of plateaus , the most recent sd - oct with this signature was identified . then
, serial eye - tracked b - scans of the region of interest were tracked back to baseline . in cases
in which the scanning protocol varied during the course of follow - up , the b - scan closest to the area of interest was matched manually and extracted for analysis .
these extracted sd - oct images were then stacked , aligned , and saved as a video file using the fiji distribution ( fiji is just image j ,
http://fiji.sc ; in the public domain ) for the qualitative analysis of the progression of the lesion ( see supplementary video ) .
at each time point , the plateau appearance on sd - oct was correlated to the appearance in cfp , nir , and faf .
three patients had en face oct and oct angiography ( octa ) imaging with 3 3-mm macular cubes ( rtvue xr avanti ; optovue , fremont , ca , usa ) .
the review software ( revue , version 2015.1.0.90 ; optovue , fremont , ca , usa ) displays flow signals superimposed in red on a cross - sectional structural sd - oct image . in a healthy eye ,
the fourth outer retinal hyperreflective band includes the rpe and brm . in a ped , by definition
in the course of this separation , either a basal lamina ( bl ) of 0.15-m thickness or a blamd of variable thickness adheres to the rpe and not to the brm .
therefore , we call the hyperreflective band that anteriorly delimits a ped the rpe - bl complex . the definition of a plateau was a distinct oct signature where a mound - like structure with a flattened apex and a wide base was observed within an area of ga after complete loss of the overlying rpe . to determine the volume of the drusenoid ped and ensuing plateau ( fig .
2 ) , the cavalieri principle of stereology was applied to sd - oct volumetric data acquired at each visit , as follows .
( 1 ) each b - scan in the volume was scaled to a 1:1 pixel aspect ratio .
the caliper function within the heidelberg eye explorer software ( version 6.3.4.0 ; heidelberg engineering ) was used to measure the area between the outer boundary of rpe+bl and the inner boundary of brm .
( 2 ) the volume between two consecutive oct slices ( hereafter called a segment ) was then determined using the following formula :
\}\begin{document}$$d\left ( { \left ( { a\_x + a\_\left ( { x + 1 } \right ) } \right)/2 } \right),$$\end{document}where d is the distance between consecutive slices in m , a is the area between the rpe+bl and brm in m , and x is the oct slice number .
( 3 ) total ped volume was calculated by summing the volumes of individual segments . the number of segments in the oct volume was ( n 1 ) , where n is the total number of slices that spanned the ped .
graphical plots of ped volume with respect to time were generated using interval data .
origin and evolution of a plateau , as revealed by nir ( a ) and oct ( b ) images .
green arrows on nir images show the levels of corresponding oct cross - sectional images .
baseline images show several elevations of the rpe with largely hyporeflective interiors . at 3 years after baseline , punctate hyperreflectivity ( red arrowhead )
, progressive loss of the onl has caused the opl ( yellow arrows ) to approach the surface of the ped .
also at 4 years , a mons hyperreflective wedge ( blue arrowheads ) appears at the ga border . at 5 years
, the downwardly deflected opl is seen to move progressively toward the edge of the plateau following the advancing border of ga .
formation of the plateau at 5 years was characterized by the loss of the overlying rpe causing marked thinning of the hyperreflective rpe - basal lamina complex now reduced to just blamd , compared with at 4 years . at 7 years
the institutional review board at university of alabama at birmingham ( uab ) approved the laboratory study , which complied with the health insurance portability and accountability act and adhered to the tenets of the declaration of helsinki .
amd eyes were identified through an ex vivo imaging screen of eyes accessioned for research purposes from nondiabetic white donors to the alabama eye bank during the period 1996 to 2012 .
median death - to - preservation time was 3:49 hours ( range , 0:4011:40 hours ) .
eyes were preserved by immersion in 1% paraformaldehyde and 2.5% glutaraldehyde in 0.1 m phosphate buffer following anterior segment excision .
after vitreous removal , maculas were photographed in color on a stereomicroscope ( smz - u ; nikon , melville , ny , usa ) . when prepared for histology ( 20112013 ) and uploaded to the project macula web site ( http://projectmacula.cis.uab.edu , in the public domain )
, eyes underwent additional multimodal ex vivo imaging . from each globe , an 8-mm - diameter full - thickness tissue punch containing the fovea and temporal portion of the optic nerve head was held in a tissue holder mounted on a spectralis , as described . a 30 20 sd - oct volume ( 143 scans , 30-m spacing ,
automated real - time averaging [ art ] = 25 ) was captured along with red - free and near - infrared reflectance scanning laser ophthalmoscopic images .
tissue punches were postfixed by osmium tannic acid paraphenylenediamine to accentuate extracellular lipid and embedded in epoxy resin ( polybed 812 ; polysciences , warrington , pa , usa ) .
sub - micrometer - thick ( 0.8 m ) sections at 25- to 30-m intervals were stained with 1% toluidine blue for polychromaticity , scanned with a 40 objective , and reviewed and photodocumented with a 60 oil - immersion objective ( numerical aperture = 1.4 ) and digital camera ( xc10 ; olympus , center valley , pa , usa ) .
images were adjusted for exposure , contrast , and sharpness ( photoshop cs6 , adobe , san jose , ca , usa ) .
patients with ga were identified from the tertiary practice of retinal specialists ( kbf , js , and ly ) if they exhibited plateau signatures in at least one eye ( study eye ) on oct imaging and if they also had at least 3 years ' follow - up with consecutive eye - tracked spectral - domain ( sd)-oct scans taken at least every 6 months
. eyes with macular neovascularization , other retinal pathology , or media opacity preventing adequate imaging were excluded .
medical records and multimodal imaging , including color fundus photography ( cfp ) , red - free photography ( rf ) , fundus autofluorescence ( faf ) , near - infrared reflectance scanning laser ophthalmoscopy ( nir ) , and sd - oct was performed on all the patients .
cfp and rf were performed with a trc-50ix flood - illuminated fundus camera ( topcon medical systems , oakland , nj , usa ) .
faf was performed with either the spectralis hra + oct ( heidelberg engineering , heidelberg , germany ) or the topcon trc-50ix fundus camera .
oct scans taken before the acquisition of eye - tracked spectralis scans were assessed qualitatively but not included in the quantitative analysis .
the sd - oct protocol used in all eyes comprised 20 horizontal raster line scans over the area of interest , ranging from 19 to 49 b - scans per eye , with each scan spaced 115 to 250 m apart , and automatic real - time averaging set between 4 and 23 . to study the origin and progression of plateaus , the most recent sd - oct with this signature was identified .
then , serial eye - tracked b - scans of the region of interest were tracked back to baseline . in cases
in which the scanning protocol varied during the course of follow - up , the b - scan closest to the area of interest was matched manually and extracted for analysis .
these extracted sd - oct images were then stacked , aligned , and saved as a video file using the fiji distribution ( fiji is just image j , http://fiji.sc ; in the public domain ) for the qualitative analysis of the progression of the lesion ( see supplementary video ) . at each time point , the plateau appearance on sd - oct was correlated to the appearance in cfp , nir , and faf .
three patients had en face oct and oct angiography ( octa ) imaging with 3 3-mm macular cubes ( rtvue xr avanti ; optovue , fremont , ca , usa ) . the review software ( revue , version 2015.1.0.90 ; optovue , fremont , ca , usa ) displays flow signals superimposed in red on a cross - sectional structural sd - oct image
in a healthy eye , the fourth outer retinal hyperreflective band includes the rpe and brm . in a ped , by definition
, the rpe is separated from the brm . in the course of this separation , either a basal lamina ( bl ) of 0.15-m thickness or a blamd of variable thickness adheres to the rpe and not to the brm .
therefore , we call the hyperreflective band that anteriorly delimits a ped the rpe - bl complex . the definition of a plateau was a distinct oct signature where a mound - like structure with a flattened apex and a wide base was observed within an area of ga after complete loss of the overlying rpe .
2 ) , the cavalieri principle of stereology was applied to sd - oct volumetric data acquired at each visit , as follows . ( 1 ) each b - scan in the volume was scaled to a 1:1 pixel aspect ratio . the caliper function within the heidelberg eye explorer software ( version 6.3.4.0 ; heidelberg engineering )
was used to measure the area between the outer boundary of rpe+bl and the inner boundary of brm .
( 2 ) the volume between two consecutive oct slices ( hereafter called a segment ) was then determined using the following formula :
\}\begin{document}$$d\left ( { \left ( { a\_x + a\_\left ( { x + 1 } \right ) } \right)/2 } \right),$$\end{document}where d is the distance between consecutive slices in m , a is the area between the rpe+bl and brm in m , and x is the oct slice number . ( 3 ) total ped volume was calculated by summing the volumes of individual segments .
the number of segments in the oct volume was ( n 1 ) , where n is the total number of slices that spanned the ped .
graphical plots of ped volume with respect to time were generated using interval data .
origin and evolution of a plateau , as revealed by nir ( a ) and oct ( b ) images .
green arrows on nir images show the levels of corresponding oct cross - sectional images .
baseline images show several elevations of the rpe with largely hyporeflective interiors . at 3 years after baseline , punctate hyperreflectivity ( red arrowhead )
, progressive loss of the onl has caused the opl ( yellow arrows ) to approach the surface of the ped . also at 4 years , a
mons hyperreflective wedge ( blue arrowheads ) appears at the ga border . at 5 years
, the downwardly deflected opl is seen to move progressively toward the edge of the plateau following the advancing border of ga .
formation of the plateau at 5 years was characterized by the loss of the overlying rpe causing marked thinning of the hyperreflective rpe - basal lamina complex now reduced to just blamd , compared with at 4 years . at 7 years , a closed ort ( pink arrowhead ) is present adjacent to the plateau .
the institutional review board at university of alabama at birmingham ( uab ) approved the laboratory study , which complied with the health insurance portability and accountability act and adhered to the tenets of the declaration of helsinki .
amd eyes were identified through an ex vivo imaging screen of eyes accessioned for research purposes from nondiabetic white donors to the alabama eye bank during the period 1996 to 2012 .
median death - to - preservation time was 3:49 hours ( range , 0:4011:40 hours ) .
eyes were preserved by immersion in 1% paraformaldehyde and 2.5% glutaraldehyde in 0.1 m phosphate buffer following anterior segment excision .
after vitreous removal , maculas were photographed in color on a stereomicroscope ( smz - u ; nikon , melville , ny , usa ) . when prepared for histology ( 20112013 ) and uploaded to the project macula web site ( http://projectmacula.cis.uab.edu , in the public domain )
, eyes underwent additional multimodal ex vivo imaging . from each globe , an 8-mm - diameter full - thickness tissue punch containing the fovea and temporal portion of the optic nerve head was held in a tissue holder mounted on a spectralis , as described . a 30 20 sd - oct volume ( 143 scans , 30-m spacing ,
automated real - time averaging [ art ] = 25 ) was captured along with red - free and near - infrared reflectance scanning laser ophthalmoscopic images .
tissue punches were postfixed by osmium tannic acid paraphenylenediamine to accentuate extracellular lipid and embedded in epoxy resin ( polybed 812 ; polysciences , warrington , pa , usa ) .
sub - micrometer - thick ( 0.8 m ) sections at 25- to 30-m intervals were stained with 1% toluidine blue for polychromaticity , scanned with a 40 objective , and reviewed and photodocumented with a 60 oil - immersion objective ( numerical aperture = 1.4 ) and digital camera ( xc10 ; olympus , center valley , pa , usa ) .
images were adjusted for exposure , contrast , and sharpness ( photoshop cs6 , adobe , san jose , ca , usa ) .
plateau signatures were observed in eight eyes of seven patients , of whom six were female .
mean age was 75 years ( range , 6089 ) and mean follow - up period was 7.7 years ( range , 3.711.6 ) .
baseline visual acuity ( va ) was logmar 0.42 ( snellen equivalent 20/52 ) ( range , 0.181 ) .
final va was logmar 1.16 ( snellen equivalent 20/289 ) ( range , 0.182.3 ) . in two eyes ,
ga spared the fovea throughout the entire course of the follow - up . at baseline
, the presence of a drusenoid ped with homogeneous hyperreflective contents and an intact overlying rpe was noted in six eyes ( 75% ; figs . 2 , 3 ) .
one eye ( patient 5 , left eye ) had an extremely large ped measuring 2.24 mm with mostly hyporeflective contents and an overlying vitelliform lesion . in another eye ( patient 4 , left eye ) , the plateau was already present at baseline .
pigmentary changes noted on cfp were also found on nir , rf , and faf in six eyes , and these corresponded to intraretinal hyperreflective foci on oct ( fig .
3 ) . overlying the ped surface , the ellipsoid zone and outer nuclear layer ( onl ) were thin , with the opl nearly apposed to the surface of the ped .
b ) at baseline and follow - up ( a , 4 years ; b , 7 years ) . (
a ) at baseline , patient 3 has a drusenoid ped with overlying pigment hyperplasia and hyperautofluorescent vitelliform material .
the oct b - scan shows intraretinal hyperreflective foci ( red arrowhead ) and loss of outer retinal bands over the apex of the ped .
four years later , a region of hypoautofluorescent ga contains a plateau that can not be appreciated in the cfp , nir , rf , or faf .
the oct b - scan shows a plateau with a shape similar to the baseline ped .
the downwardly deflecting opl ( yellow arrow ) has moved laterally as it follows the border of the advancing ga . there was marked attenuation and decreased reflectivity of the rpe - bl band .
( b ) at baseline , patient 6 has a drusenoid ped with overlying pigment hyperplasia that is located inferior to an area of ga .
bottom row : seven years later , the ga has markedly enlarged , and the ped is no longer visible on cfp , nir , rf , or faf .
the oct b - scan shows that following progressive loss of the onl , the opl ( yellow arrow ) appears draped over a plateau with a shape similar to the baseline ped .
hyporeflective areas ( blue arrowhead ) are visible within the plateau interior . during follow - up ,
onl thinning resulted in the opl approximating the surface of the ped in all cases in which the ped was present at baseline ( figs . 2 , 3 ) .
as ga progressed , this point of approximation moved toward the edges of the ped base , following the advancing border of ga ( supplementary video ) . in all eyes , there was also progressive thinning of the hyperreflective rpe - bl complex and diminution of its reflectivity , beginning at the apex of the ped and advancing toward its edges .
after rpe loss occurred over the entire ped surface , the plateau ( i.e. , a thin hyperreflective inner surface and an interior of heterogeneous and overall reduced reflectivity ) appeared . the plateau surface was highly reflective on both cross - sectional and en face oct ( fig .
4 , en face oct surface ) and showed the presence of focal defects ( fig .
4 , blue arrows ) that were usually accompanied by smaller hyperreflective foci seen beneath the hyperreflective border .
these hyperreflective foci also could be observed within the area of ga as hyperautofluorescent foci on faf and hyperreflective foci on en face oct ( fig .
4 , green arrows ) . accordingly , on en face oct , plateau contents were seen as honeycomb - like structures with hyporeflective spaces interspersed with hyperreflective foci ( fig .
4 , green arrows ) increased in abundance within the plateau interior during follow - up in all eyes as seen on cross - sectional and en face oct ( figs . 2 , 3 , 4 ) .
on both cross - sectional and en face octa , no abnormal flow signals were noted within plateaus ( fig .
( a ) a plateau located within the area of ga features internal hyporeflective areas on cross - sectional oct ( blue arrowhead ) , which appear to be organized in a honeycomb - like pattern on en face oct ( blue arrowhead ) . on cross - sectional oct ,
octa , and on oct surface ( an en face oct slab at the level of the rpe - bl atop the plateau ) , the plateau interior is bounded by a distinct hyperreflective band that continues as a thick and highly reflective rpe band .
this hyperreflective surface shows transient hyporeflective defects ( yellow arrows ) during the evolution of these lesions .
an en face oct image of the plateau surface allows better appreciation of the focal defects ( yellow arrow ) .
( b ) a plateau located within the area of ga features internal hyporeflective areas on cross - sectional oct ( blue arrowhead ) , which appear to be organized in a honeycomb - like pattern on en face oct ( blue arrowhead ) . on cross - sectional oct and octa
, a distinct hyperreflective surface surrounds the plateau interior and focal defects ( yellow arrows ) can be seen in some areas . an en face
oct taken at the plateau surface allows better appreciation of focal defects in this hyperreflective layer ( en face octa : surface yellow arrow ) .
deep to these focal defects are hyperreflective features ( faf and en face oct : green arrowheads ) that are also seen on faf as punctate hyperfluorescence within the confluent area of hypoautofluorescence .
a single plateau was seen in all eyes except for one ( patient 2 ) that had two separate plateaus ( fig .
patient 6 had one plateau in each eye . in the seven eyes with a baseline ped ,
the thinning of the hyperreflective band over the ped surface after rpe loss ( at which point we consider the plateau to be formed ) was preceded by a sharp decrease in ped volume ( fig .
subsequently , the volume of the residual plateau appeared very stable over time ( fig .
the point at which the plateaus form ( complete loss of rpe from the hyperreflective rpe - bl complex over the ped ) is shown by the dotted line .
patient 2 has two separate peds that evolve to two separate plateaus ( a , b ) .
patient 6 has a plateau in both the right eye ( r ) and the left eye ( l ) .
only patients with an intact hyperreflective rpe - bl complex baseline were included in this analysis .
when the plateau signature is first observed ( black dotted line ) , a decrease in the volume that stabilizes on further follow - up .
figure 6 shows histology of a donor eye with ga , for which local relationships and cellular processes in the interior of outer retinal corrugations were particularly clear yet also representative of other ga eyes available for review .
atrophy spared the fovea in this eye , as seen with ex vivo cfp and 488-nm autofluorescence , and small reflective puncta in the atrophic area were apparent on nir ( figs .
6e ) , blamd was seen as discontinuous , being present at three separate locations .
pigmented cells were of the subducted phenotype , that is , rpe - originated cells located external to blamd , in contact with brm . for reference , in the henle fiber layer of a normal eye , obliquely oriented mller cell processes are parallel to , and interleaved with , inner fibers of cone and rod photoreceptors . in the presence of severe photoreceptor degeneration ,
including ort in this ga eye , the trajectories of gliotic mller cell processes were oriented in many directions , as indicated by arrows in figure 6e . in one instance
, processes seemed to enter an outer retinal corrugation horizontally through an opening in the blamd and sweep the subducted cells off brm ( fig .
6d ) . persistent bl deposit and mller cell processes in ga in a 95-year - old white man .
cfp ( top row left ) shows a large area of atrophy of rpe extending to the optic nerve head but sparing the fovea .
green arrowheads approximate the level of the histologic section shown in the top far right and bottom .
( b ) ex vivo 488-nm autofluorescence shows loss of signal in the atrophic area .
( c ) ex vivo nir imaging shows small and highly reflective puncta in the atrophic area .
detailed ( d ) and panoramic ( e ) views of the atrophic area in histology .
nfl , nerve fiber layer ; gcl , ganglion cell layer ; ipl , inner plexiform layer ; dr , calcified druse .
( d ) processes from mller cells under a corrugation of persistent blamd in the atrophic area .
this corrugation overlies subducted cells of rpe origin ( teal arrowheads ) , which contain nuclei , spindle - shaped melanosomes , and lipofuscin , and are located external to blamd , on brm .
mller cell processes appear to enter the corrugation horizontally from the left ( yellow arrow ) and sweep the pigmented cells away .
( e ) blamd is discontinuous and present at three separate locations ( red arrows ) .
in the normal henle fiber layer , mller cell processes are obliquely oriented ( outer - center to inner - periphery ) , and they are parallel to , and interleaved with , inner fibers of cone and rod photoreceptors . in the presence of severe photoreceptor degeneration , the trajectories of gliotic mller cell processes and remaining photoreceptors are oriented in many directions ( yellow arrows ) .
plateau signatures were observed in eight eyes of seven patients , of whom six were female .
mean age was 75 years ( range , 6089 ) and mean follow - up period was 7.7 years ( range , 3.711.6 ) .
baseline visual acuity ( va ) was logmar 0.42 ( snellen equivalent 20/52 ) ( range , 0.181 ) .
final va was logmar 1.16 ( snellen equivalent 20/289 ) ( range , 0.182.3 ) . in two eyes ,
at baseline , the presence of a drusenoid ped with homogeneous hyperreflective contents and an intact overlying rpe was noted in six eyes ( 75% ; figs . 2 , 3 ) .
one eye ( patient 5 , left eye ) had an extremely large ped measuring 2.24 mm with mostly hyporeflective contents and an overlying vitelliform lesion . in another eye ( patient 4 , left eye ) , the plateau was already present at baseline .
pigmentary changes noted on cfp were also found on nir , rf , and faf in six eyes , and these corresponded to intraretinal hyperreflective foci on oct ( fig .
3 ) . overlying the ped surface , the ellipsoid zone and outer nuclear layer ( onl ) were thin , with the opl nearly apposed to the surface of the ped .
multimodal imaging of two patients ( a , b ) at baseline and follow - up ( a , 4 years ; b , 7 years ) .
( a ) at baseline , patient 3 has a drusenoid ped with overlying pigment hyperplasia and hyperautofluorescent vitelliform material .
the oct b - scan shows intraretinal hyperreflective foci ( red arrowhead ) and loss of outer retinal bands over the apex of the ped .
four years later , a region of hypoautofluorescent ga contains a plateau that can not be appreciated in the cfp , nir , rf , or faf .
the oct b - scan shows a plateau with a shape similar to the baseline ped .
the downwardly deflecting opl ( yellow arrow ) has moved laterally as it follows the border of the advancing ga . there was marked attenuation and decreased reflectivity of the rpe - bl band .
( b ) at baseline , patient 6 has a drusenoid ped with overlying pigment hyperplasia that is located inferior to an area of ga .
bottom row : seven years later , the ga has markedly enlarged , and the ped is no longer visible on cfp , nir , rf , or faf . the oct b - scan shows that following progressive loss of the onl , the opl ( yellow arrow ) appears draped over a plateau with a shape similar to the baseline ped .
during follow - up , onl thinning resulted in the opl approximating the surface of the ped in all cases in which the ped was present at baseline ( figs . 2 , 3 ) .
as ga progressed , this point of approximation moved toward the edges of the ped base , following the advancing border of ga ( supplementary video ) . in all eyes , there was also progressive thinning of the hyperreflective rpe - bl complex and diminution of its reflectivity , beginning at the apex of the ped and advancing toward its edges .
after rpe loss occurred over the entire ped surface , the plateau ( i.e. , a thin hyperreflective inner surface and an interior of heterogeneous and overall reduced reflectivity ) appeared . the plateau surface was highly reflective on both cross - sectional and en face oct ( fig .
4 , en face oct surface ) and showed the presence of focal defects ( fig .
4 , yellow arrows ) . on cross - sectional oct , the inner contents of the plateau consisted of round , hyporeflective areas ( fig .
4 , blue arrows ) that were usually accompanied by smaller hyperreflective foci seen beneath the hyperreflective border .
these hyperreflective foci also could be observed within the area of ga as hyperautofluorescent foci on faf and hyperreflective foci on en face oct ( fig .
4 , green arrows ) . accordingly , on en face oct , plateau contents were seen as honeycomb - like structures with hyporeflective spaces interspersed with hyperreflective foci ( fig .
4 , green arrows ) increased in abundance within the plateau interior during follow - up in all eyes as seen on cross - sectional and en face oct ( figs . 2 , 3 , 4 ) .
on both cross - sectional and en face octa , no abnormal flow signals were noted within plateaus ( fig .
( a ) a plateau located within the area of ga features internal hyporeflective areas on cross - sectional oct ( blue arrowhead ) , which appear to be organized in a honeycomb - like pattern on en face oct ( blue arrowhead ) . on cross - sectional oct ,
octa , and on oct surface ( an en face oct slab at the level of the rpe - bl atop the plateau ) , the plateau interior is bounded by a distinct hyperreflective band that continues as a thick and highly reflective rpe band .
this hyperreflective surface shows transient hyporeflective defects ( yellow arrows ) during the evolution of these lesions .
an en face oct image of the plateau surface allows better appreciation of the focal defects ( yellow arrow ) .
( b ) a plateau located within the area of ga features internal hyporeflective areas on cross - sectional oct ( blue arrowhead ) , which appear to be organized in a honeycomb - like pattern on en face oct ( blue arrowhead ) . on cross - sectional oct and octa ,
a distinct hyperreflective surface surrounds the plateau interior and focal defects ( yellow arrows ) can be seen in some areas .
an en face oct taken at the plateau surface allows better appreciation of focal defects in this hyperreflective layer ( en face octa : surface yellow arrow ) .
deep to these focal defects are hyperreflective features ( faf and en face oct : green arrowheads ) that are also seen on faf as punctate hyperfluorescence within the confluent area of hypoautofluorescence .
in seven of eight eyes , the plateau was located in the macula . in one eye ,
a single plateau was seen in all eyes except for one ( patient 2 ) that had two separate plateaus ( fig .
patient 6 had one plateau in each eye . in the seven eyes with a baseline ped ,
the thinning of the hyperreflective band over the ped surface after rpe loss ( at which point we consider the plateau to be formed ) was preceded by a sharp decrease in ped volume ( fig .
subsequently , the volume of the residual plateau appeared very stable over time ( fig .
the point at which the plateaus form ( complete loss of rpe from the hyperreflective rpe - bl complex over the ped ) is shown by the dotted line .
patient 2 has two separate peds that evolve to two separate plateaus ( a , b ) .
patient 6 has a plateau in both the right eye ( r ) and the left eye ( l ) .
only patients with an intact hyperreflective rpe - bl complex baseline were included in this analysis . when the plateau signature is first observed ( black dotted line ) , a decrease in the volume that stabilizes on further follow - up .
figure 6 shows histology of a donor eye with ga , for which local relationships and cellular processes in the interior of outer retinal corrugations were particularly clear yet also representative of other ga eyes available for review .
atrophy spared the fovea in this eye , as seen with ex vivo cfp and 488-nm autofluorescence , and small reflective puncta in the atrophic area were apparent on nir ( figs .
6e ) , blamd was seen as discontinuous , being present at three separate locations .
pigmented cells were of the subducted phenotype , that is , rpe - originated cells located external to blamd , in contact with brm . for reference , in the henle fiber layer of a normal eye , obliquely oriented mller cell processes are parallel to , and interleaved with , inner fibers of cone and rod photoreceptors . in the presence of severe photoreceptor degeneration , including ort in this ga eye , the trajectories of gliotic mller cell processes were oriented in many directions , as indicated by arrows in figure 6e . in one instance
, processes seemed to enter an outer retinal corrugation horizontally through an opening in the blamd and sweep the subducted cells off brm ( fig .
6d ) . persistent bl deposit and mller cell processes in ga in a 95-year - old white man .
( a c ) ex vivo imaging of postmortem fundus . ( a ) cfp ( top row left ) shows a large area of atrophy of rpe extending to the optic nerve head but sparing the fovea .
green arrowheads approximate the level of the histologic section shown in the top far right and bottom .
( b ) ex vivo 488-nm autofluorescence shows loss of signal in the atrophic area .
( c ) ex vivo nir imaging shows small and highly reflective puncta in the atrophic area .
detailed ( d ) and panoramic ( e ) views of the atrophic area in histology .
nfl , nerve fiber layer ; gcl , ganglion cell layer ; ipl , inner plexiform layer ; dr , calcified druse .
( d ) processes from mller cells under a corrugation of persistent blamd in the atrophic area .
this corrugation overlies subducted cells of rpe origin ( teal arrowheads ) , which contain nuclei , spindle - shaped melanosomes , and lipofuscin , and are located external to blamd , on brm .
mller cell processes appear to enter the corrugation horizontally from the left ( yellow arrow ) and sweep the pigmented cells away .
( e ) blamd is discontinuous and present at three separate locations ( red arrows ) .
in the normal henle fiber layer , mller cell processes are obliquely oriented ( outer - center to inner - periphery ) , and they are parallel to , and interleaved with , inner fibers of cone and rod photoreceptors . in the presence of severe photoreceptor degeneration , the trajectories of gliotic mller cell processes and remaining photoreceptors are oriented in many directions ( yellow arrows ) .
through a retrospective review of amd patients with long - term serial eye - tracked sd - oct , we identified a series of eyes in which drusenoid peds progressed into plateau signatures within areas of ga .
plateaus were seen on oct to contain heterogeneous reflectivity enclosed by a thin overlying hyperreflective surface . a focal defect in the hyperreflective surface , in addition to intraretinal and sub - rpe hyperreflective dots , was observed during the evolution of ped into plateau signatures .
opl subsidence was noted to begin at the surface of the ped and progress toward the edge following the areas of expanding atrophy .
there was a gradual increase in volume due to an increase in hyporeflective contents initially , followed by complete loss of the rpe and a sharp decrease in ped volume , at which time the plateau was considered to have formed .
the study of drusen life cycles and ped evolution are important to improve our understanding of the pathogenesis of amd . understanding the origins and evolution of various oct findings and correlating these findings to histology enable the in vivo study of microscopic cellular changes occurring in these eyes with progressive ga .
the development of ga has been previously reported to occur in 19% of eyes with drusenoid peds and to be associated with specific features such as subsidence of the opl and inner nuclear layer and the development of a hyporeflective wedge - shaped band within the henle fiber layer described by mons et al .
the appearance of plateaus should be distinguished from other signatures previously described in ga , such as ort , ghost drusen , hyperreflective crown - like structures , hps , and refractile drusen ( figs . 1 , 2 ) .
plateau boundaries are difficult to distinguish from the surrounding atrophy using cfp , nir , rf , and faf ( figs . 1 , 3 , 4 , 6 ) .
previous studies have reported focal areas of hyperautofluorescence within ga , and in our study these correlated with hyperreflective foci seen on en face oct ( fig .
the presence of many pigmented cells of apparent rpe origin within atrophic areas is now well - documented ( fig .
6 ) , and such cells could give rise to both autofluorescence and oct hyperreflectivity signals .
plateaus do not contain the highly refractile material as seen on cfp and nir in eyes with spherules in calcifying drusen .
1 ) have a similar overlying thin hyperreflective layer consistent with an overlying blamd drape , as previously observed in oct and in histology ( fig .
our current data support a previous study that suggested that plateaus occur at a low frequency in eyes with ga ( 7.5% ) .
the plateau is a signature of a residual structure seen on oct once the rpe has progressed into complete atrophy . in our study , after plateau formation as defined by loss of the overlying rpe layer exposing the underlying blamd that then persists , minimal changes in plateau volume were observed on subsequent follow - up , as previously seen . in all eyes of our series , a drusenoid ped was noted to precede the appearance of the plateau . in a prior description ,
plateaus were thought to evolve in the outer retina and the hyperreflective line that is likely persistent blamd was attributed to the opl .
in contrast , serial eye - tracked oct imaging in our cases shows that changes occurred in the sub - rpe compartment , defined in atrophic eyes as the space external to persistent blamd . during development of a plateau , its thin hyperreflective surface ,
having emerged after rpe atrophy , is readily distinguishable from the overlying subsiding opl ( figs . 1 , 2 ) .
plateaus are one route from drusenoid ped to atrophy , and they can be contrasted with a route via ped growth and collapse , which we recently described . growth and collapse of large ped appear to be on a continuum with similar changes described for smaller drusen
. why a ped in any one eye proceeds to a plateau or to collapse is not known at present .
our detailed description of plateaus will facilitate mechanistic thinking and guide the design of future studies with more patients to investigate associated risk factors that may have prognostic value .
evolution of a plateau entails at least four processes that are discernible at the histologic level and also in sd - oct . in
approximately chronologic order , the sequence of the evolution is as follows : loss of rpe , persistence of blamd , followed by concurrent clearing of druse contents and occupation of druse space by cellular processes .
the loss of rpe is the point at which a drusenoid ped becomes a plateau , and it is marked by a thick , roughened , and highly hyperreflective band becoming thinner , smoother , and moderately hyperreflective .
hyperreflective foci over ped destined for plateaus were noted in our series , suggestive of anterior rpe migration , as also seen over ped destined for collapse .
the development of ga is hypothesized to proceed via two main pathways of rpe fate , including migration of fully pigmented and nucleated rpe into the retina ( called sloughed and intraretinal ) and shedding of non - nucleated granule aggregates , possibly apoptotic remnants , into underlying blamd .
our previous studies correlated intraretinal hyperreflective foci seen on in vivo and ex vivo oct with anteriorly migrating rpe cells .
thus , we have good reason to suspect that the spherical hyperreflective intraretinal foci directly internal to the plateaus are also migrating rpe cells . the thin , moderately hyperreflective band draped over plateaus
blamd is a stereotypically structured thick layer of basement membrane material that was initially used by sarks et al . to stage amd eyes and
a basement membrane is a defining feature of an epithelium , as is the rpe .
it is crossed by lipoprotein particles en route to brm and choriocapillaris for apparent egress to the systemic circulation .
when blamd is thick enough to be visible , it is observable as a hyporeflective layer .
after the rpe atrophies and can no longer cast a shadow , blamd emerges as a moderately hyperreflective line of overall horizontal orientation that is sometimes corrugated .
outer retinal corrugations were proposed to be contraction folds of persistent blamd created by loss of neovascular tissue or drusen / basal linear deposit previously located in the sub - rpe space . here
we extend this model to large drusenoid ped , on the grounds that corrugations and plateaus represent a continuum of oct signatures involving persistent blamd .
further , we propose on the basis of prior pathology literature a possible mechanism for how sub - rpe material may be cleared and the shape of persistent blamd established .
our clinical series showed that most of the peds that evolved into plateaus were drusenoid , and druse contents ( e.g. , lipids , proteins , and minerals ) appear to be removed , replaced , or both , over time .
the cells and secreted factors ( e.g. , metalloproteinases , lipases ) responsible for druse removal are not established .
mller cells are candidates , because previous studies have shown that mller cell processes may protrude into the sub - rpe space , extend over brm , and break up persistent blamd .
gliosis indicated by intense glial fibrillary acidic protein immunoreactivity is prominent in areas of severe photoreceptor loss and rpe atrophy , and mller cells remain when all photoreceptors have died . in our exemplar histologic case ,
we hypothesize that plateau and corrugation formation requires extension of mller cell processes through defects in the rpe / blamd complex ( figs . 4 , 6 ) that occur during the life cycle of drusenoid peds .
we further hypothesize that increasing hyporeflectivity observed within the sub - rpe space during the progression of drusenoid peds represents a change in composition of the sub - rpe material that encourages the entry of these processes .
the strength of this research is the long follow - up period for our patient cohort , with serial eye - tracked sd - oct imaging for up to 7 years and notably dating back to before the development of ga in all but one eye .
another strength is the availability of up to nine separate imaging technologies , each specifically highlighting different tissue features in any one case and the availability of one illustrative histology case .
limitations include the small number of eyes and the absence of direct correlation of in vivo imaging to postmortem histology ; hence , although our hypothesis of mller cells involvement in plateau formation is credible and testable , whether these cellular activities are directly visible in oct is currently unknown .
our technique of volume measurement may be subject to variations according to oct b - scan density between patients , but for our purpose of studying progression on a per - patient basis , this limitation was acceptable . to minimize inconsistencies
, all patients in our study had eye - tracked scans at each follow - up visit with similar density scan patterns available for analysis .
our study adds value to existing literature on ped by suggesting a role of mller cell gliosis , elongation , and elaboration in the pathogenesis of amd , which should be further explored in future studies .
it is important for clinicians to be aware of plateaus and to distinguish them from oct findings related to neovascular amd , such as vascularized peds or signs of exudation , as octa imaging of three eyes in our study showed no evidence of neovascular flow within plateaus ( fig .
plateau structures should not be confused with areas of subretinal fluid and treatment is not indicated for this finding .
finally , the distinctive reflectivity of persistent blamd in outer retinal corrugations , and , now , plateaus , provides strong motivation for renaming the fourth outer retinal hyperreflective band of normal eyes , which is currently called the rpe - brm complex , to the rpe - blam - brm complex ( abbreviated as rbb ) to accommodate the appearance of ped and persistent blamd in eyes with amd .
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purposehistologic details of progression routes to geographic atrophy ( ga ) in amd are becoming available through optical coherence tomography ( oct ) .
we studied the origins and evolution of an oct signature called plateau in eyes with ga and suggested a histologic correlate.methodsserial eye - tracked oct scans and multimodal imaging were acquired from eight eyes of seven patients with ga and plateau signatures over a mean follow - up of 7.7 years ( range , 3.711.6 ) .
the histology of unrelated donor eyes with amd was reviewed.resultsdrusenoid pigment epithelial detachment ( ped ) on oct imaging progressed into wide - based mound - like signatures with flattened apices characterized by a hyporeflective yet heterogeneous interior and an overlying hyperreflective exterior , similar to outer retinal corrugations previously ascribed to persistent basal laminar deposit ( blamd ) but larger .
these new signatures are described as plateaus .
an initial increase of the ped volume and hyporeflectivity of its contents was followed by a decrease in ped volume and thinning of an overlying hyperreflective band attributable to the loss of the overlying rpe leaving persistent blamd .
both imaging and histology revealed persistent blamd with defects through which gliotic mller cell processes pass.conclusionsplateaus can be traced back to drusenoid peds on oct imaging .
we hypothesize that during progressive rpe atrophy , mller cell extension through focal defects in the residual persistent blamd may contribute to the heterogeneous internal reflectivity of these entities .
the role of mller cell activation and extension in the pathogenesis of amd should be explored in future studies .
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Methods
Study Cohort
Imaging Protocol and Analysis
SD-OCT Terminology
Quantification of Volume Changes During Progression
Histology Study
Results
Patient Characteristics
Baseline Features
Characteristics Seen During Progression of Drusenoid PED into the Plateau
Changes in Volume of the Drusenoid PED and Plateau Signature With Time
Illustrative Histologic Case
Discussion
Supplementary Material
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during the past 2 decades , laparoscopic surgery has gained worldwide popularity resulting from its well - known characteristics of less postoperative pain , fewer complications , earlier discharge , and better cosmesis .
smaller skin incisions in laparoscopic surgery were related to fewer unwanted side effects , including less postoperative pain . however , in recent studies comparing conventional and single - incision laparoscopic surgical procedures , the postoperative pain scores were not different .
keyless abdominal rope - lifting surgery ( kars ) is a novel , gasless ( isobaric ) , single - incision laparoscopic surgical technique used in various gynecologic operations and cholecystectomies . besides being a single - incision laparoscopic technique
, kars does not require carbon dioxide ( co2 ) use to inflate the abdominal wall and the intra - abdominal pressure is not elevated during the operation .
in addition , postoperative shoulder pain seems to be more frequently observed after laparoscopic surgical procedures in which the intra - abdominal pressure is elevated to between 12 and 18 mm hg by use of co2 . in this study we aimed to compare the postoperative pain levels for kars and conventional multiport laparoscopy performed for adnexal cysts .
although kars has been performed in our institute since the first half of 2010 , the study included benign ovarian cysts operated on between november 2011 and june 2013 .
beginning in november 2011 , intraoperative and postoperative pain management for all operations performed in our unit was standardized ; thus the uniform management approach allowed us to collect and analyze the data from different surgical techniques .
the study was approved by the local institutional ethical committee of kafkas university school of medicine .
the included women had cystic masses including simple cysts with or without septation , endometriomas , and benign cystic teratomas .
most of the participants complained of groin pain , menstrual bleeding disorders , dysmenorrhea , and infertility .
emergency cases including patients with hemodynamic instability , severe abdominal pain , and inaccurate preoperative diagnoses were excluded .
malignant cysts were excluded based on the patient 's history , clinical findings , ultrasonography and magnetic resonance imaging , and levels of cancer antigen 125 , alpha fetoprotein , carcinoembryonic antigen , and cancer antigen 199 .
women who were diagnosed with an adnexal cyst and in whom a laparoscopic operation was indicated were invited to choose one of the operative techniques .
because kars was performed only in our center , we could not design a randomized trial .
thus the groups of this cross - sectional prospective clinical study were formed according to the women 's choice as the kars or conventional co2 laparoscopy groups .
initial visual analog scale ( vas ) scores of 5.7 1.42 and 6.3 1.42 in patients undergoing kars and conventional laparoscopy , respectively , indicated that we needed at least 30 operations in each group to perform a study with a statistical power of 80% at an level of .05 .
the demographic and physical characteristics of the participating patients included their age , gravidity , parity , height , weight , and body mass index , as well as the number of abortions , ectopic pregnancies , and offspring .
oral intake was prohibited starting at 11 pm , and a 135-ml solution containing 19 g of sodium dihydrogen phosphate and 7 g of disodium hydrogen phosphate was administered rectally at 6 am on the operation day .
we used the operative laparoscopy study group 's classification system for staging intra - abdominal adhesions : 0 , none ; 1 , filmy and avascular ; 2 , dense and vascular ; and 3 , binding and cohesive .
the abdominal access time in kars patients included the time needed for the construction of the pathway into the abdominal cavity and the placement of the lifting ropes . in conventional laparoscopy patients ,
the abdominal access time included the time needed for the creation of the co2 pneumoperitoneum at 14 mm hg of pressure and the insertion of the 3 intra - abdominal access ports .
all included patients were considered class 1 or 2 according to the american society of anesthesiologists classification and received a standard anesthetic management regimen during the operations .
we used propofol , 1.5 to 2.5 mg / kg , to induce anesthesia and facilitated intubation with rocuronium , 0.4 to 0.6 mg / kg .
oxygen was supplemented before and after the intubation at rates of 100% and 50% ( mixed with the operating theater 's air ) , respectively .
we used 2% sevoflurane and 50 g of fentanyl for anesthesia maintenance . to replace the fluid deficit , we used sodium chloride or ringer lactate solution at 10 ml / kg on intravenous insertion at 2 ml / kg per hour .
unexpected bleeding was managed by use of additional crystalloid solution at 3 ml / kg per hour .
atropine , 1 to 1.2 mg intravenously , was used to reverse the neuromuscular blockade .
the postoperative pain management and vas pain score recordings were initiated in the obstetrics and gynecology service .
all patients , regardless of initial vas pain score , received 50 mg of meperidine intramuscularly at the beginning of the postoperative period and 75 mg of diclofenac sodium during the second postoperative hour .
vas pain scores were recorded at the beginning and second , fourth , and 24th hours of the postoperative period .
beginning from the fourth postoperative hour , patients with vas pain scores of > 4 points received an additional dose of meperidine , 25 mg intramuscularly .
kars has common features of conventional laparoscopy and laparotomy . during the creation of the abdominal access pathway ,
the umbilical fold is lifted with 2 clamps bilaterally and a third clamp at the center of the umbilicus .
depending on the need for multiple instrument use or the depth and thickness of the subcutaneous tissue , the length of the incision may be adjusted smaller or larger .
after the skin incision , the subcutaneous tissue is dissected bluntly with the tip of a fine instrument until the underlying fascia is reached .
the opening is bluntly widened with the moderate stretching force of 2 fine retractors . at this stage
, the surgeon can examine the intra - abdominal viscera by using his or her index or little finger to identify possible adhesions .
the fascia at the entry site is lifted by 2 stitches placed at the lower and upper borders at the 6- and 12-o'clock positions ( figure 1 ) to guide the rest of the process .
while the 2 stitches elevate the entry site , the intra - abdominal viscera is observed for possible injuries and adhesions by use of the introduced telescope .
the lower and upper borders of the fascia underlying the umbilical incision are sutured at the 6- and 12-o'clock positions .
the veress needle is unloaded from its cannula , and one tip of a no .
the loaded cannula is introduced into the elevated entry site under direct or telescopic vision .
the tip of the cannula is inserted toward the abdominal wall , 1 to 2 cm below the entry , under direct eye vision , and the tip is slid just above the peritoneum .
it is turned to the right 6 to 7 cm to avoid injury to the epigastric vessels , and the abdominal wall is pierced from the inside toward the outside at approximately 5 cm below the umbilical entry . during this procedure , the tip of the veress cannula should always be maintained in an upward direction to avoid an accidental bowel injury .
the tip of the suture is unloaded from the cannula outside the abdominal wall , and the unloaded cannula is withdrawn back from the entry .
the other tip of the suture is loaded into the cannula , and the same steps are repeated . however , this time , the tip of the cannula is taken outside the abdominal cavity , 5 cm below the first tip
then , an assistant elevates the abdominal wall , and the surgeon ties the sutures over the prepared sterile retractor located between the pubis and umbilicus ( figure 3 ) .
the entry - site sutures are stretched during introduction of the telescope into the abdominal cavity to prevent staining of the optic of the telescope .
the laparoscopic hand instruments are used through the same single incision ( figure 3 ) .
there is no need to use trocars ( keyless ) to prevent gas leakage , and any surgical instrument ( a laparoscopic hand instrument or a conventional surgical hand instrument ) that can pass through the incision can be used .
if the intra - abdominal operative space is not adequate to perform the operation , additional lifting sutures can be placed at the sites at which further elevations are required .
lifting of a particular area ( eg , protrusion of the peritoneal folds or adipose tissue into the operative field , particularly in overweight patients ) can be managed by a suture just penetrating the skin and tied to the same retractor .
if the operation is limited to the pelvis , the retractor used does not limit the surgeon 's motions .
however , if an additional operation is anticipated at the upper side of the abdominal cavity , the placement of the retractor should be changed .
the fascial layer is closed with continuous unlocked sutures while the lifting sutures assist the elevation of the fascial layer .
3 - 0 or 4 - 0 intracutaneous absorbable sutures and hidden into the umbilical fold .
one month after the operation , the umbilical incision and the scars of the veress cannula are nearly indemonstrable ( figure 4 ) .
the umbilical incision and the scars of the veress cannula are nearly indemonstrable 1 month after surgery .
co2 laparoscopy is performed with the use of a 10-mm port ( infraumbilical port for the telescope ) and two 5-mm ports ( lateral ports for the hand instruments ) placed after the creation of co2 pneumoperitoneum using the veress needle . at the end of the surgical procedures ,
all fascial - layer incisions of the 10-mm ports are sutured before the placement of skin sutures ; however , only the skin incisions of 5-mm ports are closed with no . 3 - 0 or 4
statistical analysis was performed with spss software , version 16.0 ( spss , chicago , illinois ) .
the distribution of variables was studied by use of kurtosis , skewness , and shapiro - wilk tests .
normally distributed variables of the 2 groups were compared by use of the student t test , and non normally distributed variables were compared by use of the mann - whitney u test .
the test was used to assess the probability of existence of shoulder pain in the 2 groups .
a logistic regression model was created to study the confounding factors that influenced the perception of shoulder pain .
the hosmer - lemeshow test showed that the model fit ( = 13 762 , p = .88 , n = 77 ) .
kars has common features of conventional laparoscopy and laparotomy . during the creation of the abdominal access pathway ,
the umbilical fold is lifted with 2 clamps bilaterally and a third clamp at the center of the umbilicus .
depending on the need for multiple instrument use or the depth and thickness of the subcutaneous tissue , the length of the incision may be adjusted smaller or larger . after the skin incision , the subcutaneous tissue is dissected bluntly with the tip of a fine instrument until the underlying fascia is reached .
the opening is bluntly widened with the moderate stretching force of 2 fine retractors . at this stage
, the surgeon can examine the intra - abdominal viscera by using his or her index or little finger to identify possible adhesions .
the fascia at the entry site is lifted by 2 stitches placed at the lower and upper borders at the 6- and 12-o'clock positions ( figure 1 ) to guide the rest of the process .
while the 2 stitches elevate the entry site , the intra - abdominal viscera is observed for possible injuries and adhesions by use of the introduced telescope .
the lower and upper borders of the fascia underlying the umbilical incision are sutured at the 6- and 12-o'clock positions .
the veress needle is unloaded from its cannula , and one tip of a no .
the loaded cannula is introduced into the elevated entry site under direct or telescopic vision .
the tip of the cannula is inserted toward the abdominal wall , 1 to 2 cm below the entry , under direct eye vision , and the tip is slid just above the peritoneum .
it is turned to the right 6 to 7 cm to avoid injury to the epigastric vessels , and the abdominal wall is pierced from the inside toward the outside at approximately 5 cm below the umbilical entry . during this procedure , the tip of the veress cannula should always be maintained in an upward direction to avoid an accidental bowel injury .
the tip of the suture is unloaded from the cannula outside the abdominal wall , and the unloaded cannula is withdrawn back from the entry .
the other tip of the suture is loaded into the cannula , and the same steps are repeated . however , this time , the tip of the cannula is taken outside the abdominal cavity , 5 cm below the first tip .
then , an assistant elevates the abdominal wall , and the surgeon ties the sutures over the prepared sterile retractor located between the pubis and umbilicus ( figure 3 ) .
the entry - site sutures are stretched during introduction of the telescope into the abdominal cavity to prevent staining of the optic of the telescope .
the laparoscopic hand instruments are used through the same single incision ( figure 3 ) .
there is no need to use trocars ( keyless ) to prevent gas leakage , and any surgical instrument ( a laparoscopic hand instrument or a conventional surgical hand instrument ) that can pass through the incision can be used .
if the intra - abdominal operative space is not adequate to perform the operation , additional lifting sutures can be placed at the sites at which further elevations are required .
lifting of a particular area ( eg , protrusion of the peritoneal folds or adipose tissue into the operative field , particularly in overweight patients ) can be managed by a suture just penetrating the skin and tied to the same retractor .
if the operation is limited to the pelvis , the retractor used does not limit the surgeon 's motions .
however , if an additional operation is anticipated at the upper side of the abdominal cavity , the placement of the retractor should be changed .
the fascial layer is closed with continuous unlocked sutures while the lifting sutures assist the elevation of the fascial layer .
3 - 0 or 4 - 0 intracutaneous absorbable sutures and hidden into the umbilical fold .
one month after the operation , the umbilical incision and the scars of the veress cannula are nearly indemonstrable ( figure 4 ) .
the umbilical incision and the scars of the veress cannula are nearly indemonstrable 1 month after surgery .
kars has common features of conventional laparoscopy and laparotomy . during the creation of the abdominal access pathway ,
the umbilical fold is lifted with 2 clamps bilaterally and a third clamp at the center of the umbilicus .
depending on the need for multiple instrument use or the depth and thickness of the subcutaneous tissue , the length of the incision may be adjusted smaller or larger . after the skin incision , the subcutaneous tissue is dissected bluntly with the tip of a fine instrument until the underlying fascia is reached .
the opening is bluntly widened with the moderate stretching force of 2 fine retractors . at this stage
, the surgeon can examine the intra - abdominal viscera by using his or her index or little finger to identify possible adhesions .
the fascia at the entry site is lifted by 2 stitches placed at the lower and upper borders at the 6- and 12-o'clock positions ( figure 1 ) to guide the rest of the process .
while the 2 stitches elevate the entry site , the intra - abdominal viscera is observed for possible injuries and adhesions by use of the introduced telescope .
the lower and upper borders of the fascia underlying the umbilical incision are sutured at the 6- and 12-o'clock positions .
the veress needle is unloaded from its cannula , and one tip of a no .
the loaded cannula is introduced into the elevated entry site under direct or telescopic vision .
the tip of the cannula is inserted toward the abdominal wall , 1 to 2 cm below the entry , under direct eye vision , and the tip is slid just above the peritoneum .
it is turned to the right 6 to 7 cm to avoid injury to the epigastric vessels , and the abdominal wall is pierced from the inside toward the outside at approximately 5 cm below the umbilical entry . during this procedure , the tip of the veress cannula should always be maintained in an upward direction to avoid an accidental bowel injury .
the tip of the suture is unloaded from the cannula outside the abdominal wall , and the unloaded cannula is withdrawn back from the entry .
the other tip of the suture is loaded into the cannula , and the same steps are repeated . however , this time , the tip of the cannula is taken outside the abdominal cavity , 5 cm below the first tip .
then , an assistant elevates the abdominal wall , and the surgeon ties the sutures over the prepared sterile retractor located between the pubis and umbilicus ( figure 3 ) .
the entry - site sutures are stretched during introduction of the telescope into the abdominal cavity to prevent staining of the optic of the telescope .
the laparoscopic hand instruments are used through the same single incision ( figure 3 ) .
there is no need to use trocars ( keyless ) to prevent gas leakage , and any surgical instrument ( a laparoscopic hand instrument or a conventional surgical hand instrument ) that can pass through the incision can be used .
if the intra - abdominal operative space is not adequate to perform the operation , additional lifting sutures can be placed at the sites at which further elevations are required .
lifting of a particular area ( eg , protrusion of the peritoneal folds or adipose tissue into the operative field , particularly in overweight patients ) can be managed by a suture just penetrating the skin and tied to the same retractor .
if the operation is limited to the pelvis , the retractor used does not limit the surgeon 's motions .
however , if an additional operation is anticipated at the upper side of the abdominal cavity , the placement of the retractor should be changed .
the fascial layer is closed with continuous unlocked sutures while the lifting sutures assist the elevation of the fascial layer .
3 - 0 or 4 - 0 intracutaneous absorbable sutures and hidden into the umbilical fold .
one month after the operation , the umbilical incision and the scars of the veress cannula are nearly indemonstrable ( figure 4 ) .
the umbilical incision and the scars of the veress cannula are nearly indemonstrable 1 month after surgery .
co2 laparoscopy is performed with the use of a 10-mm port ( infraumbilical port for the telescope ) and two 5-mm ports ( lateral ports for the hand instruments ) placed after the creation of co2 pneumoperitoneum using the veress needle . at the end of the surgical procedures ,
all fascial - layer incisions of the 10-mm ports are sutured before the placement of skin sutures ; however , only the skin incisions of 5-mm ports are closed with no . 3 - 0 or 4 - 0 intracutaneous absorbable sutures .
statistical analysis was performed with spss software , version 16.0 ( spss , chicago , illinois ) .
the distribution of variables was studied by use of kurtosis , skewness , and shapiro - wilk tests .
normally distributed variables of the 2 groups were compared by use of the student t test , and non normally distributed variables were compared by use of the mann - whitney u test .
the test was used to assess the probability of existence of shoulder pain in the 2 groups .
a logistic regression model was created to study the confounding factors that influenced the perception of shoulder pain .
the hosmer - lemeshow test showed that the model fit ( = 13 762 , p = .88 , n = 77 ) .
during a 20-month period , 77 women with benign ovarian cysts were operated on by kars ( n = 32 ) or conventional co2 laparoscopy ( n = 45 ) .
the demographic data , physical characteristics , and preoperative findings of the participating women were summarized in table 1 .
the demographic findings , surgical histories , and preoperative hematologic statuses of the groups did not differ significantly ( p > .05 ) .
demographic data , physical characteristics , and preoperative findings of participating women in both groups data are presented as mean standard deviation unless otherwise indicated .
conventional multiport laparoscopy performed after creation of pneumoperitoneum . student t test ( used for normal distribution ) . mann - whitney test ( used for non - normal distribution ) .
minor bleeding and unintended cyst ruptures were the only observed intraoperative complications . in none of the cases was a blood transfusion or postoperative intensive care admission needed .
we did not need to convert to conventional laparoscopy or laparotomy in the kars group or to laparotomy or mini - laparotomy in the conventional laparoscopy group .
simple oral analgesic use after the 24th hour of the postoperative period was adequate for postoperative pain relief in both groups .
most of the intraoperative and postoperative findings were similar in the 2 groups ( p > .05 ) ; however , the abdominal cavity access and total operative times in the kars group were significantly longer than those in the conventional laparoscopy group ( p < .05 ) .
intraoperative and postoperative findings according to operative technique data are presented as mean standard deviation unless otherwise indicated .
the vas pain scores at the beginning and second , fourth , and 24th hours of the postoperative period were significantly lower in the kars group ( p < .05 ) , although the differences in mean vas scores never exceeded 1 point .
in addition , the women in the kars group needed less additional analgesia in comparison with those in the conventional laparoscopy group ( p < .05 ) .
moreover , a higher number and percentage of the patients in the conventional co2 laparoscopy group had shoulder pain ( p < .05 ) .
postoperative pain scores and additional analgesic requirements according to operative techniques data are presented as mean standard deviation unless otherwise indicated .
conventional multiport laparoscopy performed after creation of pneumoperitoneum . student t test ( used for normal distribution ) . mann - whitney test ( used for non - normal distribution ) . the test analyzing shoulder pain showed that postoperative shoulder pain was significantly less frequent in the kars group ( = 5.304 , p = .021 , n = 77 ) .
logistic regression for postoperative shoulder pain predicted that shoulder pain was affected only by the operation type and duration ( table 4 ) .
although the operation duration was longer in the kars group , the frequency of shoulder pain was higher in the conventional laparoscopy group .
the total operative time correlated positively with the abdominal access and rope - lifting process time , weight and body mass index of the women , adhesion score , cyst diameter , cyst rupture rate , length of hospital stay , and shoulder pain frequency ( p < .05 ) . in the conventional laparoscopy group ,
the total operative time correlated positively with the abdominal access time , weight and body mass index of the women , adhesion score , cyst diameter , cyst rupture rate , length of hospital stay , and shoulder pain frequency ( p < .05 ) .
in comparison with conventional co2 laparoscopy , kars performed to treat benign ovarian cysts causes less postoperative abdominal and shoulder pain .
, our study is the first comparing the postoperative pain scores and additional analgesic requirements of a gasless single - incision laparoscopic technique with conventional multiport co2 laparoscopy .
postoperative pain scores during single - incision and conventional multiport co2 laparoscopic surgical procedures were compared previously ; however , the abdominal viscera was inflated by using co2 in all these studies . in our study all the operations for ovarian cysts were performed by the same surgical team under the same operating theater conditions .
although the study included a control group , the participants determined which operative technique they would undergo ; thus the study lacks the power of randomization .
the vas pain scores measured pain severity at the beginning and second , fourth , and 24th hours of the postoperative period ; however , the pain scores between the fourth and 24th hours were missing .
postoperative shoulder pain was evaluated by its presence or absence ; however , its severity was not evaluated .
pain sensation is subjective and may even differ for the same individual under different conditions .
it develops motivations to avoid damaging conditions , to be cautious about the damaged tissue , and to prevent future conditions related to pain . from this point of view , we did not gather information about the past pain experiences of the patients .
although the frequencies of previous operations were similar in both groups , we can not truly argue that the pain sensation , perception , and definition of the participants were similar .
gynecologic operations including myomectomy , hysterectomy , ovarian cyst resection , colposuspension , and radical hysterectomy were performed by various surgeons . however , most of the published articles dealt with the feasibility , complications , operative times , and cosmesis of the procedures .
in addition , although the previous surgical techniques consisted of gasless laparoscopic procedures , they had multiple abdominal entry sites .
gargiulo and nezhat published an article describing the details of 3 new minimally invasive surgical approaches , including single - incision laparoscopic surgery .
single - incision laparoscopic surgery seemed to yield more intraoperative complications because of the poor visualization and difficulty in maintaining pneumoperitoneum .
gargiulo and nezhat concluded that the improved postoperative pain with single - incision laparoscopic surgical procedures was not proven in comparison with the conventional multiport co2 laparoscopies .
similarly , murji et al conducted a meta - analysis of randomized controlled trials and high - quality observational studies with > 2000 patients and failed to ascertain the impact of single - incision laparoscopy surgery on postoperative pain because of the paucity of data and lack of uniform collection .
however , both articles reviewed the results of studies including surgical procedures performed under elevated intra - abdominal pressure created by the insufflation of co2 . in our study
in addition , postoperative shoulder pain experienced more frequently after co2 laparoscopies may increase the severity of perceived pain .
thus the gasless ( isobaric ) nature of our technique may be an explanation for the contradiction with the findings of the previous publications .
recently , gerbershagen et al studied postoperative pain scores on the first postoperative day in 70 764 german patients .
surprisingly , they found that patients had reported high pain scores after many minor surgical procedures , and contrary to this finding , many major abdominal surgical procedures had caused comparatively less pain because of sufficient epidural analgesia . in our study
we used a standardized pain management protocol in both groups ; thus our results suggest that the single - incision gasless laparoscopic technique yielded better pain scores than conventional co2 laparoscopy .
in addition , at the 24th hour of the postoperative period , 59.3% of the women in the kars group and 35.5% of the women in the conventional multiport co2 laparoscopy group had vas pain scores of 0 points . moreover , none of the scores in either group were > 4 points .
previous studies comparing the postoperative pain scores after single - incision and conventional laparoscopies mostly included cholecystectomies and showed similar pain scores for the same surgical procedures . in the prospective , randomized , multicenter trial conducted by marks et al
, the postoperative pain scores did not differ between single - incision and multiport laparoscopies .
however , a substantial increase in the incidence of hernia with cholecystectomies performed by single - incision laparoscopic surgery was found . in their study
the closure techniques for the access sites were not briefly defined , and one of the centers unavoidably lost more than one third of its patients to follow - up . in addition
, there were contradictory findings in other publications dealing with postoperative hernia formation after single - incision laparoscopies .
moreover , all of the included operations had been performed after the creation of pneumoperitoneum by using co2 .
better pain scores after kars may be the result of the avoidance of co2 pneumoperitoneum .
although single - incision laparoscopic surgery was found to be feasible , safe , and reproducible in gynecology patients with benign and cancerous diseases , only a few articles suggested better postoperative pain scores , and some of them were case reports or pilot studies . in 2011 fagotti et al published their randomized controlled trial comparing postoperative pain after conventional laparoscopy and laparoendoscopic single - site surgery ( less ) for benign adnexal disease .
they concluded that less was more advantageous than conventional multiport laparoscopy in terms of postoperative pain and the need for rescue analgesia .
although we had similar postoperative findings in terms of pain , the need for rescue analgesics , and length of hospital stay , we also had different findings .
the women included in the previous study had a higher median age ( 49 years in the less group and 42 years in the conventional laparoscopy group ) than the women in our study ( 33.5 years in the kars group and 38 years in the conventional laparoscopy group ) .
the median body mass index , major cyst diameter , and previous abdominal operation rate and the mean operative time in both groups in our study were higher than the values in both groups in the previous study . whereas fagotti et al showed a lower bleeding rate in the less group , we observed similar bleeding rates in the kars and conventional multiport laparoscopy groups . in comparison with conventional multiport laparoscopy ,
fagotti et al showed significantly lower pain scores in the less group at the second and fourth postoperative hours ; however , we showed lower pain scores in the kars group initially postoperatively and at the second , fourth , and 24th hours of the postoperative period .
in addition , we showed significantly less postoperative shoulder pain in the kars group , which may result from the gasless nature of kars . in 2011
prasad et al published their study comparing the postoperative pain scores after single - incision and conventional laparoscopic cholecystectomies .
they observed similar pain scores in both groups and noticed that pain scores decreased after the operative times decreased in the single - incision group .
although the decreased pain scores were also lower than the pain scores in the conventional laparoscopy group , the difference was not significant . in our study , although the kars group had significantly longer operative times ( by a mean of 16 minutes ) , the postoperative pain scores and additional analgesic requirements were significantly lower .
our results suggest that kars yields better pain scores than conventional multiport co2 laparoscopy and that fewer additional analgesics are required after kars .
however , one should note that our study included the results of surgical procedures performed for ovarian cysts . to increase the level of evidence , randomized prospective studies including various operations are needed .
in addition , the gasless single - incision technique should be compared with single - incision laparoscopic surgical procedures using co2 to create pneumoperitoneum .
in comparison with conventional co2 laparoscopy , kars performed to treat benign ovarian cysts causes less postoperative abdominal and shoulder pain .
to our knowledge , our study is the first comparing the postoperative pain scores and additional analgesic requirements of a gasless single - incision laparoscopic technique with conventional multiport co2 laparoscopy .
postoperative pain scores during single - incision and conventional multiport co2 laparoscopic surgical procedures were compared previously ; however , the abdominal viscera was inflated by using co2 in all these studies . in our study all the operations for ovarian cysts were performed by the same surgical team under the same operating theater conditions .
although the study included a control group , the participants determined which operative technique they would undergo ; thus the study lacks the power of randomization .
the vas pain scores measured pain severity at the beginning and second , fourth , and 24th hours of the postoperative period ; however , the pain scores between the fourth and 24th hours were missing .
postoperative shoulder pain was evaluated by its presence or absence ; however , its severity was not evaluated .
pain sensation is subjective and may even differ for the same individual under different conditions .
it develops motivations to avoid damaging conditions , to be cautious about the damaged tissue , and to prevent future conditions related to pain . from this point of view , we did not gather information about the past pain experiences of the patients .
although the frequencies of previous operations were similar in both groups , we can not truly argue that the pain sensation , perception , and definition of the participants were similar .
gynecologic operations including myomectomy , hysterectomy , ovarian cyst resection , colposuspension , and radical hysterectomy were performed by various surgeons .
however , most of the published articles dealt with the feasibility , complications , operative times , and cosmesis of the procedures .
in addition , although the previous surgical techniques consisted of gasless laparoscopic procedures , they had multiple abdominal entry sites .
gargiulo and nezhat published an article describing the details of 3 new minimally invasive surgical approaches , including single - incision laparoscopic surgery .
single - incision laparoscopic surgery seemed to yield more intraoperative complications because of the poor visualization and difficulty in maintaining pneumoperitoneum .
gargiulo and nezhat concluded that the improved postoperative pain with single - incision laparoscopic surgical procedures was not proven in comparison with the conventional multiport co2 laparoscopies .
similarly , murji et al conducted a meta - analysis of randomized controlled trials and high - quality observational studies with > 2000 patients and failed to ascertain the impact of single - incision laparoscopy surgery on postoperative pain because of the paucity of data and lack of uniform collection .
however , both articles reviewed the results of studies including surgical procedures performed under elevated intra - abdominal pressure created by the insufflation of co2 . in our study
in addition , postoperative shoulder pain experienced more frequently after co2 laparoscopies may increase the severity of perceived pain .
thus the gasless ( isobaric ) nature of our technique may be an explanation for the contradiction with the findings of the previous publications .
recently , gerbershagen et al studied postoperative pain scores on the first postoperative day in 70 764 german patients .
surprisingly , they found that patients had reported high pain scores after many minor surgical procedures , and contrary to this finding , many major abdominal surgical procedures had caused comparatively less pain because of sufficient epidural analgesia . in our study
we used a standardized pain management protocol in both groups ; thus our results suggest that the single - incision gasless laparoscopic technique yielded better pain scores than conventional co2 laparoscopy .
in addition , at the 24th hour of the postoperative period , 59.3% of the women in the kars group and 35.5% of the women in the conventional multiport co2 laparoscopy group had vas pain scores of 0 points . moreover , none of the scores in either group were > 4 points .
previous studies comparing the postoperative pain scores after single - incision and conventional laparoscopies mostly included cholecystectomies and showed similar pain scores for the same surgical procedures . in the prospective , randomized , multicenter trial conducted by marks et al , the postoperative pain scores did not differ between single - incision and multiport laparoscopies . however , a substantial increase in the incidence of hernia with cholecystectomies performed by single - incision laparoscopic surgery was found . in their study
the closure techniques for the access sites were not briefly defined , and one of the centers unavoidably lost more than one third of its patients to follow - up .
in addition , there were contradictory findings in other publications dealing with postoperative hernia formation after single - incision laparoscopies .
moreover , all of the included operations had been performed after the creation of pneumoperitoneum by using co2 .
better pain scores after kars may be the result of the avoidance of co2 pneumoperitoneum .
although single - incision laparoscopic surgery was found to be feasible , safe , and reproducible in gynecology patients with benign and cancerous diseases , only a few articles suggested better postoperative pain scores , and some of them were case reports or pilot studies . in 2011 fagotti et al published their randomized controlled trial comparing postoperative pain after conventional laparoscopy and laparoendoscopic single - site surgery ( less ) for benign adnexal disease .
they concluded that less was more advantageous than conventional multiport laparoscopy in terms of postoperative pain and the need for rescue analgesia .
although we had similar postoperative findings in terms of pain , the need for rescue analgesics , and length of hospital stay , we also had different findings .
the women included in the previous study had a higher median age ( 49 years in the less group and 42 years in the conventional laparoscopy group ) than the women in our study ( 33.5 years in the kars group and 38 years in the conventional laparoscopy group ) .
the median body mass index , major cyst diameter , and previous abdominal operation rate and the mean operative time in both groups in our study were higher than the values in both groups in the previous study . whereas fagotti et al showed a lower bleeding rate in the less group , we observed similar bleeding rates in the kars and conventional multiport laparoscopy groups . in comparison with conventional multiport laparoscopy ,
fagotti et al showed significantly lower pain scores in the less group at the second and fourth postoperative hours ; however , we showed lower pain scores in the kars group initially postoperatively and at the second , fourth , and 24th hours of the postoperative period .
in addition , we showed significantly less postoperative shoulder pain in the kars group , which may result from the gasless nature of kars . in 2011
prasad et al published their study comparing the postoperative pain scores after single - incision and conventional laparoscopic cholecystectomies .
they observed similar pain scores in both groups and noticed that pain scores decreased after the operative times decreased in the single - incision group .
although the decreased pain scores were also lower than the pain scores in the conventional laparoscopy group , the difference was not significant . in our study
, although the kars group had significantly longer operative times ( by a mean of 16 minutes ) , the postoperative pain scores and additional analgesic requirements were significantly lower .
our results suggest that kars yields better pain scores than conventional multiport co2 laparoscopy and that fewer additional analgesics are required after kars .
however , one should note that our study included the results of surgical procedures performed for ovarian cysts . to increase the level of evidence , randomized prospective studies including various operations are needed .
in addition , the gasless single - incision technique should be compared with single - incision laparoscopic surgical procedures using co2 to create pneumoperitoneum .
kars seems to cause less postoperative pain during the management of benign ovarian cysts in comparison with conventional multiport co2 laparoscopy .
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background and objectives : keyless abdominal rope - lifting surgery is a novel , gasless , single - incision laparoscopic surgical technique . in this study we aimed to compare the postoperative pain from keyless abdominal rope - lifting surgery with carbon dioxide laparoscopy performed for benign ovarian cysts.methods:during a 20-month period , 77 women underwent surgery for a benign ovarian cyst .
keyless abdominal rope - lifting surgery and conventional carbon dioxide laparoscopy techniques were used for the operations in 32 women and 45 women , respectively .
the 2 operative techniques were compared with regard to demographic characteristics ; preoperative , intraoperative , and postoperative data including early postoperative pain scores ; and frequency of shoulder pain and analgesic requirements.results:data regarding demographic characteristics , preoperative findings , cyst diameters and rupture rates , intra - abdominal adhesions , intraoperative blood loss , and postoperative hospital stay did not differ between groups ( p > .05 ) .
however , the mean operative and abdominal access times were significantly longer in the keyless abdominal rope - lifting surgery group ( p < .05 ) .
visual analog scale pain scores at initially and at the second , fourth , and 24th hours of the postoperative period were significantly lower in the keyless abdominal rope - lifting surgery group ( p < .05 ) .
similarly , keyless abdominal rope - lifting surgery caused significantly less shoulder pain and additional analgesic use ( p < .05).conclusion : keyless abdominal rope - lifting surgery seems to cause less pain in the management of benign ovarian cysts in comparison with conventional carbon dioxide laparoscopy .
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INTRODUCTION
METHODS
Keyless Abdominal Rope-Lifting Surgery
Abdominal access.
Rope-lifting process.
Wound closure.
CO
Statistical Analysis
RESULTS
DISCUSSION
Principal Findings
Strengths of Study
Limitations of Study
Comparison With Previous Studies
CONCLUSION
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workers ' health may be protected by his / her own preventive behavior , despite numerous health hazards in the workplace .
however , worker behavior is constrained by various workplace pressures such as working conditions , including what is considered to be accepted working behavior and overall morale of the working group .
based on the employment contract , every employer has a certain amount of control / power and the expectation that workers will follow work - related requests .
this may lead to adverse health effects in workers ; therefore , the government has implemented regulations to protect worker health , such as the occupational safety and health ( osh ) act .
adverse health effects induced by unexpected incidents may present as occupational injuries , but occupational diseases can appear due to long term continuous exposure to harmful chemicals , dust , noise , etc . to prevent occupational diseases , it is important to manage worker health via health monitoring for early detection of abnormal conditions .
it is very important for worker health management to monitor the level of worker exposure to hazards in the workplace ; for example , health check - ups including absorption of hazard materials , so called ' biological monitoring ' .
noise levels in the workplace should also be measured in order to prevent worker 's hearing loss .
if a worker is exposed to high levels of noise , he / she should be transferred to another work area without noise or provided with proper personal protective equipment ( ppe ) such as earplugs , etc .
as above , information needed for workers ' health management can be classified into three categories according to 3 questions : 1 ) what is the exact nature of the occupational hazards ?
3 ) what health problems are induced by these hazards ? these questions should be answered in order to manage workers ' health effectively and efficiently .
health management for workers is accomplished by two different strategies : 1 ) focusing on reduction of exposure to hazard factors in the workplace based on risk assessment using exposure information ; 2 ) the provision of health care services and compensation for workers with adverse health effects .
recently , psychosocial factors such as work - related stressors have been included as hazard factors in the workplace , and health promotion activities have become an important component of worker health management before symptoms and signs of occupational diseases manifest . in this article , we reviewed the various kinds of surveillance systems for occupational diseases in korea and resultant data , and suggest reformation of the workers ' health management system .
the workers ' compensation insurance system is one of the old systems and ensures a worker 's life and his / her family is secure after work - related accident .
workers ' compensation insurance has been continuously developing since 1960 ; in particular , its coverage has expanded from large - sized companies to small businesses .
now it is mandatory for all employees to be covered by workers ' compensation insurance . during the early period of workers ' compensation insurance ,
only traditional occupational diseases requiring the recuperation of 11 or more days were compensated , but now work - related diseases such as musculoskeletal disorder , cerebrovascular attack and cardiovascular attack requiring the recuperation of 4 or more days , may also be compensated .
industrial accident is defined in the industrial accident compensation insurance act , and occupational diseases are included as industrial accidents .
occupational injuries and illnesses can be covered by workers ' compensation insurance as social insurance managed by the korea workers ' compensation and welfare service ( comwel ) , supervised and funded by the ministry of employment and labor ( moel ) korea government .
work relatedness refers to work or the working environment causing occupational injuries and illnesses or occupational injuries and illness happening during work .
data on occupational diseases compensated as industrial accidents are collected and analyzed by the korea occupational safety and health agency ( kosha ) . the osh act
the proportion of reported cases is very small ( under 1% of total occupational diseases ) .
kosha collects compensated cases from comwel and reported cases from the moel , and produces official statistics on all occupational diseases . until now
, some workers have been excluded from workers ' compensation insurance :
self - employed , unpaid family workerscivil servants , teachers , soldiersagriculture , hunting , fishery , and forestry workplaces with less than 5 workers
self - employed , unpaid family workers civil servants , teachers , soldiers agriculture , hunting , fishery , and forestry workplaces with less than 5 workers civil servants , as government employees , teachers and soldiers are covered by other insurance systems , while there is no compensation system for self - employed and unpaid family workers and workers of small companies in agriculture , hunting , fishery and forestry .
since 2000 , the number of occupational diseases has increased continuously from 4,051 in 2000 to 11,472 in 2007 . however , after 2007 occupational disease cases decreased . in particular , occupational diseases accounted for 8,721 cases in 2009 , representing a decrease of 1,013 persons ( 10.4% ) compared to the previous year 's 9,734 persons in 2008 ( 1 ) ( fig .
the number of non - fatal work - related diseases stood at 6,626 in 2009 , representing a decrease of 944 ( 12.5% ) compared to the previous year 's 7,570 persons .
the increasing trend in the number of pneumoconiosis cases reversed to a decreasing trend in 2005 .
non - fatal work - related diseases increased continuously until 2007 , but recently statistics have shown decreasing trend .
specifically , brain and heart disease frequency have decreased since 2004 ( 1 ) ( table 1 ) .
the number of fatal occupational diseases increased until 2003 , but has since shifted to a decreasing trend .
there is also a steadily increasing trend in fatal pneumoconiosis , while brain and heart disease cases have decreased .
yearly trends in size of occupational diseases as industrial accidents in korea may be reflected by changes in institutions of industrial accident compensation .
in particular , brain and heart disease and musculoskeletal disorders have increased since the 1990s and 2000s , respectively .
recently , these work - related diseases were the most of compensated cases except for coal worker 's pneumoconiosis ( table 1 , 2 ) .
another method of investigation of the magnitude and profile of occupational diseases is the national survey method ; however , this method is less developed . since 1998 , there were three kinds of nationwide surveys : first , is the national workers ' health interview survey ; second , is the national survey on workplace safety and health management ; and third , is the working conditions survey ( wcs ) .
the first one was done only one time in 1998 for workers in manufacturing sectors .
the second one was done a total of 5 times between 2000 and 2008 , and the sixth survey was done in 2009 .
the third one was conducted in 2006 and focused on psychosocial factors of workers ' health in the workplace .
the working conditions survey is using the same questionnaire as the fifth survey of the european founplace wcs in order to compare korea and the eu .
every survey has included some questions about worker 's experiences of occupational disease . according to the wcs
, the magnitude of occupational diseases experienced as symptoms and signs is detailed hereafter : among the various symptoms and signs , the highest prevalence rate is that of musculoskeletal disorders ( 18.1% ) ; the second most frequent is that of stress ( 17.9% ) ; back pain is the third ( 16.8% ) ; and overall fatigue is the fourth ( 16.7% ) .
the prevalence rate of stress is the highest one among male workers , but musculoskeletal disorders are the highest among female workers ( 2 ) ( table 3 ) .
workers ' compensation insurance has been continuously developing since 1960 ; in particular , its coverage has expanded from large - sized companies to small businesses .
now it is mandatory for all employees to be covered by workers ' compensation insurance . during the early period of workers ' compensation insurance ,
only traditional occupational diseases requiring the recuperation of 11 or more days were compensated , but now work - related diseases such as musculoskeletal disorder , cerebrovascular attack and cardiovascular attack requiring the recuperation of 4 or more days , may also be compensated .
industrial accident is defined in the industrial accident compensation insurance act , and occupational diseases are included as industrial accidents .
occupational injuries and illnesses can be covered by workers ' compensation insurance as social insurance managed by the korea workers ' compensation and welfare service ( comwel ) , supervised and funded by the ministry of employment and labor ( moel ) korea government .
work relatedness refers to work or the working environment causing occupational injuries and illnesses or occupational injuries and illness happening during work .
data on occupational diseases compensated as industrial accidents are collected and analyzed by the korea occupational safety and health agency ( kosha ) . the osh act
the proportion of reported cases is very small ( under 1% of total occupational diseases ) .
kosha collects compensated cases from comwel and reported cases from the moel , and produces official statistics on all occupational diseases .
until now , some workers have been excluded from workers ' compensation insurance :
self - employed , unpaid family workerscivil servants , teachers , soldiersagriculture , hunting , fishery , and forestry workplaces with less than 5 workers
self - employed , unpaid family workers civil servants , teachers , soldiers agriculture , hunting , fishery , and forestry workplaces with less than 5 workers civil servants , as government employees , teachers and soldiers are covered by other insurance systems , while there is no compensation system for self - employed and unpaid family workers and workers of small companies in agriculture , hunting , fishery and forestry .
since 2000 , the number of occupational diseases has increased continuously from 4,051 in 2000 to 11,472 in 2007 . however , after 2007 occupational disease cases decreased . in particular ,
occupational diseases accounted for 8,721 cases in 2009 , representing a decrease of 1,013 persons ( 10.4% ) compared to the previous year 's 9,734 persons in 2008 ( 1 ) ( fig .
the number of non - fatal work - related diseases stood at 6,626 in 2009 , representing a decrease of 944 ( 12.5% ) compared to the previous year 's 7,570 persons .
the increasing trend in the number of pneumoconiosis cases reversed to a decreasing trend in 2005 .
non - fatal work - related diseases increased continuously until 2007 , but recently statistics have shown decreasing trend . specifically , brain and heart disease frequency have decreased since 2004 ( 1 ) ( table 1 ) .
the number of fatal occupational diseases increased until 2003 , but has since shifted to a decreasing trend .
there is also a steadily increasing trend in fatal pneumoconiosis , while brain and heart disease cases have decreased .
yearly trends in size of occupational diseases as industrial accidents in korea may be reflected by changes in institutions of industrial accident compensation . in particular , brain and heart disease and musculoskeletal disorders
recently , these work - related diseases were the most of compensated cases except for coal worker 's pneumoconiosis ( table 1 , 2 ) .
another method of investigation of the magnitude and profile of occupational diseases is the national survey method ; however , this method is less developed . since 1998 , there were three kinds of nationwide surveys : first , is the national workers ' health interview survey ; second , is the national survey on workplace safety and health management ; and third , is the working conditions survey ( wcs ) .
the first one was done only one time in 1998 for workers in manufacturing sectors .
the second one was done a total of 5 times between 2000 and 2008 , and the sixth survey was done in 2009 .
the third one was conducted in 2006 and focused on psychosocial factors of workers ' health in the workplace .
the working conditions survey is using the same questionnaire as the fifth survey of the european founplace wcs in order to compare korea and the eu .
according to the wcs , the magnitude of occupational diseases experienced as symptoms and signs is detailed hereafter : among the various symptoms and signs , the highest prevalence rate is that of musculoskeletal disorders ( 18.1% ) ; the second most frequent is that of stress ( 17.9% ) ; back pain is the third ( 16.8% ) ; and overall fatigue is the fourth ( 16.7% ) .
the prevalence rate of stress is the highest one among male workers , but musculoskeletal disorders are the highest among female workers ( 2 ) ( table 3 ) .
these institutions are examine workers by the request of employers based on the osh act mandatorily .
for example , health examinations are regulated by government in order to protect worker health through early detection of occupational diseases .
health examinations for worker health protection are classified into two different types : special health examination and general health examination .
the special health examinations are comprised of health screening tests for early detection of selective occupational diseases in workers exposed to specific hazard factors such as noise , dust , organic solvent , etc . the general health examination is a mass screening test for finding health problems including occupational diseases of workers without exposure to hazard factors .
the health examination is focused on early detection of occupational diseases , and its results are used as information for workplace management in order to protect worker health .
cases diagnosed as ' suspicious cases of occupational disease ' are recommended to be transferred to non - hazardous workplaces . some of them may be compensated based on diagnosis of occupational diseases through the workers ' compensation insurance system
. detection rate of ' suspicious cases of occupational diseases ' in the special health examination was 0.46% in 2008 , an increase from 2006 .
the detection rate of ' cases observed for occupational disease ' was 12.85% in 2008 with an increasing trend .
the health examination is defined as a screening test for occupational diseases , so there is no confirmatory diagnosis in the health examination process . '
cases to be observed for occupational diseases ' are the result of an examination showing marginal figures for ' suspicious cases for occupational diseases ' . based on results from the health examination ,
suspicious cases will be recommended to undergo medical treatment , change jobs or tasks for reduction of exposure to hazard workplace , etc .
cases to be observed are not in need of any immediate intervention ; however , these cases may be also recommended similar treatment , such as wearing personal protective equipment ( ppe ) , changing jobs or tasks , etc . among suspicious cases and cases
to be observed detected by the special health examination , hearing loss due to noise was the most prevalent ( 3 ) ( table 4 ) .
detection rate of ' suspicious cases of occupational diseases ' during the general health examination was 0.001% in 2008 , and has been very steady since 2004 . in contrast , the detection rate of ' suspicious cases of general diseases ' has shown fluctuations between 4 and 5% in the last ten years ( 3 ) ( table 5 ) .
health examinations are very effective in finding asymptomatic chronic diseases , such as pneumoconiosis , hearing loss and some poisonings .
however , it can barely detect symptomatic diseases with no signs , like asthma , and easily curable diseases like dermatitis because of the characteristics of the periodic screening test .
therefore , statistics from the health examination include mostly pneumoconiosis and noise - induced hearing loss ( 4 ) ( table 4 ) .
the working environment measurement institution , regulated by the osh act , is in charge of assessing and , ultimately , reducing the level of worker exposure to hazard factors in the workplace .
every employer should do working environment measurement in order to identify hazard factors in the workplace and the level of worker exposure to these factors .
the special health examination is based on working environment measurement , such as selection of the examinee .
if the level of worker exposure is higher than the occupational exposure level in korea , the employer should immediately act to reduce it .
the magnitude of occupational diseases and associated fatality rate in korea have gradually increased since 2007 .
the trend in fatality rate can be reflected by the trend in fatal pneumoconiosis and brain and heart disease , as these two categories of occupational diseases predominate total occupational diseases .
most pneumoconiosis cases were coal workers ' pneumoconiosis ; however , nowadays most coal mines have closed due to restructuring of the energy production system in korea .
other dominant fatal occupational diseases , such as brain and heart diseases , will also decrease because general health programs have focused on prevention of hypertension , one of the most important causal factors of brain and heart diseases in workers . in the general population , the morbidity rate of brain and heart diseases has shown a recent downward trend .
one is the causal factors of occupational diseases for prevention , and the other is affected workers of occupational diseases in order to rehabilitate them through recovery of the labor force .
occupational diseases are induced by worker exposure to various hazard factors in the workplace , such as physical , chemical , biological and psychosocial factors .
workers with abnormal conditions can detect acute occupational diseases through self - diagnosis just after exposure to high levels of hazard factors , such as lead , mercury , etc .
however , it is not so easy to identify abnormal conditions of chronic occupational diseases , such as asbestos - induced diseases .
furthermore , exposure to low levels of hazard factors may have long - term cumulative effects on worker health ( fig .
if there is no evidence of work - relatedness , no ill - health can be confirmed as an occupational disease .
work - relatedness may be classified into two different types : direct and indirect causality .
first , based on direct causality between work or work environment and ill - health , it is easy to determine work - relatedness , but it is very difficult to evaluate indirect work - relatedness .
the same condition diagnosed as occupational disease by a physician can also be categorized as non - work - related because a particular hazard factor can not be detected in the working history .
final determination of work - related illness or death in any individual case requires detailed occupational and non - occupational information ( 5 ) . in the united states
, there exists occupational health surveillance , such as death certificates , hospital discharge data , physician reports , laboratory reports , workers ' compensation reports , national surveys , employer surveillance programs and occupational health clinics ( 5 ) .
since 1998 , there were some suggestions for the introduction of a surveillance system for occupational diseases in korea because worker health examinations and work environment measurements had limitations for management of occupational diseases ( 6 , 7 ) .
the magnitude of occupational diseases in korea is assessed mainly by the workers ' compensation system .
statistics might exclude unclaimed occupational diseases and those of workers from the public sector and workers who are not covered by compensation insurance ( 4 ) .
this means that non - compensated cases can be neglected and latent cases can always emerge as compensated cases in the immediate future .
moreover , statistics based on compensated cases of occupational diseases are not stable because different acceptance criteria for work - relatedness have been applied by the korea labor welfare corporation ( former name of comwel ) and judiciary branch ( 8) . in korea , there are two different kinds of health insurance : national health insurance and workers ' compensation insurance .
every worker with occupational ill - health can be covered by either of these two .
if some occupational diseases cases are covered by the national health insurance system , official statistics on occupational diseases can not include these cases . in order to determine the correct number , all occupational diseases covered by the two different insurances
unfortunately , the national health insurance system has no screening and confirming system for occupational diseases .
occupational disease compensation will focus on the treatment , compensation , and rehabilitation as secondary or tertiary prevention .
national surveys of worker health can provide useful information about ill - health workers without treatment or compensation .
this information can be used to prevent occupational diseases by detection of at - risk workers as a target population for management of occupational diseases .
the primary prevention of occupational diseases and injuries depends more directly on hazard surveillance , since the identification of hazards enables their reduction prior to the onset of ill - health .
occupational health surveillance is less well developed than its health counterpart due to scarcity of data sources and , with the exception of the occupational safety and health administration and the national institute for occupational safety and health surveys in the usa , the proprietary and inaccessible nature of hazard data collected by employers ( 5 ) .
occupational health surveillance entails the systematic monitoring of health events and exposures in working populations in order to prevent and control occupational hazards and their associated diseases and injuries ( 5 ) .
medical surveillance refers to the ongoing application of medical tests and procedures to individual workers who may be at risk for occupational morbidity to determine whether an occupational disorder may be present ( 5 ) .
if an individual or population is exposed to a toxin with known effects , and the tests and procedures are highly targeted to detect the likely presence of one or more effects in these persons , then this surveillance activity is more aptly described as medical screening ( 9 ) . in occupational health , there are two kinds of surveillance activities , one is public health and medical surveillance and the other is hazard surveillance .
the main purpose of occupational health surveillance is to identify the incidence and prevalence of known occupational diseases and injuries ( 5 ) .
a second broad function is to identify individual cases of occupational disease and injury in order to find and evaluate other individuals from the same workplaces who may be at risk for similar disease and injury .
another purpose of case identification may be to assure that the affected individual receives appropriate clinical follow - up , an important consideration in view of the scarcity of clinical occupational medicine specialists ( 10 , 11 ) .
finally , occupational health surveillance is an important means of discovering new associations between occupational agents and accompanying diseases . discovery of rare diseases , patterns of common diseases , or suspicious exposure - disease associations through surveillance activities in the workplace can provide vital leads for a more conclusive scientific evaluation of the problem and possible verification of new occupational diseases ( 5 ) .
periodic medical screening and surveillance examinations are another frequent service provided by occupational medicine physicians .
the term screening focuses on the individual ; screening tests are tools for secondary prevention with the goal of early diagnosis and treatment of disease in exposed workers .
true ' surveillance ' aggregates information about individuals in order to examine patterns within a population . properly applied ,
surveillance is a tool for primary prevention through the identification and elimination of the cause of disease ( 12 ) .
screening examinations may be target - organ specific or substance specific , and thus tend to be more focused than preplacement evaluations ( 12 - 14 ) .
based on considerable information , occupational disease management can be accomplished in order to protect and promote worker health .
many kinds of information are required to suggest the etiology of poor worker health , such as hazard , exposure level and related information in workplace .
the present system of surveillance for management of occupational diseases mainly depends upon the workers ' compensation and health examination systems .
these two systems can not cover all kinds of occupational diseases . in order to focus on primary prevention of occupational diseases , various data sources on hazards and exposure levels in workplaces
especially , prevention of occupational diseases may be focused on the reduction of worker exposure to the occupational hazards .
the workers ' compensation insurance system reviews each claim case for evaluating its work - relatedness .
this means that hazard factors and worker exposure to those hazard factors may be assessed .
these review data can be some of the most important information for prevention of ill - health by surveillance .
most of the current surveillance system in korea depends upon the osh act . from the viewpoint of surveillance , the occupational management system in korea is well designed except for the national survey system . in the future , national surveys for detection of occupational hazards and ill - health outcomes of workers should be developed .
the existing surveillance system of occupational disease can be improved by providing more refined information through statistical analysis of surveillance data .
recently , data mining has been used in order to estimate the national burden of occupational disease with other international statistics . in korea
, this technique can be also introduced for development of the management system of occupational diseases .
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the management system of occupational diseases in korea can be assessed from the perspective of a surveillance system .
workers ' compensation insurance reports are used to produce official statistics on occupational diseases in korea .
national working conditions surveys are used to monitor the magnitude of work - related symptoms and signs in the labor force .
a health examination program was introduced to detect occupational diseases through both selective and mass screening programs .
the working environment measurement institution assesses workers ' exposure to hazards in the workplace .
government regulates that the employer should do health examinations and working conditions measurement through contracted private agencies and following the occupational safety and health act .
it is hoped that these institutions may be able to effectively detect and monitor occupational diseases and hazards in the workplace . in view of this , the occupational management system in korea is well designed , except for the national survey system . in the future , national surveys for detection of hazards and ill - health outcomes in workers should be developed .
the existing surveillance system for occupational disease can be improved by providing more refined information through statistical analysis of surveillance data .
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INTRODUCTION
OCCUPATIONAL DISEASES SURVEILLANCE ACTIVITIES IN KOREA
Workers' compensation insurance report
National survey report
HEALTH EXAMINATION SYSTEM
MONITORING SYSTEM FOR WORKER HAZARD EXPOSURE
DISCUSSION
CONCLUSION
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their marbling has been increased over many decades to meet domestic consumer preferences . in both countries ,
highly marbled beef is greatly prized for traditional meat cooking methods such as sukiyaki for japanese and gogigui for korean . because of these demands , the use of heifers and steers instead of bulls , intensive feeding system , and genetic ability of wagyu and hanwoo cattle have resulted in greater fat deposition in these breeds compared to european breeds . as intramuscular fat ( imf ) improves beef quality at least in juiciness and flavor ( hornsterin and wasserman , 1987 ; wheeler et al . , 1994 )
it is assessed in abattoirs by meat graders in various countries , including usa , australia , japan , and korea . like other kinds of foods , meat has three functions : 1 ) it provides nutrition ; 2 ) it provides deliciousness ; and 3 ) it prevents disease .
although beef has these three functions , the main food in both japan and korea is boiled rice while beef is a side dish .
therefore , these two countries have developed the quality of beef rather than its quantity .
this is quite different from foreign countries where meat is consumed as a main dish . in japanese and korean cuisine , soft and delicious beef with imf and a good red color
in the past , fat was not given a good image in its role towards human health , although fat is an important energy resource for human .
recently , fat has been reported to have fewer adverse effects on health than carbohydrates , especially simple carbohydrates .
in fact , meat has played a crucial role in human evolution of a healthy and well balanced diet ( pereira and vicente , 2013 ) .
furthermore , meat plays a pivotal role in nutritious diets . high quality marbled beef not only has excellent eating quality , but also contains a lot of beneficial fatty acids ( troy et al . , 2016 ) . in this regard ,
the current paper reviews the characteristics and health benefit of highly marbled beef from wagyu and hanwoo cattle .
it has well known that wagyu cattle have high potential of accumulating imf and producing highly marbled beef .
hanwoo cattle are also known for their high imf for marbled beef similar to wagyu beef .
highly marbled wagyu loin contains more than 40% of imf , sometimes more than 60% ( horii et al . , 2009 ) , while quality grade 1 hanwoo , the highest quality grade , has approximately 28% of imf in longissimus thoracis muscle ( hwang and joo , 2016 ) .
wagyu cattle include four types of japanese cattle : the black , brown , short horn , and polled breeds .
numerous studies have investigated the meat quality , quantity , and muscle physiology of crossbreed wagyu ( japanese black cattle ) in foreign countries ( cafe et al . , 2006 ; cafe et al . , 2009 ; greenwood et al . , 2006 ; greenwood et al . , 2009 ; may et al . , 1993 ) .
they are identified by different coat color : brown ( major hanwoo ) , black face ( heukwoo ) , black ( jeju heukwoo ) , and tiger color ( chickso ) ( jo et al . , 2012 ) .
in this review , wagyu and hanwoo are used to describe the japanese black breed and the brown coat color hanwoo , respectively .
all four types of wagyu cattle have played important roles locally and in the history of mixed farming .
they also played important roles in the synergies between cattle and crops , especially rice .
farmers gradually began to replace the role of cattle as draft animals and started to use industrial fertilizers approximately 50 years ago . in recent years , japanese wagyu cattle have been raised more specifically for beef production .
the famous brand name wagyu not only includes the japanese black cattle produced in japan , but also includes animals or even cross - bred japanese black cattle produced in foreign countries such as australia and the united states .
similarly , the utilization of hanwoo cattle as an edible meat had been minimal for long time .
full - scale production of hanwoo as meat - type cattle has started since the 1970s . because hanwoo cattle have maintained stable traits through pure breeding , the current blood lineage is very valuable .
it is mainly spread in the korean peninsula ( kim and lee , 2000 ) .
recently , hanwoo beef has been reported to have highly marbled imf similar to wagyu beef .
especially , hanwoo beef has relatively thin muscle fiber and minimal content of connective tissues ( kim et al . , 1994 ) .
it has less subcutaneous fat depth with greater ossification scores and marbling scores than those of australian angus ( cho et al . , 2005 ) .
in 2013 , a total of 2.64 million heads of cattle were fed for beef production in japan .
approximately 1.71 million heads were japanese black cattle ( maff , 2013 ) , and approximately 873,400 were holstein cattle .
, the number of farmers producing beef was 613,000 , but 86.5% of these farmers fed less than 50 heads of cattle .
the mean body weight and carcass weight of beef at slaughter ( 26 - 30 mon of age ) were 725 kg and 470 kg , respectively .
high performance marbled beef production has caused japanese black cattle to comprise the greatest share of japan s wagyu cattle population ( albrecht et al . , 2011 ;
recently , the imf percentage of beef from japanese black cattle has an average value of greater than 30% ( albrecht et al . , 2011 ; horii et al . ,
the total number of slaughter cattle was 1,007,000 , including 883,593 hanwoo cattle , 66,485 holstein cows , and 56,923 holstein heifers and bulls ( kape , 2015 ) .
the number of cattle farming households was 99,858 , including 89,403 hanwoo farmers ( kape , 2015 ) . during the last decade ,
the number of households raising hanwoo cattle has drastically decreased from 186,000 households in 2006 to 89,403 households in 2015 ( kape , 2016 ) .
the average live and carcass weights of hanwoo cattle at slaughter ( 26 - 30 mon of age ) were 719 kg and 430 kg , respectively ( kape , 2015 ) .
wagyu carcasses are evaluated by accredited graders from the japan meat grading association ( jmga ) in accordance with beef carcass grading standards .
first established in 1988 , the present grading system assigns both yield grade ( a , b , and c ) and meat quality grade ( 1 , 2 , 3 , 4 , and 5 ) ( jmga , 2014 ) .
in korea , all cattle carcasses should be evaluated by korean carcass grading system . established in 1992 ,
the korean carcass grading system presently has three levels of yield grade ( yg ) for meat amount ( a , b , and c ) and five levels of meat quality grade ( qg ) ( 1 , 1 , 1 , 2 , and 3 ) ( kape , 2016 ) . for beef quality grading in japan ,
all cattle carcasses are graded at the 6 to 7 rib section at least one hour after ribbing .
the following four items are independently evaluated : beef marbling ; meat color and brightness ; meat firmness and texture ; and fat color , luster , and quality . meat quality grade of the carcass is then assigned according to the lowest grade of these four items .
korean beef quality grading is also estimated based on several factors , including marbling score , meat color , fat color , firmness and texture of lean meat , and maturity of the exposed loin muscle at the 13 rib interface .
this means that marbling score is the most dominating determinant in korea because korean consumers have an extraordinary preference for high marbled meats . in 1988 , wagyu marbling levels were assigned by the beef marbling standard ( bms ) using a plastic model made from silicone resin .
this standard was calculated based on the circumference and area percentage of marbling particles in the rib eye section ( longissimus thoracis ) . in october 2008 , a new marbling standard using carcass photographs replaced the 1988 standard . in march 2014 , an even newer marbling standard
1 ) . graders now determine the bms number ( 1 to 12 ) by comparing the actual carcass marbling to the standard photograph of marbling . during this process
, any larger inclusions of fat at the periphery of the rib eye are not considered as marbling according to the japanese grading system .
the bms of korean carcass grading system has been changed by the addition of marbling number . in 1992 , when the carcass grading system was established for the first time in korea , the bms had only 5 numbers ( 1 to 5 ) with 3 qg ( 1 , 2 , and 3 ) . however , in 1997 , new qg 1 was added with new bms no . 6 and no .
furthermore , in 2004 , another new qg 1 was added with new bms no . 8 and no .
hanwoo beef with qg 1 or 1 is considered as a premium class of beef in korea .
of hanwoo cattle slaughtered in 2015 , 10.0% were qg 1 , 26.4% were qg 1 , and 31.4% were qg 1 ( kape , 2016 ) .
the plentiful marbling of wagyu and hanwoo beef has attracted attention . in both japan and korea ,
the value of cattle carcasses is determined by a qg which considers marbling as a decisive determinant . since the liberalization of beef importation
, marbling has been greatly emphasized to differentiate domestic beef from imported beef ( hirooka , 2014 ; hwang et al . , 2010 ) .
the high content of imf can improve the texture and juiciness of hanwoo beef and thereby its acceptability ( jung et al . , 2016 ) .
korean consumers prefer qg 1 or 1 beef because of its high imf content ( kim et al . , 1999 ) .
2015 ) have demonstrated that an increase in crude fat content ( range 23.8 - 48.6% ) can increase the tenderness , juiciness , and fattiness .
however , they also reported that an increase in crude fat content can reduce the crude protein content and slightly reduce the content of umami components such as nucleic acid and glutamic acid .
it is well known that imf content varies depending on feeding time , finishing diet , and breed type . to produce high qg beef ,
great attention has been paid to more accumulation of imf in wagyu and hanwoo muscle .
one of good strategy to increase imf content in beef muscle is to extend slaughtering age . although the marbling score is increased and reached a plateau at about 24 mon of age ( choi et al . ,
2002 ) , the slaughtering age of hanwoo has been extended to increase the bms score ( jo et al . , 2012 ) .
in korea , the marketing age of hanwoo has been extended to an average of 31 mon with weight of 719 kg to fatten the cattle ( kape , 2015 ) .
however , average daily gain is decreased due to increased slaughtering weight ( paek et al . , 1993 ) .
recently , cattle in china are fed for unusually long periods of time before slaughter as wagyu and hanwoo .
this might have contributed to their high imf and oleic acid contents ( smith , 2016 ; tanaka , 1985 ) .
it is clear that imf increases with feeding time for grain - fed and pasture - fed cattle .
however , the rate of imf increase in grain - fed cattle is faster than that in pasture - fed cattle ( smith et al . , 2009 ) .
it has been reported that wagyu fed on a high - concentration diet have higher expression of adipogenic transcription factors in the subcutaneous and intramuscular adipocytes than those fed on a high - rough - age diet ( yamada and nakanishi , 2012 ) .
the imf content and the numbers of preadipocytes and adipocytes are reported to be higher in wagyu than those in angus ( duarte et al . , 2013 ) .
( 2009 ) have reported that the imf contents in the longissimus muscle of wagyu , german angus , belgain blue , and holstein friesian are 23.3% , 4.4% .
the wagyu and european cattle breeds did not differ in their mechanisms of postnatal fat accretion .
however , they differed in their efficiency of accretion of imf ( gotoh et al . , 2009 ) .
for every 1% increase of imf in the longissimus muscle , the increase amounts of subcutaneous adipose tissue in wagyu , holstein friesian , german angus , and belgain blue were 3.0 , 4.3 , 7.9 , and 10.7 kg , respectively ( gotoh et al . , 2009 ) .
although imf content is the most dominating determinant of beef quality , the imf content is not the only parameter that decides the quality grade of beef carcass .
marbling is called shimo - furi in japanese and sang - gang in korean .
it literally means frosting . in japan , marbling with a fine appearance resembling frost is highly valued , but coarse marbling is not ( motoyama et al . ,
recently , korea also began to discriminate between fine and coarse marbling in hanwoo beef .
this marbling quality contributes to the tenderness of beef because imf deposits are found mainly between muscle fiber bundles , resulting in the disorganization of perimysium connective tissue ( nishimura , 2015 ; sasaki et al . , 2012 ) .
therefore , the sensory of tenderness could be qualitatively affected by histological difference in marbling due to difference in tissue disorganization extent .
there are several types of fatty acids : 1 ) monounsaturated fatty acids ( mufa ) , 2 ) polyunsaturated fatty acids ( pufa ) , and 3 ) saturated fatty acid ( sfa ) .
pufa such as linoleic acid , -linolenic acid ( n-3 ) , -linolenic acid ( n-6 ) , arachidonic acid , and so on contain many important compounds such as essential fatty acids .
the fatty acid that has the highest amount in beef is oleic acid ( c18:1n-9 ) .
it has been reported that fatty acid compositions are different depending on breeds ( smith et al . , 2006
the fatty acid compositions in highly marbled wagyu and hanwoo are considerably different from those in other cattle breeds .
highly marbled wagyu beef has a higher percentage of mufa within fat compared with other breeds ( yang et al . , 1999a ) .
( 2006 ) have investigated oleic acid concentrations in the subcutaneous adipose tissues of wagyu , hanwoo , australian crossbred , angus ( corn - fed ) , angus ( hay - fed ) , and angus ( weaned ) and found that they are 52.9% , 47.3% , 39.8% , 39.8% , 34.6% , and 32.9% , respectively .
a higher percentage of mufa will lead to a lower fat - melting point which contributes to the softness of beef fat and favorable beef flavor .
it may decrease the circulating concentration of ldl cholesterol in consumers ( melton et al .
, 1982 ; rudel et al . , 1995 ; smith , 1994 ) . therefore , fatty acid compositions of beef have recently become important in the beef industry , especially in highly marbled wagyu and hanwoo cattle
( 1995 ) have investigated the effect of breed type ( including japanese black ) and sex on fatty acid compositions of subcutaneous and intramuscular lipids in finishing steers and heifers of pure japanese black and holstein as well as crossbred japanese black , holstein , japanese brown , and charolais .
they have reported that the japanese black is genetically predisposed to producing carcass lipids containing higher concentrations of mufa than holstein , japanese brown , or charolais steers ( zembayashi et al . , 1995 ) .
1992 ) have also concluded that beef from purebred wagyu cattle raised in japan is rich in mufa .
( 2011 ) have compared intramuscular fatty acid composition of longissimus muscle in 26-month - old japanese black steers and holstein steers reared and fattened using a standard fattening system ( table 1 ) . in the longissimus muscle of japanese black steers ,
a higher percentage of unsaturated fatty acid was found than that in holstein steers ( gotoh et al . , 2014 )
2011 ) have also compared the imf content and fatty acid compositions of 21 major skeletal muscles using the same animals .
muscles from the japanese black cattle contained a greater proportion of numerous fatty acids , particularly mufa such as c16:1 , c18:1 , and c20:1 compared to fatty acids in holstein cattle .
in japanese black cattle , the proportion of sfa including c18:0 was much lower compared to that in holstein cattle .
sfa : saturated fatty acids , mufa : monosaturated fatty acids , pufa : polysaturated fatty acids .
2005 ) have investigated the fatty acid compositions of hanwoo and australian angus beef and found a significant difference in fatty acid compositions between these two cattle breeds ( table 2 ) .
especially , angus beef had significantly higher n-3 pufa while hanwoo beef contained greater n-6 pufa in three different muscles ( cho et al . , 2005 ) .
the difference in fatty acid composition might be attributed to the influence of different diets , forage , and grain feeding , although fatty acid profile in ruminants is not a direct reflection of the dietary fatty acid composition due to hydrogenation by rumen microorganism ( enser et al . , 1998 ) .
sfa : saturated fatty acids , usfa : unsaturated fatty acids , mufa : monosaturated fatty acids , pufa : polysaturated fatty acids .
f - ratio statistic : * if p<0.05 , * * if p<0.01 , * * * if p<0.001 .
therefore , it can be easily anticipated that hanwoo beef has a fatty acid profile similar to that of high concentratefed animals ( jo et al . , 2012 ) .
recently , hwang and joo ( 2016 ) have evaluated the fatty acid profile of ten muscles from high marbled ( qg 1 ) and low marbled ( qg 2 ) hanwoo carcass and found significant differences in fat content and fatty acid composition among 10 muscles and between high and low marbled hanwoo beef . in particular , high marbled hanwoo muscles had significantly higher proportion of mufa due to higher oleic acid ( c18:1 ) proportion , while low marbled hanwoo muscles had higher proportion of sfa due to higher proportion of stearic acid ( c18:0 ) ( hwang and joo , 2016 ) .
stearoyl - coa desaturase ( scd ) was first identified and reported as one of the genes associated with beef fatty acid composition ( taniguchi et al . ,
the composition of fatty acids stored in fat depots reflects the earlier action of scd on substrates such as stearic acid and palmitic acid ( kim and ntambi , 1999 ) .
1999b ) have reported interesting correlations between scd enzyme activity and fatty acid composition in bovine adipose tissue .
although the adipogenic mechanism is extremely complicated , several genes have been identified and confirmed as either associated with or responsible for the fatty acid composition in wagyu cattle ( gotoh et al . , 2014 ) .
it is generally accepted that the concentration of oleic acid in beef adipose tissue is dependent on scd expression and activity .
wagyu cattle are genetically disposed to produce more oleic acid ( smith et al . , 2006 ) .
very high heritability has been reported for oleic acid in wagyu cattle ( nogi et al . , 2011 ) .
higher levels of concentrated feed in the later fattening period can lead to higher mufa concentration in the subcutaneous adipose tissues of wagyu steer ( kimura et al . ,
interest in beef fat and fatty acids has been increasing , especially in highly marbled beef such as wagyu and hanwoo because fatty acids composition in the diet have impact on human health .
consumption of fat and cholesterol has been reported to be linked to cardiovascular disease , obesity , and cancer ( micha et al . , 2010 ;
consequently , reduction of total fatty acid intake and replacement of sfa with pufa have been recommended .
ulbricht and southgate ( 1991 ) have demonstrated that stearic acid has no effect on plasma cholesterol level and that oleic acid can lower serum cholesterol similar to pufa .
furthermore , pavan and duckett ( 2013 ) have suggested that a higher proportion of oleic acid in beef is desirable because the consumption of high - oleic acid ground beef can increase hdl - cholesterol concentration ( gilmore et al . , 2011 ) . according to smith ( 2016 ) ,
the amount of fat consumed in a typical portion of beef will not increase risk factors for cardiovascular disease .
clinical trials have demonstrated that ground beef containing elevated oleic acid can increase the concentration of hdl - cholesterol or at least has no negative effect on the concentration of hdl - cholesterol . in earlier research on oleic acid , the major mufa in beef , grundy et al .
( 1988 ) have found that it can lower ldl - cholesterol without affecting beneficial hdl - cholesterol .
( 2014 ) have reported that mufa can normalize or improve lipid metabolism and maintain the balance in cardiac muscle .
these have implied that mufa have little effect on total cholesterol and that they are heart - healthy dietary fat that can lower ldl - cholesterol and increase hdl - cholesterol ( lahey et al . , 2014 ) .
this effect is repeatable when natural foods are used to supplement diets with oleic acid . in this
regard , smith ( 2016 ) have concluded that beef cattle should be raised under production conditions to increase the concentration of oleic acid in their edible tissue , i.e. , by grain feeding over extended periods of time .
it is obvious that consumer in the world has an overwhelmingly negative attitude toward animal fats , especially saturated fat in meat for the last several decades ( ngapo and dransfield , 2006 ; williams and droulez , 2010 ) .
according to higgs ( 2000 ) , the per capita decline in beef consumption in the us and other western countries has been attributed in large part to animal fat phobia .
consumers have been warned to reduce saturated fat in their diet and to avoid meat cuts containing high fat content .
these health recommendations are obviously in conflict with the health of highly marbled wagyu and hanwoo beef .
many research studies have shown that the imf of wagyu and hanwoo beef contains a lot of mufa that could prevent arteriosclerosis .
researches have also demonstrated that high - oleic acid ground beef may reduce risk factors for cardiovascular disease ( adams et al .
thus , although some consumers in japan and korea consider highly marbled wagyu and hanwoo beef as being unhealthy , there is no scientific evidence to indicate that beef that is high in oleic acid will increase risk factors for diseases ( smith , 2016 ) . consequently , the role of animal fats in the diet should be re - evaluated because scientists around the globe increasingly doubt the validity of the so called diet - heart hypothesis
it is now generally accepted that diets with low fat , high carbohydrate failed to curb obesity ( drewnowski , 2015 ) . on the other hand ,
more recent functional medicine research studies have suggested that the intake of fat has positive effect on human health ( saito , 2016 ) .
it is essential to consume fats containing good quality fatty acids while reducing the consumption of food high in simple carbohydrates .
excessive intake of simple carbohydrates is detrimental to health because they have negative effects on the body ( yu et al . , 2013 ) .
in this regard , inclusion of high fat foods with superior sensory properties in a balanced diet such as highly marbled wagyu and hanwoo beef is likely to gain wider acceptance as a well - being food in the near future .
in japan and korea , highly marbled wagyu and hanwoo cattle are greatly prized for traditional meat cooking methods .
many researches have shown that wagyu and hanwoo cattle have high potential of accumulating imf and producing highly marbled beef .
the beef quality grading system in both countries is primarily determined by marbling score with bms and additionally adjusted by other carcass traits .
literature suggests that imf content varies on the basis of feeding time , finishing diet , and breed type .
great attention has been paid to more accumulation of imf to produce high quality grade beef .
the rate of imf increase in grain - fed cattle is faster than that in pasture - fed cattle .
highly marbled wagyu and hanwoo beef have higher proportions of mufa due to higher concentrations of oleic acid .
they are heart - healthy dietary fat because they can lower ldl - cholesterol while increasing hdl - cholesterol .
clinical trials have also indicated that highly marbled beef does not increase ldl - cholesterol and that beef high in oleic acid can consistently increase hdl - cholesterol . finally , literatures have concluded that high - oleic acid beef such as wagyu and hanwoo beef may reduce risk factors for cardiovascular diseases .
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this review addresses the characteristics and health benefit of highly marbled wagyu and hanwoo beef .
marbling of wagyu and hanwoo beef has been increased in japan and korea to meet domestic consumer preferences .
wagyu and hanwoo cattle have high potential of accumulating intramuscular fat ( imf ) and producing highly marbled beef .
the imf content varies depending on the feeding of time , finishing diet , and breed type .
imf increases when feeding time is increased .
the rate of imf increase in grain - fed cattle is faster than that in pasture - fed cattle .
fatty acid composition are also different depending on breeds .
highly marbled wagyu and hanwoo beef have higher proportions of monounsaturated fatty acid ( mufa ) due to higher concentrations of oleic acid .
mufas have little effect on total cholesterol .
they are heart - healthy dietary fat because they can lower low - density lipoprotein ( ldl)-cholesterol while increasing high - density lipoprotein ( hdl)-cholesterol .
clinical trials have indicated that highly marbled beef does not increase ldl - cholesterol .
this review also emphasizes that high oleic acid beef such as wagyu and hanwoo beef might be able to reduce risk factors for cardiovascular disease .
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Introduction
Wagyu and Hanwoo Cattle
Carcass Grading of Wagyu and Hanwoo
Marbling of Wagyu and Hanwoo Beef
Fatty Acid Composition of Wagyu and Hanwoo Beef
Health Implications of Highly Marbled Wagyu and Hanwoo Beef
Conclusions
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parental drug misuse and its effects on children and families are policy priorities in the uk and elsewhere ( flavin & paltrow , 2010 ; hm government , 2010 ) .
polydrug use is the norm among drug users in the uk , and benzodiazepine use is common ( bird & robertson , 2011 ; department of health ( england ) , 2007 ; hay , gannon , casey , & mckeganey , 2009 ; jones , mogali , & comer , 2012 ) . increased risks and harm , and a range of adverse effects are associated with long - term benzodiazepine use ( lader , 2011 , 2014 ; obrien , 2005 ) . however , existing research on drug use and parenting has tended to focus on opioids , opioid substitution therapy ( ost ) , or crack cocaine ( radcliffe , 2009 , 2011 ; rhodes et al . , 2010 ) , and in the case of pregnancy , also alcohol , nicotine , marijuana and amphetamines ( behnke & smith , 2013 ) . in this paper ,
we examine accounts provided by polydrug - dependent parents about their use of , and dependence on , benzodiazepines as compared to ost .
benzodiazepine use is highly prevalent among drug users worldwide ( jones et al . , 2012 ) .
opioid - dependent and ost patients use benzodiazepines to prolong and enhance the effects of opiates ( jaffe et al . , 2004 ) , and increased rates of dependence are associated with co - occurring psychosocial problems ( jones et al . , 2012 ) .
weizman , gelkopf , melamed , adelson , and bleich ( 2003 ) estimated a lifetime prevalence of benzodiazepine dependence in the opiate - dependent population ( in or out of treatment ) ranging from 6194% .
benzodiazepines were originally marketed in the 1960s for the relief of anxiety , stress and insomnia ( who , 1996 ) and remain one of the most widely used psychoactive drugs ( lader , 2011 ) .
their use within the general population is common , particularly among females ( obrien , 2005 ) .
the gendered cultural meanings of diazepam ( valium ) , a well - known benzodiazepine , was cemented in the 1966 rolling stones song mother s little helper .
benzodiazepines are recommended for the short - term treatment of anxiety and insomnia ( baldwin et al . , 2013 ) .
tolerance and withdrawal effects can develop after only a few weeks of treatment ( lader , 2011 ) .
adverse effects include sedation , impaired cognitive and psychomotor functioning , and increased likelihood of accidents and injuries ( lader , 2011 ; obrien , 2005 ) .
withdrawal effects vary in severity and duration , and include anxiety , insomnia , depression , impaired memory , muscle spasms and tension , and rarely , seizures and psychosis ( lader , 2011 ) .
combined benzodiazepine and opioid use is associated with increased sedation , decreased psychomotor performance ( lintzeris , mitchell , bond , nestor , & strang , 2007 ; lintzeris & nielsen , 2010 ) , poorer psychosocial adjustment and general health , heightened risk - taking behaviour , and increased risk of overdose ( bird & robertson , 2011 ) . however , evidence is limited ( jones et al . , 2012 ) , and
few studies have focused on the combined effects of opioid and benzodiazepine dependence on pregnancy or parenting ( acmd , 2003 ) , despite evidence to suggest that both drugs are problematic in these contexts . whilst there is evidence that parental opioid use can compromise parenting capacity and pregnancy outcomes ( acmd , 2003 ) , the health and wellbeing of children and families can also be negatively affected by social and structural factors such as poverty , poor housing , unemployment , parental stress , domestic violence and criminal justice involvement ( hepburn , 2004 ; klee , jackson , & lewis , 2002 ) .
however research indicates that despite this complexity , there is a tendency among service users and service providers to focus on drug use alone ( banwell & bammer , 2006 ; chandler et al . , 2013 ) , and in most cases the primary drug of misuse ( winklbaur et al . , 2008 ) .
while this focus may relate to concerns regarding the effects of specific drugs per se on parenting capacity and the immediate safety of children , it could also result in a limited view of drug dependence that neglects to consider polydrug use and the impact of socio - economic and structural inequalities . while the use of opioids and ost is stigmatising , especially in the context of pregnancy and parenthood ( holland , forrester , williams , & copello , 2013 ) , use of benzodiazepines in the context of mothering has historically been characterised in a less problematic ( though not entirely positive ) manner ( metzl , 2003 ) .
we found no published literature which compared benzodiazepine and opioid dependence , from the perspective of parents , or expectant parents .
therefore the aim of our study was to address this gap in the evidence and to undertake a comparative thematic analysis of the ways in which parents accounted for their use of opioids , ost and benzodiazepines during and after pregnancy .
this was a longitudinal , qualitative study involving 19 opioid - dependent service users over the course of approximately one year in order to examine changes over time in participants experiences with healthcare services , drug use and parenting .
the truth , but rather as a resource through which to explore how parenting and drug use are understood , negotiated and mutually constructed ( bury , 2001 ; rhodes , bernays , & houmoller , 2010 ) .
the study was carried out in scotland in an area with a mixed socio - economic profile , containing areas of significant deprivation .
ost services for drug - dependent patients are delivered via primary care and specialist community - based substance misuse services .
there are local guidelines both for the management of substance misuse in pregnancy ( whittaker , 2003 ) , and parental substance use ( elbeg , 2013 ) .
notably , a large number of patients in the area are prescribed benzodiazepines in addition to ost .
participants , recruited via healthcare practitioners , were interviewed at around 2832 weeks gestation ; 23 months postnatal ; and 69 months postnatal .
inclusion criteria were opioid - dependence , being an expectant or recent parent , and > 18 years .
all were unemployed and white , reflecting the population of opioid - dependent adults in the study area .
five participants were first - time parents , fourteen other participants had between one and three children , aged 219 .
all were receiving ost at the first interview , primarily methadone , with a minority prescribed buprenorphine or dihydrocodeine .
eleven reported heroin use in the 12 months prior to the first interview and thirteen reported benzodiazepine use during the study period . in five cases , diazepam was prescribed long - term alongside ost .
this reflects the pattern of poly - drug use and prescribing practices in this geographical setting ( bird & robertson , 2011 ) . in total ,
semi - structured interviews , lasting between 45120 minutes , addressed participants social background , drug use , experiences with parenting , and involvement with services .
this was supplemented by close reading of transcripts and thematic coding by authors ac and aw ( ritchie , spencer , & oconnor , 2003 ) .
the present paper is based on analysis of two content codes : benzodiazepine use and methadone practice .
accounts were compared and contrasted and analytic themes were generated and agreed by the research team .
participants gave informed consent and were provided with a 20 voucher for each interview completed .
a common theme across parents accounts of all drug use was that their substance use helped to support their attempts to engage in normal daily life , providing a better context in which to undertake parenting .
positive meanings were sometimes ascribed to ost which was portrayed as enabling parents to manage their opioid - dependence in a legal , safer and relatively more socially acceptable manner ( chandler et al . , 2013 ) .
this was particularly the case for parents who had only recently ceased use of illicit opioids:[t]he only way to have a normal life [ ] and keep a normal life to normality , to being able to live again is a methadone prescription .
( alison , 3 months postnatal )
[ t]he only way to have a normal life [ ] and keep a normal life to normality , to being able to live again is a methadone prescription .
( alison , 3 months postnatal ) however , parents accounts more often emphasised the barriers and problems ost posed . particularly in the post - natal period , the structures around ost prescribing ( e.g. dispensing requirements ) were highlighted as having a negative impact on parents attempts to engage in a
normal life or recovery - orientated activities such as training and employment : im no wanting to stay on daily supervised , especially to try and go back to work , to go back to college .
( laura , 9 months postnatal )
i m no wanting to stay on daily supervised , especially to try and go back to work , to go back to college .
( laura , 9 months postnatal ) this was particularly the case for those participants who were required to have their use of ost supervised where methadone was consumed in the pharmacy under the surveillance of the dispensing pharmacist ( and any onlookers ) .
such practices were framed as especially problematic for parents : sometimes when i see ones in the chemist wi their weans [ children ] and i think they should nt be bringing their wean oot they re standing waiting for their meth and i try and sit in a seat away fae them .
( melanie , antenatal ) when you re being on supervised and that , because at the chemist i m at , they ve no got a bit that s blocked off , so you re standing at [ the locality ] swallowing your methadone in the middle of the shop flair .
so you get folk looking at you [ ] whether i ve got [ daughter ] wi me or no , so it s just , there s the junkie , look at her .
( caitlin , 9 months postnatal )
sometimes when i see ones in the chemist wi their weans [ children ] and i think they should nt be bringing their wean oot they re standing waiting for their meth and i try and sit in a seat away fae them .
( melanie , antenatal ) when you re being on supervised and that , because at the chemist i m at , they ve no got a bit that s blocked off , so you re standing at [ the locality ] swallowing your methadone in the middle of the shop flair .
so you get folk looking at you [ ] whether i ve got [ daughter ] wi me or no , so it s just ,
( caitlin , 9 months postnatal ) both melanie and caitlin s accounts highlight the role of stigma in shaping the experiences of parents in ost programmes .
in contrast , accounts of benzodiazepine use and dependence tended to be framed more positively , and crucially without reference to stigma .
most participants described using benzodiazepines in constructive ways : treating anxiety states , mood enhancers , providing practical and emotional support for parenting , enabling them to engage in daily routines and household tasks , and ultimately providing a context in which
carol , for instance , argued that her use of illicit diazepam was controlled , and helped her manage household chores.yes , like that song ,
mother s little helper , that s what i like to think of it as , just sort of gets me going , puts me in a good mood .
( carol , 6 months postnatal)similar justifications for illicit use were given by two fathers in the study .
here , paul s account refers to his ( unsuccessful ) attempts to acquire a prescription for benzodiazepines : i said to him [ general practitioner ] i m not taking them to get out of my face [ intoxicated ] .
i said i just take the two at night to help me get to sleep .
( paul , antenatal )
yes , like that song , mother s little helper , that s what i like to think of it as , just sort of gets me going , puts me in a good mood .
( carol , 6 months postnatal ) i said to him [ general practitioner ] i m not taking them to get out of my face [ intoxicated ] .
i said i just take the two at night to help me get to sleep .
( paul , antenatal ) participant s explanations for their continued use of benzodiazepines often centred on the supposed functional benefits of the drug in helping parent s cope with day - to - day life .
in contrast , accounts about ost were more likely to be framed around emphasising reduction and abstinence , narratives which were largely absent when it came to benzodiazepines .
this is most evident in the accounts of parents who were dependent on both benzodiazepines and ost . in nicola s final interview she reported that she was committed to reducing her methadone , contrasting starkly with her account of her benzodiazepine prescription : im fed up being stuck on something , where i wake up with my daughter , feeling like crap , and the first thing i dae is have to swallow medicine and have a cup of tea , to feel normal [ ] aye , i m not ready for [ reducing ] diazepam . that s like mother s little helper [ ] they help me , to want to get up in time to do stuff and everything .
( nicola , 10 months postnatal )
i m fed up being stuck on something , where i wake up with my daughter , feeling like crap , and the first thing i dae is have to swallow medicine and have a cup of tea , to feel normal [ ] aye , i m not ready for [ reducing ] diazepam . that s like mother s little helper [ ] they help me , to want to get up in time to do stuff and everything .
( nicola , 10 months postnatal ) divergent orientations towards methadone and benzodiazepine reduction were also discussed with tricia . in earlier interviews
, tricia had rejected the idea of reducing any of her prescriptions , however , like nicola , at 10 months postnatal she reported reducing her methadone , but not her benzodiazepine dosage : i feel like i m still a junkie because i need this green liquid [ methadone ] every day , it s crap , but i mean i dinnae feel like that with my valium [ ] aye i need to stay there [ on the same dose of benzodiazepine ] the now , i dinnae ken [ do nt know ] i would get all nervous .
i do need my valium and if they said to me they were going to start cutting me i would panic [ ] but they re not , they re just happy at me being stable just now .
( tricia , 10 months postnatal )
i feel like i m still a junkie because i need this green liquid [ methadone ] every day , it s crap , but
i mean i dinnae feel like that with my valium [ ] aye i need to stay there [ on the same dose of benzodiazepine ] the now , i dinnae ken [ do nt know ] i would get all nervous .
i do need my valium and if they said to me they were going to start cutting me i would panic [ ] but they re not , they re just happy at me being stable just now .
( tricia , 10 months postnatal ) rhetoric about stability , abstinence and dependence , therefore , were employed by participants differently according to the particular substance they were discussing .
accounts of substance use during pregnancy were for women especially more similar . in the majority of cases , women indicated that they wanted to reduce their dosage of both ost and benzodiazepine .
each of these substances were framed as problematic during pregnancy , largely centring on potential harm to the unborn baby.ive forgotten the other one , stop smoking , come off that , like the valium , and the methadone [ ] and i said it would be a bit much for me doing all of that [ ] but it s no too much for me coming down from the methadone and the valium [ ] . because it s going to be helping the baby and me .
( iona , antenatal )
i ve forgotten the other one , stop smoking , come off that , like the valium , and the methadone [ ] and i said it would be a bit much for me doing all of that [ ] but it s no too much for me coming down from the methadone and the valium [ ] . because it s going to be helping the baby and me .
( iona , antenatal ) women stressed a keen awareness that use of a range of substances might have negative effects on their babies .
those women dependent on multiple substances ( the majority ) described a careful balance of trying to stabilise or reduce one or more prescriptions , whilst avoiding relapse to illicit drug use . a clear contrast between parents accounts of opioid and benzodiazepine use and dependence was that the latter were often framed as a legitimate method of managing anxiety , negative emotions and past trauma .
as such , while women described reducing their benzodiazepine use during pregnancy , as with nicola s account here , once the pregnancy ended , the need to reduce ( based on perceived potential harm to the baby ) also ceased.ive been on them maist of my life , and that s to stop paranoia , it s to stop me going ootside , and feel like everybody s looking at me , and agitated , anxiety , everything , so they know i ll no be coming off them .
i ve already cut doon to ten milligrams , when i was pregnant , and nae mair , that s it .
i ll sort the meth [ ost ] , and then see aboot that [ diazepam ] in the later future .
( nicola , 10 months post - natal )
i ve been on them maist of my life , and that s to stop paranoia , it s to stop me going ootside , and feel like everybody s looking at me , and agitated , anxiety , everything , so they know i ll no be coming off them .
i ve already cut doon to ten milligrams , when i was pregnant , and nae mair , that s it .
i ll sort the meth [ ost ] , and then see aboot that [ diazepam ] in the later future .
( nicola , 10 months post - natal ) while nicola reported recommencing her reduction of ost at 10 months postnatal , she maintained that she would not be doing the same with her benzodiazepine prescription .
carrie described regular use of illicit benzodiazepines to manage anxiety states , and support her parenting .
as with tricia and nicola above , she justified her use of benzodiazepines , and need for a prescription of benzodiazepines , on therapeutic grounds , whilst simultaneously affirming a desire to reduce her methadone prescription : ive been taking valium , but like the drug worker says , instead of getting it fae the streets , cos you do nt know what s in it , she s going to give me a prescription [ ] so i m not sitting all thingy [ pulls agitated face ] around the kids and stuff .
cos i m a bit , still a bit , like anxious i think that s the word .
but i m down to 19ml [ methadone ] noo , so i ve been coming doon a ml a week .
( carrie , 4 months postnatal )
i ve been taking valium , but like the drug worker says , instead of getting it fae the streets , cos you do nt know what s in it , she s going to give me a prescription [ ] so i m not sitting all thingy [ pulls agitated face ] around the kids and stuff .
cos i m a bit , still a bit , like anxious i think that s the word .
but i m down to 19ml [ methadone ] noo , so i ve been coming doon a ml a week .
( carrie , 4 months postnatal ) when participants described benzodiazepine use as therapeutic , their accounts framed this as reasonable , responsible and more importantly , acceptable .
carrie emphasises that she was seeking , and expected to be given , a prescription for benzodiazepine , indicating that her dependence was validated by healthcare staff .
another participant , michael , maintained that the illicit benzodiazepines he bought were obtained from a reputable source and were trustworthy in terms of pharmaceutical content .
these narratives supported participants arguments that their benzodiazepine use , whether illicit or prescribed , was controlled and unproblematic .
thus , participants constructed benzodiazepine use and dependence as a legitimate means to help them regulate problem emotional states which might otherwise interfere with their ability to live a normal life and effectively parent their children . benzodiazepine use and dependence for women
was frequently normalised : women described using prescribed and illicit benzodiazepines to manage anxiety , perform household chores or regulate sleep patterns .
in contrast , while men in the study described using benzodiazepines for similar reasons , their accounts indicated that this was seen as problematic .
one participant framed this as explicitly related to gender : ive got all these people , telling me what s right , and what s wrong , and i take valium now and again , so , therefore , i m not a good parent .
how many bloody mothers oot there , do you ken that take valium ? does that stop them from being good parents [ ] ? where s their just cause [ ] ?
where s the argument , like , because i chose to be on valium when i was young , he must be f***ed up . to me , i would like to think i m reasonably intelligent .
but , fae their angle , i m no , they must think i m a total madman , which i m no. ( stuart , 8 months postnatal )
i ve got all these people , telling me what s right , and what s wrong , and
i take valium now and again , so , therefore , i m not a good parent .
how many bloody mothers oot there , do you ken that take valium ? does that stop them from being good parents [ ] ? where s their just cause [ ] ? where s the argument , like , because i chose to be on valium when i was young , he must be f***ed up . to me
but , fae their angle , i m no , they must think i m a total madman , which i m no. ( stuart , 8 months postnatal ) stuart alludes to the existence of gendered stereotypes about benzodiazepine dependence suggesting that use among women and mothers is accepted , but that among men and fathers is seen as dangerous or evidence of severe personality dysfunction . while the other men in the study did not make this gendered link explicit , the four male participants who reported illicit use of benzodiazepines provided similar accounts in other ways .
all asserted the therapeutic benefits of benzodiazepines , described unsuccessful attempts to acquire prescriptions and subsequently affirmed a desire or need to cease their illicit use of benzodiazepines .
while it is impossible to say based on this limited sample , these accounts allude to the existence of gendered patterns of prescribing , with healthcare practitioners seeming more ready to legitimise women s use of , and dependence on , benzodiazepines .
benzodiazepine use among men was also portrayed as problematic by three women in the study who described illicit use of benzodiazepines by their partners .
one participant suggested that her partner s benzodiazepine use was associated with aggression and domestic abuse , while the others clearly indicated that benzodiazepine use for their partners was more concerning than their use of other substances , particularly when combined with alcohol .
this parallels findings of another study with young offenders ( forsyth , khan , & mckinlay , 2011).what i m doing , i m just pottering about the house quite happy , tidying up , sorting things out , and they [ benzodiazepines ] make me feel happy , and i go to the shops and chat away to people , and it gives me a bit more confidence , but with him they make him angry , like i do nt know , it s weird .
( carol , 9 months postnatal )
what i m doing , i m just pottering about the house quite happy , tidying up , sorting things out , and they [ benzodiazepines ] make me feel happy , and i go to the shops and chat away to people , and it gives me a bit more confidence , but with him they make him angry , like i do nt know , it s weird .
( carol , 9 months postnatal ) in contrast , while women occasionally noted adverse effects of benzodiazepine use , this was generally focused on pregnancy and risks to the unborn baby .
the potential harms associated with benzodiazepine use , including the potential for cognitive impairment and acute withdrawal symptoms , appeared little understood or acknowledged in most participant accounts .
this was a longitudinal , qualitative study involving 19 opioid - dependent service users over the course of approximately one year in order to examine changes over time in participants experiences with healthcare services , drug use and parenting .
the truth , but rather as a resource through which to explore how parenting and drug use are understood , negotiated and mutually constructed ( bury , 2001 ; rhodes , bernays , & houmoller , 2010 ) .
the study was carried out in scotland in an area with a mixed socio - economic profile , containing areas of significant deprivation .
ost services for drug - dependent patients are delivered via primary care and specialist community - based substance misuse services .
there are local guidelines both for the management of substance misuse in pregnancy ( whittaker , 2003 ) , and parental substance use ( elbeg , 2013 ) .
notably , a large number of patients in the area are prescribed benzodiazepines in addition to ost .
participants , recruited via healthcare practitioners , were interviewed at around 2832 weeks gestation ; 23 months postnatal ; and 69 months postnatal .
inclusion criteria were opioid - dependence , being an expectant or recent parent , and > 18 years .
all were unemployed and white , reflecting the population of opioid - dependent adults in the study area .
five participants were first - time parents , fourteen other participants had between one and three children , aged 219 .
all were receiving ost at the first interview , primarily methadone , with a minority prescribed buprenorphine or dihydrocodeine .
eleven reported heroin use in the 12 months prior to the first interview and thirteen reported benzodiazepine use during the study period . in five cases , diazepam was prescribed long - term alongside ost .
this reflects the pattern of poly - drug use and prescribing practices in this geographical setting ( bird & robertson , 2011 ) . in total ,
semi - structured interviews , lasting between 45120 minutes , addressed participants social background , drug use , experiences with parenting , and involvement with services .
this was supplemented by close reading of transcripts and thematic coding by authors ac and aw ( ritchie , spencer , & oconnor , 2003 ) .
the present paper is based on analysis of two content codes : benzodiazepine use and methadone practice .
accounts were compared and contrasted and analytic themes were generated and agreed by the research team .
participants gave informed consent and were provided with a 20 voucher for each interview completed .
a common theme across parents accounts of all drug use was that their substance use helped to support their attempts to engage in normal daily life , providing a better context in which to undertake parenting .
positive meanings were sometimes ascribed to ost which was portrayed as enabling parents to manage their opioid - dependence in a legal , safer and relatively more socially acceptable manner ( chandler et al . , 2013 ) .
this was particularly the case for parents who had only recently ceased use of illicit opioids:[t]he only way to have a normal life [ ] and keep a normal life to normality , to being able to live again is a methadone prescription .
( alison , 3 months postnatal )
[ t]he only way to have a normal life [ ] and keep a normal life to normality , to being able to live again is a methadone prescription .
( alison , 3 months postnatal ) however , parents accounts more often emphasised the barriers and problems ost posed . particularly in the post - natal period ,
the structures around ost prescribing ( e.g. dispensing requirements ) were highlighted as having a negative impact on parents attempts to engage in a
normal life or recovery - orientated activities such as training and employment : im no wanting to stay on daily supervised , especially to try and go back to work , to go back to college .
( laura , 9 months postnatal )
i m no wanting to stay on daily supervised , especially to try and go back to work , to go back to college .
( laura , 9 months postnatal ) this was particularly the case for those participants who were required to have their use of ost supervised where methadone was consumed in the pharmacy under the surveillance of the dispensing pharmacist ( and any onlookers ) .
such practices were framed as especially problematic for parents : sometimes when i see ones in the chemist wi their weans [ children ] and i think they should nt be bringing their wean oot they re standing waiting for their meth and i try and sit in a seat away fae them .
( melanie , antenatal ) when you re being on supervised and that , because at the chemist i m at , they ve no got a bit that s blocked off , so you re standing at [ the locality ] swallowing your methadone in the middle of the shop flair .
so you get folk looking at you [ ] whether i ve got [ daughter ] wi me or no , so it s just , there s the junkie , look at her .
( caitlin , 9 months postnatal )
sometimes when i see ones in the chemist wi their weans [ children ] and i think they should nt be bringing their wean oot
they re standing waiting for their meth and i try and sit in a seat away fae them .
( melanie , antenatal ) when you re being on supervised and that , because at the chemist i m at , they ve no got a bit that s blocked off , so you re standing at [ the locality ] swallowing your methadone in the middle of the shop flair .
so you get folk looking at you [ ] whether i ve got [ daughter ] wi me or no , so it s just , there s the junkie , look at her .
( caitlin , 9 months postnatal ) both melanie and caitlin s accounts highlight the role of stigma in shaping the experiences of parents in ost programmes .
in contrast , accounts of benzodiazepine use and dependence tended to be framed more positively , and crucially without reference to stigma .
most participants described using benzodiazepines in constructive ways : treating anxiety states , mood enhancers , providing practical and emotional support for parenting , enabling them to engage in daily routines and household tasks , and ultimately providing a context in which
carol , for instance , argued that her use of illicit diazepam was controlled , and helped her manage household chores.yes , like that song ,
mother s little helper , that s what i like to think of it as , just sort of gets me going , puts me in a good mood .
( carol , 6 months postnatal)similar justifications for illicit use were given by two fathers in the study .
here , paul s account refers to his ( unsuccessful ) attempts to acquire a prescription for benzodiazepines : i said to him [ general practitioner ] i m not taking them to get out of my face [ intoxicated ] .
i said i just take the two at night to help me get to sleep .
( paul , antenatal )
yes , like that song , mother s little helper , that s what i like to think of it as , just sort of gets me going , puts me in a good mood .
( carol , 6 months postnatal ) i said to him [ general practitioner ] i m not taking them to get out of my face [ intoxicated ] .
i said i just take the two at night to help me get to sleep .
( paul , antenatal ) participant s explanations for their continued use of benzodiazepines often centred on the supposed functional benefits of the drug in helping parent s cope with day - to - day life .
in contrast , accounts about ost were more likely to be framed around emphasising reduction and abstinence , narratives which were largely absent when it came to benzodiazepines .
this is most evident in the accounts of parents who were dependent on both benzodiazepines and ost . in nicola s final interview she reported that she was committed to reducing her methadone , contrasting starkly with her account of her benzodiazepine prescription :
im fed up being stuck on something , where i wake up with my daughter , feeling like crap , and the first thing i dae is have to swallow medicine and have a cup of tea , to feel normal [ ] aye , i m not ready for [ reducing ] diazepam . that s like mother s little helper [ ] they help me , to want to get up in time to do stuff and everything .
( nicola , 10 months postnatal )
i m fed up being stuck on something , where i wake up with my daughter , feeling like crap , and the first thing i dae is have to swallow medicine and have a cup of tea , to feel normal [ ] aye , i m not ready for [ reducing ] diazepam . that s like mother s little helper [ ] they help me , to want to get up in time to do stuff and everything .
( nicola , 10 months postnatal ) divergent orientations towards methadone and benzodiazepine reduction were also discussed with tricia . in earlier interviews
, tricia had rejected the idea of reducing any of her prescriptions , however , like nicola , at 10 months postnatal she reported reducing her methadone , but not her benzodiazepine dosage : i feel like i m still a junkie because i need this green liquid [ methadone ] every day , it s crap , but i mean i dinnae feel like that with my valium [ ] aye i need to stay there [ on the same dose of benzodiazepine ] the now , i dinnae ken [ do nt know ] i would get all nervous .
i do need my valium and if they said to me they were going to start cutting me i would panic [ ] but they re not , they re just happy at me being stable just now .
( tricia , 10 months postnatal )
i feel like i m still a junkie because i need this green liquid [ methadone ] every day , it s crap , but
i mean i dinnae feel like that with my valium [ ] aye i need to stay there [ on the same dose of benzodiazepine ] the now , i dinnae ken [ do nt know ] i would get all nervous .
i do need my valium and if they said to me they were going to start cutting me i would panic [ ] but they re not , they re just happy at me being stable just now .
( tricia , 10 months postnatal ) rhetoric about stability , abstinence and dependence , therefore , were employed by participants differently according to the particular substance they were discussing . accounts of substance use during pregnancy were for women especially more similar . in the majority of cases
, women indicated that they wanted to reduce their dosage of both ost and benzodiazepine .
each of these substances were framed as problematic during pregnancy , largely centring on potential harm to the unborn baby.ive forgotten the other one , stop smoking , come off that , like the valium , and the methadone [ ] and i said it would be a bit much for me doing all of that [ ] but it s no too much for me coming down from the methadone and the valium [ ] . because it s going to be helping the baby and me .
( iona , antenatal )
i ve forgotten the other one , stop smoking , come off that , like the valium , and the methadone [ ] and i said it would be a bit much for me doing all of that [ ] but it s no too much for me coming down from the methadone and the valium [ ] . because it s going to be helping the baby and me .
( iona , antenatal ) women stressed a keen awareness that use of a range of substances might have negative effects on their babies .
those women dependent on multiple substances ( the majority ) described a careful balance of trying to stabilise or reduce one or more prescriptions , whilst avoiding relapse to illicit drug use .
a common theme across parents accounts of all drug use was that their substance use helped to support their attempts to engage in normal daily life , providing a better context in which to undertake parenting .
positive meanings were sometimes ascribed to ost which was portrayed as enabling parents to manage their opioid - dependence in a legal , safer and relatively more socially acceptable manner ( chandler et al . , 2013 ) .
this was particularly the case for parents who had only recently ceased use of illicit opioids:[t]he only way to have a normal life [ ] and keep a normal life to normality , to being able to live again is a methadone prescription .
( alison , 3 months postnatal )
[ t]he only way to have a normal life [ ] and keep a normal life to normality , to being able to live again is a methadone prescription .
( alison , 3 months postnatal ) however , parents accounts more often emphasised the barriers and problems ost posed . particularly in the post - natal period ,
the structures around ost prescribing ( e.g. dispensing requirements ) were highlighted as having a negative impact on parents attempts to engage in a
normal life or recovery - orientated activities such as training and employment : im no wanting to stay on daily supervised , especially to try and go back to work , to go back to college .
( laura , 9 months postnatal )
i m no wanting to stay on daily supervised , especially to try and go back to work , to go back to college .
( laura , 9 months postnatal ) this was particularly the case for those participants who were required to have their use of ost supervised where methadone was consumed in the pharmacy under the surveillance of the dispensing pharmacist ( and any onlookers ) .
such practices were framed as especially problematic for parents : sometimes when i see ones in the chemist wi their weans [ children ] and i think they should nt be bringing their wean oot they re standing waiting for their meth and i try and sit in a seat away fae them .
( melanie , antenatal ) when you re being on supervised and that , because at the chemist i m at , they ve no got a bit that s blocked off , so you re standing at [ the locality ] swallowing your methadone in the middle of the shop flair .
so you get folk looking at you [ ] whether i ve got [ daughter ] wi me or no , so it s just , there s the junkie , look at her .
( caitlin , 9 months postnatal )
sometimes when i see ones in the chemist wi their weans [ children ] and i think they should nt be bringing their wean oot
they re standing waiting for their meth and i try and sit in a seat away fae them .
( melanie , antenatal ) when you re being on supervised and that , because at the chemist i m at , they ve no got a bit that s blocked off , so you re standing at [ the locality ] swallowing your methadone in the middle of the shop flair .
so you get folk looking at you [ ] whether i ve got [ daughter ] wi me or no , so it s just , there s the junkie , look at her .
( caitlin , 9 months postnatal ) both melanie and caitlin s accounts highlight the role of stigma in shaping the experiences of parents in ost programmes .
in contrast , accounts of benzodiazepine use and dependence tended to be framed more positively , and crucially without reference to stigma .
most participants described using benzodiazepines in constructive ways : treating anxiety states , mood enhancers , providing practical and emotional support for parenting , enabling them to engage in daily routines and household tasks , and ultimately providing a context in which
carol , for instance , argued that her use of illicit diazepam was controlled , and helped her manage household chores.yes , like that song ,
mother s little helper , that s what i like to think of it as , just sort of gets me going , puts me in a good mood .
( carol , 6 months postnatal)similar justifications for illicit use were given by two fathers in the study .
here , paul s account refers to his ( unsuccessful ) attempts to acquire a prescription for benzodiazepines : i said to him [ general practitioner ] i m not taking them to get out of my face [ intoxicated ] .
i said i just take the two at night to help me get to sleep .
( paul , antenatal )
yes , like that song , mother s little helper , that s what i like to think of it as , just sort of gets me going , puts me in a good mood .
( carol , 6 months postnatal ) i said to him [ general practitioner ] i m not taking them to get out of my face [ intoxicated ] .
i said i just take the two at night to help me get to sleep .
( paul , antenatal ) participant s explanations for their continued use of benzodiazepines often centred on the supposed functional benefits of the drug in helping parent s cope with day - to - day life .
in contrast , accounts about ost were more likely to be framed around emphasising reduction and abstinence , narratives which were largely absent when it came to benzodiazepines .
this is most evident in the accounts of parents who were dependent on both benzodiazepines and ost . in nicola s final interview she reported that she was committed to reducing her methadone , contrasting starkly with her account of her benzodiazepine prescription :
im fed up being stuck on something , where i wake up with my daughter , feeling like crap , and the first thing i dae is have to swallow medicine and have a cup of tea , to feel normal [ ] aye , i m not ready for [ reducing ] diazepam . that s like mother s little helper [ ] they help me , to want to get up in time to do stuff and everything .
( nicola , 10 months postnatal )
i m fed up being stuck on something , where i wake up with my daughter , feeling like crap , and the first thing i dae is have to swallow medicine and have a cup of tea , to feel normal [ ] aye , i m not ready for [ reducing ] diazepam . that s like mother s little helper [ ] they help me , to want to get up in time to do stuff and everything .
( nicola , 10 months postnatal ) divergent orientations towards methadone and benzodiazepine reduction were also discussed with tricia . in earlier interviews
, tricia had rejected the idea of reducing any of her prescriptions , however , like nicola , at 10 months postnatal she reported reducing her methadone , but not her benzodiazepine dosage : i feel like i m still a junkie because i need this green liquid [ methadone ] every day , it s crap , but i mean i dinnae feel like that with my valium [ ] aye i need to stay there [ on the same dose of benzodiazepine ] the now , i dinnae ken [ do nt know ] i would get all nervous .
i do need my valium and if they said to me they were going to start cutting me i would panic [ ] but they re not , they re just happy at me being stable just now .
( tricia , 10 months postnatal )
i feel like i m still a junkie because i need this green liquid [ methadone ] every day , it s crap , but
i mean i dinnae feel like that with my valium [ ] aye i need to stay there [ on the same dose of benzodiazepine ] the now , i dinnae ken [ do nt know ] i would get all nervous .
i do need my valium and if they said to me they were going to start cutting me i would panic [ ] but they re not , they re just happy at me being stable just now .
( tricia , 10 months postnatal ) rhetoric about stability , abstinence and dependence , therefore , were employed by participants differently according to the particular substance they were discussing . accounts of substance use during pregnancy were for women especially more similar . in the majority of cases
, women indicated that they wanted to reduce their dosage of both ost and benzodiazepine .
each of these substances were framed as problematic during pregnancy , largely centring on potential harm to the unborn baby.ive forgotten the other one , stop smoking , come off that , like the valium , and the methadone [ ] and i said it would be a bit much for me doing all of that [ ] but it s no too much for me coming down from the methadone and the valium [ ] . because it s going to be helping the baby and me .
( iona , antenatal )
i ve forgotten the other one , stop smoking , come off that , like the valium , and the methadone [ ] and i said it would be a bit much for me doing all of that [ ] but it s no too much for me coming down from the methadone and the valium [ ] . because it s going to be helping the baby and me .
( iona , antenatal ) women stressed a keen awareness that use of a range of substances might have negative effects on their babies .
those women dependent on multiple substances ( the majority ) described a careful balance of trying to stabilise or reduce one or more prescriptions , whilst avoiding relapse to illicit drug use .
a clear contrast between parents accounts of opioid and benzodiazepine use and dependence was that the latter were often framed as a legitimate method of managing anxiety , negative emotions and past trauma .
as such , while women described reducing their benzodiazepine use during pregnancy , as with nicola s account here , once the pregnancy ended , the need to reduce ( based on perceived potential harm to the baby ) also ceased.ive been on them maist of my life , and that s to stop paranoia , it s to stop me going ootside , and feel like everybody s looking at me , and agitated , anxiety , everything , so they know i ll no be coming off them .
i ve already cut doon to ten milligrams , when i was pregnant , and nae mair , that s it .
i ll sort the meth [ ost ] , and then see aboot that [ diazepam ] in the later future .
( nicola , 10 months post - natal )
i ve been on them maist of my life , and that s to stop paranoia , it s to stop me going ootside , and feel like everybody s looking at me , and agitated , anxiety , everything , so they know i ll no be coming off them .
i ve already cut doon to ten milligrams , when i was pregnant , and nae mair , that s it .
i ll sort the meth [ ost ] , and then see aboot that [ diazepam ] in the later future .
( nicola , 10 months post - natal ) while nicola reported recommencing her reduction of ost at 10 months postnatal , she maintained that she would not be doing the same with her benzodiazepine prescription .
carrie described regular use of illicit benzodiazepines to manage anxiety states , and support her parenting .
as with tricia and nicola above , she justified her use of benzodiazepines , and need for a prescription of benzodiazepines , on therapeutic grounds , whilst simultaneously affirming a desire to reduce her methadone prescription : ive been taking valium , but like the drug worker says , instead of getting it fae the streets , cos you do nt know what s in it , she s going to give me a prescription [ ] so i m not sitting all thingy [ pulls agitated face ] around the kids and stuff . just to calm me
cos i m a bit , still a bit , like anxious i think that s the word .
but i m down to 19ml [ methadone ] noo , so i ve been coming doon a ml a week .
( carrie , 4 months postnatal )
i ve been taking valium , but like the drug worker says , instead of getting it fae the streets ,
cos you do nt know what s in it , she s going to give me a prescription [ ] so i m not sitting all thingy [ pulls agitated face ] around the kids and stuff . just to calm me
cos i m a bit , still a bit , like anxious i think that s the word .
but i m down to 19ml [ methadone ] noo , so i ve been coming doon a ml a week .
( carrie , 4 months postnatal ) when participants described benzodiazepine use as therapeutic , their accounts framed this as reasonable , responsible and more importantly , acceptable .
carrie emphasises that she was seeking , and expected to be given , a prescription for benzodiazepine , indicating that her dependence was validated by healthcare staff .
another participant , michael , maintained that the illicit benzodiazepines he bought were obtained from a reputable source and were trustworthy in terms of pharmaceutical content .
these narratives supported participants arguments that their benzodiazepine use , whether illicit or prescribed , was
thus , participants constructed benzodiazepine use and dependence as a legitimate means to help them regulate problem emotional states which might otherwise interfere with their ability to live a normal life and effectively parent their children . benzodiazepine use and dependence for women
was frequently normalised : women described using prescribed and illicit benzodiazepines to manage anxiety , perform household chores or regulate sleep patterns .
in contrast , while men in the study described using benzodiazepines for similar reasons , their accounts indicated that this was seen as problematic .
one participant framed this as explicitly related to gender : ive got all these people , telling me what s right , and what s wrong , and i take valium now and again , so , therefore , i m not a good parent .
how many bloody mothers oot there , do you ken that take valium ? does that stop them from being good parents [ ] ? where s their just cause [ ] ? where s the argument , like , because i chose to be on valium when i was young , he must be f***ed up . to me
but , fae their angle , i m no , they must think i m a total madman , which i m no. ( stuart , 8 months postnatal )
i ve got all these people , telling me what s right , and what s wrong , and i take valium now and again , so , therefore , i m not a good parent .
how many bloody mothers oot there , do you ken that take valium ? does that stop them from being good parents [ ] ? where s their just cause [ ] ? where s the argument , like , because i chose to be on valium when i was young , he must be f***ed up . to me
but , fae their angle , i m no , they must think i m a total madman , which i m no. ( stuart , 8 months postnatal ) stuart alludes to the existence of gendered stereotypes about benzodiazepine dependence suggesting that use among women and mothers is accepted , but that among men and fathers is seen as dangerous or evidence of severe personality dysfunction . while the other men in the study did not make this gendered link explicit , the four male participants who reported illicit use of benzodiazepines provided similar accounts in other ways .
all asserted the therapeutic benefits of benzodiazepines , described unsuccessful attempts to acquire prescriptions and subsequently affirmed a desire or need to cease their illicit use of benzodiazepines . while it is impossible to say based on this limited sample , these accounts allude to the existence of gendered patterns of prescribing , with healthcare practitioners seeming more ready to legitimise women s use of , and dependence on , benzodiazepines .
benzodiazepine use among men was also portrayed as problematic by three women in the study who described illicit use of benzodiazepines by their partners .
one participant suggested that her partner s benzodiazepine use was associated with aggression and domestic abuse , while the others clearly indicated that benzodiazepine use for their partners was more concerning than their use of other substances , particularly when combined with alcohol .
this parallels findings of another study with young offenders ( forsyth , khan , & mckinlay , 2011).what i m doing , i m just pottering about the house quite happy , tidying up , sorting things out , and they [ benzodiazepines ] make me feel happy , and i go to the shops and chat away to people , and it gives me a bit more confidence , but with him they make him angry , like i do nt know , it s weird .
( carol , 9 months postnatal )
what i m doing , i m just pottering about the house quite happy , tidying up , sorting things out , and they [ benzodiazepines ] make me feel happy , and i go to the shops and chat away to people , and it gives me a bit more confidence , but with him they make him angry , like i do nt know , it s weird .
( carol , 9 months postnatal ) in contrast , while women occasionally noted adverse effects of benzodiazepine use , this was generally focused on pregnancy and risks to the unborn baby .
the potential harms associated with benzodiazepine use , including the potential for cognitive impairment and acute withdrawal symptoms , appeared little understood or acknowledged in most participant accounts .
benzodiazepine use and dependence for women was frequently normalised : women described using prescribed and illicit benzodiazepines to manage anxiety , perform household chores or regulate sleep patterns .
in contrast , while men in the study described using benzodiazepines for similar reasons , their accounts indicated that this was seen as problematic .
one participant framed this as explicitly related to gender : ive got all these people , telling me what s right , and what s wrong , and i take valium now and again , so , therefore , i m not a good parent .
how many bloody mothers oot there , do you ken that take valium ? does that stop them from being good parents [ ] ? where s their just cause [ ] ?
where s the argument , like , because i chose to be on valium when i was young , he must be f***ed up . to me , i would like to think i m reasonably intelligent .
but , fae their angle , i m no , they must think i m a total madman , which i m no. ( stuart , 8 months postnatal )
i ve got all these people , telling me what s right , and what s wrong , and i take valium now and again , so , therefore , i m not a good parent .
how many bloody mothers oot there , do you ken that take valium ? does that stop them from being good parents [ ] ? where s their just cause [ ] ? where s the argument , like
, because i chose to be on valium when i was young , he must be f***ed up . to me , i would like to think i m reasonably intelligent .
but , fae their angle , i m no , they must think i m a total madman , which i m no. ( stuart , 8 months postnatal ) stuart alludes to the existence of gendered stereotypes about benzodiazepine dependence suggesting that use among women and mothers is accepted , but that among men and fathers is seen as dangerous or evidence of severe personality dysfunction . while the other men in the study did not make this gendered link explicit , the four male participants who reported illicit use of benzodiazepines provided similar accounts in other ways .
all asserted the therapeutic benefits of benzodiazepines , described unsuccessful attempts to acquire prescriptions and subsequently affirmed a desire or need to cease their illicit use of benzodiazepines .
while it is impossible to say based on this limited sample , these accounts allude to the existence of gendered patterns of prescribing , with healthcare practitioners seeming more ready to legitimise women s use of , and dependence on , benzodiazepines .
benzodiazepine use among men was also portrayed as problematic by three women in the study who described illicit use of benzodiazepines by their partners .
one participant suggested that her partner s benzodiazepine use was associated with aggression and domestic abuse , while the others clearly indicated that benzodiazepine use for their partners was more concerning than their use of other substances , particularly when combined with alcohol .
this parallels findings of another study with young offenders ( forsyth , khan , & mckinlay , 2011).what i m doing , i m just pottering about the house quite happy , tidying up , sorting things out , and they [ benzodiazepines ] make me feel happy , and i go to the shops and chat away to people , and it gives me a bit more confidence , but with him they make him angry , like i do nt know , it s weird .
( carol , 9 months postnatal )
what i m doing , i m just pottering about the house quite happy , tidying up , sorting things out , and they [ benzodiazepines ] make me feel happy , and i go to the shops and chat away to people , and it gives me a bit more confidence , but with him they make him angry , like i do nt know , it s weird .
( carol , 9 months postnatal ) in contrast , while women occasionally noted adverse effects of benzodiazepine use , this was generally focused on pregnancy and risks to the unborn baby .
the potential harms associated with benzodiazepine use , including the potential for cognitive impairment and acute withdrawal symptoms , appeared little understood or acknowledged in most participant accounts .
this longitudinal qualitative study explored accounts of poly - drug use within the context of pregnancy and parenting , highlighting differential understandings and practices surrounding benzodiazepine and opioid use .
our findings illuminate a number of issues with implications for theory , policy , prevention and education .
the accounts in our study resonate with others ( klee , 1998 ; radcliffe , 2011 ) in demonstrating the morally difficult nature of being a drug - using parent , and the extent to which stigma can play an important role in the day - to - day lives of parents .
our findings suggest that while drug use , in general , is seen as incompatible with parenting , there are clear indications that some drugs , and types of addiction , are more acceptable than others .
crucially , our analysis indicates important differences in the way opioid and benzodiazepine use are understood and narrated within the context of parenthood . unlike benzodiazepine use
, participants in this study often described ost in negative terms even when they acknowledged that ost was helpful in treating opioid dependence .
this dominant negative discourse on ost by drug users themselves , has been described elsewhere ( harris & mcelrath , 2012 ; lloyd , 2013 ; radcliffe & stevens , 2008 ) , and highlights the unique stigma attached to ost for the treatment of opioid dependence ( keane , 2013 ) .
participants described using benzodiazepines in constructive ways to enhance social and emotional functioning and engagement in normal life appealing to a self - medicating hypothesis of addiction to explain and justify continued use ( jones , et al . , 2012 ) .
these meanings served to legitimise benzodiazepine dependence , contrasting sharply with participants alignment of ost with
junkie identities , as documented elsewhere ( keane , 2013 ; radcliffe & stevens , 2008 ) . in the context of parenthood ,
parents were more likely to describe a desire to reduce opioid rather than benzodiazepine use , and there was evidence that illicit benzodiazepine use was considered less harmful than opioid use .
this is noteworthy , because alongside this complacency were occasional notes of concern , particularly in relation to benzodiazepine use by men , and its link with increased aggression and domestic abuse ( reported by some women participants ) . accounts from this study provide a tentative indication of the way gender may shape understandings of benzodiazepine use .
the long - term use of benzodiazepines by mothers was framed as being therapeutic and unproblematic .
in contrast , while men reported using benzodiazepine for similar reasons , their accounts indicated that practitioners did not legitimise this and prescriptions were denied .
these findings sit uneasily with the dominant recovery agenda in uk drug policy ( valentine & treloar , 2013 ; wardle , 2012 ) , where abstinence from all drug use is assumed to be both desirable and necessary for social reintegration .
it may be that in practice recovery refers to particular categories of drugs , with the hierarchy of acceptability identified among service users in this study being apparently mirrored in prescribing practices and approaches taken to treating addiction by clinicians ( anthierens , habraken , petrovic , & christiaens , 2007 ) .
thus , while opioid - dependent individuals may express a desire to reduce their opioid prescriptions , this is not necessarily reflected in their orientation towards other drug use , particularly benzodiazepines .
as such , the claim that opioid - dependent individuals would prefer abstinence - based treatment approaches may result from the unique stigma attached to opioids and may not apply to all drug use , or all drugs of dependence ( lloyd , 2013 ; mckeganey , morris , neale , & robertson , 2004 ; neale , nettleton , & pickering , 2011 ) .
our findings suggest a need for further preventative measures and targeted education to address benzodiazepine use and dependence within the context of pregnancy and parenting , especially in populations of parents engaged in ost programmes where stability on opioids may detract from issues associated with benzodiazepine use and dependence . reviews of benzodiazepine prescribing ( sirdifield et al . , 2013 ) ,
identify a variety of reasons for inconsistent practice , and conclude that improved education and training , provision of non - pharmacological interventions , and enhanced communication with patients is required .
our findings support these recommendations , especially the need for enhanced education and training of both clinicians and patients , and extra provision of non - pharmacological interventions to promote the psychological and social functioning of parents on ost .
this was an in - depth , qualitative exploration of the accounts of nineteen drug - dependent parents on ost in a specific geographical area .
as such , our findings and conclusions should be read as indicative and suggestive rather than definitive .
further qualitative exploration in different geographical areas and populations is needed , as it is likely that local or cultural contexts may shape the types of narratives expressed in respect of the range of substances consumed .
our findings may have limited applicability to opioid dependent parents who are not engaged in ost programmes .
however , the contrasting accounts depicted here underline the importance of qualitative research in illuminating understandings about the meanings that drugs of addiction have for those who use them ( neale , allen , & coombes , 2005 ) .
although long - term benzodiazepine use and dependence among opioid - dependent populations has been a topic of concern for some decades ( who , 1996 ) and is associated with increased risks and harm ( lader , 2011 , 2014 ) , research regarding the impact on pregnancy , parenting and family life has been limited ( acmd , 2003 ) .
our findings suggest that parents frame benzodiazepine use and dependence as largely unproblematic , less stigmatising and more legitimate than ost .
we suggest these divergent accounts can be understood as drawing on differing cultural meanings attached to ost and benzodiazepine use , which are reflected in policy and practice . as such , prevention ,
education and policy initiatives should pay greater attention to the kind of narratives and strategies that are employed in addressing benzodiazepine use and dependence within opioid dependent populations , especially in respect of the parenting and child welfare agenda .
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aimsto explore the ways in which opioid - dependent parents accounted for their use of opioids and benzodiazepines during and after pregnancy.methodslongitudinal qualitative interviews [ n = 45 ] with 19 opioid - dependent adults recruited in scotland , uk , were held during the antenatal and post - natal period .
interviews focused on parenting and parenting support within the context of problem drug use and were analysed using a narrative informed , thematic analysis .
findingsthe majority of participants described using benzodiazepines in addition to opioids .
almost all indicated a desire to stop or reduce opioid use , whereas cessation or reduction of benzodiazepines was rarely prioritised .
in stark contrast to opioid dependence , benzodiazepine dependence was portrayed as unproblematic , therapeutic and acceptable in the context of family life . whereas opioid dependence was framed as stigmatising , benzodiazepine use and dependence was normalised .
an exception was benzodiazepine use by men which was occasionally associated with aggression and domestic abuse.conclusionsdrug-dependent parents attach different meanings to opioid and benzodiazepine use and dependence in the context of parenthood .
divergent meanings , and stigma , may impact on stated commitment to stability or recovery from dependent drug - use .
attention should be paid to the way in which policy and practice regarding ost and benzodiazepines reflects this divergence .
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Introduction
Methods
Study design and setting
Recruitment, sample and data collection
Analysis
Ethics
Findings
Benzodiazepine and opioid use for normality and normal life.
Therapeutic qualities of benzodiazepine and opioid use
Benzodiazepines: mothers little helper; fathers ruin?
Discussion
Limitations
Conclusion
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compounds 14 , 7 , 8 , and 11 were
synthesized following our previously reported synthetic routes .
saturated derivatives 5 and 6 , unique to this work , were synthesized via the hydrogenation
of diastereomers 1 and 2 , respectively .
palmyrolide alcohol ( 9 ) and acid ( 10 )
were
prepared exploiting the known acid - catalyzed ring opening of 1 , followed by treating the resultant
aldehyde with either nabh4 ( for 9 ) or naclo2 ( for 10 ) .
alcohol 12 could be accessed
in a similar manner from 14-epi - palmyrolide ( 2 ) . in order to detect an interaction of ( )-palmyrolide
a and
its analogues with voltage - gated sodium channels ( vgscs )
, we examined
the ability of these compounds to antagonize veratridine - stimulated
na influx in murine primary cerebrocortical neurons .
we
previously demonstrated that veratridine is a partial agonist at neurotoxin
site 2 on the vgsc subunit .
murine
cerebrocortical neurons were loaded with the na - binding
benzofuran isophthalate ( sbfi ) , and the ability of palmyrolide analogues
to block veratridine - induced elevation of neuronal [ na]i was determined .
sodium influx was monitored with sbfi ( 340/380 ) responses to veratridine
in the absence and presence of ( )-palmyrolide a and its analogues .
the compounds shown are ( a ) ( )-palmyrolide a ( 1 ) ; ( b ) 17,18-dihydropalmyrolide a ( 5 ) ; ( c ) ( + ) -palmyrolide
a ( 3 ) ; ( d ) 17,18-dihydro-14-epi - palmyrolide
a ( 6 ) ; ( e ) [ c-5(r),c-7(s),c-14(r)]-palmyrolide ( 8) ; and ( f )
[ c-5(r),c-7(s),c-14(s)]-palmyrolide ( 7 ) .
data shown are from a representative
experiment performed in 2 or 3 replicates for each concentration .
in total 47 experiments
nonlinear regression analysis of concentration response
data for active analogues of ( )-palmyrolide a. a three - parameter
logistic fit of the palmyrolide a analogue inhibition of the response
to veratridine is shown for each active compound .
the compounds shown
are ( a ) ( )-palmyrolide a ( 1 ) ; ( b ) 17,18-dihydropalmyrolide
a ( 5 ) ; ( c ) ( + ) -palmyrolide a ( 3 ) ; ( d ) 17,18-dihydro-14-epi - palmyrolide a ( 6 ) ; ( e ) [ c-5(r),c-7(s),c-14(r)]-palmyrolide ( 8) ; and ( f ) [ c-5(r),c-7(s),c-14(s)]-palmyrolide ( 7 ) . data points
shown represent the mean sem of 25 experiments performed
with 210 replicates each ( a f ) .
values represent mean sem
of 25 experiments each performed with 210 replicates . naturally occurring ( )-palmyrolide
a is a neuroactive macrolide
that was previously shown to block veratridine - induced sodium influx
in neuro-2a cells ( ic50 = 5.2 m ) and to display significant inhibition of ca oscillation in murine cerebrocortical neurons , with an ic50 value of 3.70 m ( 2.295.98 m , 95% ci ) .
we have confirmed and extended these original
findings by demonstrating that synthetic ( )-palmyrolide a
( 1 , figures 2a and 3a ) and its enantiomer ( 3 , figures 2c and 3c ) produced concentration - dependent
antagonism of veratridine - induced increase in neuronal [ na]i .
the concentration response curves for these
compounds were best fit by a three - parameter logistic equation .
this
analysis yielded an ic50 value ( 95% confidence interval )
of 2.1 m ( 1.23.1 , 95% ci ) , which was in good agreement
with the ic50 value reported earlier for natural ( )-palmyrolide
a. we found that ( )-palmyrolide
a ( 1 ) and macrolide analogues 5 and 3 were approximately 39-fold more potent than the
other analogues tested ( figures 2a
the observation that saturated
congener 5 showed a comparable ic50 to synthetic
( )-palmyrolide a suggests that the enamide double bond is
not essential for activity .
it is moreover noteworthy that acyclic
analogues of ( )-palmyrolide a with the natural absolute configuration
were inactive as blockers of veratridine - stimulated na influx .
these data indicate that the three - dimensional molecular
shape of ( )-palmyrolide a was crucial for the interaction
with vgscs . in order to support these observations and to better
understand
how structure relates to biological function , an exhaustive nmr and
computational analysis was next conducted on 1 and three
diastereomers ( i.e. , 2 , 7 , and 8) .
saturated derivatives 5 and 6 exist
as a mixture of rotational isomers and precluded precise nmr assignments
from being made .
chemical shift assignments of ( )-palmyrolide
a ( 1 ) and synthetic diastereomers 2 , 7 , and 8 were made using dqf - cosy , hsqc , hmbc ,
and h2bc spectra .
the chemical shifts of these four diastereomeric compounds follow
the same general trends but differ slightly , indicating that the conformations
of the macrolide ring are different . for example , in each diastereomer
a different methylene group exhibited degenerate chemical shifts for
its two protons .
stereospecific assignments were made for most of
the prochiral atoms that possessed nondegenerate shifts .
the noesy
spectra provided 74 nonredundant distance restraints for 1 , 83 for 2 , 84 for 7 , and 77 for 8 .
h coupling in the h nmr spectra
and from the heteronuclear couplings measured by the hetloc experiments
( see supporting information for all restraints ) .
applying the distance and torsion restraints during simulated annealing
calculations produced fairly well - defined ensembles of structures .
table 2 summarizes the inputs
and results of the structure calculations . to test the specificity
of the restraints and their ability to discriminate between the four
diastereomers ,
restrained simulated annealing calculations were performed
for all 16 combinations of starting structure and restraint set . if
the restraints correctly discriminated between conformations , then
the correct combination should have the least restraint violations
and the lowest energy .
for all four structures we found the best restraint
set for each starting structure was the correct one , namely , the restraints
derived from nmr spectra of that compound ( table 3 ) .
this confirms that the restraint sets were able to correctly
discriminate between the diastereomers .
the four ensembles generally
have the same overall conformation ,
with the major differences being due to the orientations of the substitutents .
all four diastereomers have a cisoid configuration
of the n - methyl group ( n - ch3 ) and the
adjacent carbonyl despite the starting structures having a transoid configuration .
examining the trajectories reveals
that the configuration converts early in the simulated annealing as
the restraints take effect .
the driving force in these simulations
appears to be noes between h-18 and the h-2 methylene group . to quantify differences between the ensembles , we calculated root - mean - square
deviations ( rmsd s ) between the representative structures of
each ensemble ( table 4 ) .
the rmsd s
were calculated using the differences in the positions of the carbon ,
nitrogen , and oxygen atoms of the macrolide ring after aligning the
structures to minimize these differences .
representative structures
were selected as the structure having the smallest rmsd among all
other structures in its ensemble .
the rmsd s between all four
representative structures were similar ( table 4 ) , with 2 having the lowest values overall , suggesting
that this ensemble is the most central in comparison
to the other diastereomers .
visual inspection of the ensembles
shows that the macrolide ring
is relatively flat , but the orientation of the tert - butyl and methyl groups alters the global shape ( figure 4 ) . in 1
the tert - butyl
and methyl groups lie close to the plane of the ring , but in 7 the tert - butyl group projects almost perpendicular
to the plane of the ring , and in 8 the two methyl groups
project in opposite directions above and below the ring . examining
the surface electrostatic potential of the representative structures
( figure 5 ) we find that 8 is the
most similar to the active compound 1 . however , the c-14
methyl group in 1 projects to the side , whereas in 8 it is situated above the macrolide ring . taken together ,
these differences between diastereomers may account , at least in part ,
for the observed differences in biological activity .
in addition ,
due to the highly flexible nature of the macrolide , as evidenced through
computational modeling , the minimized ligand ensembles reported here
may not necessarily reflect the structure these molecules adopt when
bound to their molecular target .
further consideration and investigations
into structure activity relationships are ongoing and will
be reported in due course .
number of diastereotopic hydrogens
for which stereospecific assignments were obtained , and total number
of protons with nondegenerate chemical shifts .
the three numbers reported for each
combination of starting structure and restraint set are number of
selected structures , average amber energy , and average violation energy .
the italic cells indicate the best set of restraints for each starting
structure , with respect to amber energy and violation energy , and
also correspond to the correct pairing .
the final row is the sum of the
three rmsd s above , a measure of the total difference from
the other structures .
the four structures in the top row are shown in
a similar orientation to figure 4 . in the lower
row
the structures have been rotated 180 about the horizontal
axis to show the rear face .
the electrostatic potential ranges from
red ( 2 kcal / mole ) through white ( 0 ) to blue ( + 2 kcal / mole ) .
these
findings suggest that both synthetic ( )-palmyrolide
a and its enantiomer function with equal potency as vgsc antagonists
to block veratridine - induced sodium influx . with regard to identification
of the pharmacophore of this natural product
, it appears that the
intact macrolide is required since acyclic versions were inactive
as inhibitors of vgscs .
moreover , analogues lacking the enamide double
bond were of high potency in this assay , and thus this functionality
appears unnecessary for blocking vgscs . finally ,
using detailed nmr
and computational approaches with the natural product and three diastereomeric
analogues , it was found that the c-5c-7 anti / c-7c14 syn arrangement of stereocenters
produces a unique combination of three - dimensional shape and electrostatic
potential that is responsible for the potent biological activity of
the natural product . in an effort to understand
the similar biological
activity found for the natural stereoisomer and its enantiomer , continued
investigations in this area will focus on uncovering the specific
molecular target and associated binding site , which may also assist
in future analogue development of novel sodium channel blocking analgesics
derived from palmyrolide a.
unless otherwise noted ,
reactions were performed in flame - dried glassware under an atmosphere
of dry nitrogen .
reaction solvents ( ch2cl2 ,
thf , and et2o ) were purified before use in a solvent purification
system under a flow of dry nitrogen .
all other solvents and reagents
were purchased from commercial suppliers and used as received , unless
otherwise specified .
thin - layer chromatography ( tlc ) was performed
using plates precoated with silica gel 60 f-254 ( 250 m )
and visualized by uv light , kmno4 , or anisaldehyde stains ,
followed by heating .
ir samples were prepared by evaporation
from chcl3 or ch2cl2 on nacl plates
and run on a perkinelmer spectrum one ft - ir spectrometer . for the
synthetic studies ,
h and c nmr spectra were
recorded at 300 and 75 mhz ( oxford magnet with a varian 300 console ) ,
at 400 and 100 mhz ( oxford magnet with a varian unity 400 console ) ,
and at 600 and 150 mhz ( magnex magnet with a bruker avance
iii 600
console ) , respectively , and are reported relative to residual solvent
peak ( h 7.26 and c 77.0 for h and c in cdcl3 ) .
high - resolution
mass spectra were obtained using positive electrospray ionization
on a bruker 12 t apex - qe fticr - ms with an apollo ii ion source at
the cosmic laboratory facility at old dominion university , va . to a solution of ( s , e)-6-iodo-2-methylhex-5-enoic
acid ( 0.096 g , 0.37 mmol ) in thf ( 3.7 ml )
was added diisopropylethylamine
( 0.100 ml , 0.60 mmol ) followed by the yamaguchi reagent ( 0.071 ml ,
0.45 mmol ) .
the mixture was allowed to stir at room temperature for
3 h before being concentrated in vacuo .
the resultant
mixed anhydride was dissolved in toluene ( 7.4 ml ) and then added ,
via cannula , to a flask containing ( 5r,7r)-7-hydroxy-5,8,8-trimethylnonanamide ( 0.051 g , 0.23 mmol ) and dmap
( 0.046 g , 0.37 mmol ) . after stirring at room temperature ( rt ) for
12 h
, the reaction mixture was diluted with ch2cl2 , washed with a saturated aqueous solution of nahco3 ,
dried over mgso4 , and concentrated .
purification via flash
column chromatography ( 1:1 hexanes / etoac ) afforded 0.088 g ( 81% ) of
the coupled product as a colorless oil : [
]d22.5 + 13.3 ( c 1.98 ,
chcl3 ) ; ir ( neat , thin film ) 3429 , 3351 , 3203 ,
2962 , 2934 , 2868 , 1725 , 1665 , 1607 , 1461 , 1380 , 1366 , 1259 , 1181 ,
1119 , 1067 , 957 , 935 cm ; h nmr ( 300
mhz , cdcl3 ) 6.49 ( dt , j = 7.2 ,
14.4 hz , 1 h ) , 6.02 ( d , j = 14.4 hz , 1h ) , 5.88 ( bs ,
1 h ) , 5.46 ( bs , 1 h ) , 4.79 ( dd , j = 4.5 , 6.3 hz ,
1 h ) , 2.45 ( sextet , j = 6.9 hz , 1 h ) , 2.232.15
( m , 2 h ) , 2.122.05 ( m , 2 h ) , 1.861.69 ( m , 2 h ) , 1.611.25
( m , 6 h ) , 1.17 ( d , j = 7.2 hz , 3 h ) , 1.110.99
( m , 1 h ) , 0.910.87 ( overlapping doublet / singlet , 12 h ) ; c nmr ( 75 mhz , cdcl3 ) 176.5 , 175.8 , 145.6 ,
79.0 , 75.5 , 39.3 , 37.7 , 35.7 , 34.79 , 34.77 , 33.9 , 32.3 , 29.2 , 26.1 ,
22.8 , 21.1 , 17.5 ; hresims m / z 474.1471
[ m + na ]
( calcd for c19h34ino3na , 474.1476 ) . to the coupled product ( 0.088 g , 0.19
mmol ) in thf ( 19.0 ml )
was added cui ( 0.005 g , 0.03 mmol ) and cs2co3 ( 0.100 g , 0.30 mmol ) .
the mixture was first
degassed using nitrogen ( 10 min ) before n , n-dimethylethylenediamine ( 0.050 ml , 0.46 mmol )
was added .
the rubber septum was quickly replaced with a glass stopper ,
and the reaction mixture heated to 60 c for 19 h. after cooling
to rt , the reaction mixture was diluted with etoac , filtered through
a short plug of silica , and concentrated .
the crude product was purified
via flash column chromatography ( 8:2 7:3 1:1 hexanes / etoac )
to yield the n - h enamide intermediate ( 0.022 g ) ,
which was immediately dissolved in thf ( 0.7 ml ) and cooled to 0 c .
to this
was added nah ( 0.014 g , 0.34 mmol , 60% in mineral oil ) in
a single portion .
the reaction mixture was allowed to stir for 20
min before mei ( 0.100 ml ) was added dropwise , followed by removal
of the ice water bath . after being allowed to stir at rt for 20 min ,
the reaction mixture was again cooled to 0 c before being quenched
with h2o , extracted using etoac , dried over mgso4 , and concentrated .
the crude product was purified via flash column
chromatography ( 9:1 hexanes / etoac ) to yield 14-epi - palmyrolide a ( 0.016 g , 25% over two steps ) as a colorless oil :
[ ]d24.5 11.4 ( c 0.58 , chcl3 ) ; ir ( neat ,
thin film ) 2959 , 2927 , 2873 , 1728 , 1675 , 1646 , 1466 , 1413 ,
1384 , 1366 , 1333 , 1298 , 1240 , 1205 , 1193 , 1171,1121 , 933 cm ; h nmr ( 400 mhz , cdcl3 ) 6.63 ( d , j = 13.6 hz , 1 h ) , 4.92 ( ddd , j = 5.2 ,
8.8 , 13.6 hz , 1 h ) , 4.85 ( dd , j = 2.0 , 9.6 , 1 h ) ,
3.05 ( s , 3 h ) , 2.602.42 ( m , 3 h ) , 2.32 ( dt , j = 7.2 , 13.6 hz , 1 h ) , 2.242.17 ( m , 1 h ) , 2.031.95
( m , 1 h ) ,
1.841.74 ( m , 1 h ) , 1.681.54 ( m , 2 h ) , 1.491.30
( m , 4 h ) , 1.26 ( d , j = 7.2 hz , 3 h ) , 1.030.93
( m , 1 h ) , 0.89 ( d , j = 6.4 hz , 3 h ) , 0.86 ( s , 9 h ) ; c nmr ( 100 mhz , cdcl3 ) 175.4 , 172.8 , 129.8 ,
110.2 , 76.5 , 37.2 , 36.6 , 35.4 , 34.3 , 33.6 , 31.0 , 29.8 , 29.5 , 27.3 ,
25.9 , 24.9 , 20.0 , 19.2 ; hresims m / z 360.2504 [ m + na ] ( calcd for c20h35no3na , 360.2509 ) .
to a solution
of ( )-palmyrolidea ( 0.008 g , 0.022 mmol ) in anhydrous meoh
( 1 ml ) was added 10% palladium on carbon ( 0.005 g ) .
the atmosphere
in the flask was replaced with hydrogen , and the reaction mixture
was allowed to stir at rt for 18 h before being filtered through a
short plug of silica .
after rinsing several times with ether , the
filtrate was concentrated and purified via column chromatography ( 8:2
hexanes / etoac ) to afford compound 5 ( 0.006 g , 77% ) as
a mixture of rotational isomers ( 2:1 ratio ) : [ ]d23 + 35.6 ( c 0.48 , chcl3 ) ; ir ( neat , thin film )
2954 , 2931 , 2871 , 1720 , 1651 , 1269 , 1250 , 1073 cm ; h nmr ( 300 mhz , cdcl3 , mixture of rotational
isomers with mostly overlapping peaks , making the assignment of major
and minor components difficult ) ( major isomer ) 4.82 ( d , j = 10.0 hz , 1 h ) , 3.30 ( t , j = 5.0 hz ,
1 h ) , 2.98 ( s , 3 h ) , 2.532.65 ( m , 2 h ) , 2.212.33 ( m ,
2 h ) , 1.211.73 ( m , 14 h ) , 1.18 ( d , j = 6.9
hz , 3 h ) , 0.94 ( d , j = 5.9 hz , 3 h ) , 0.86 ( s , 9 h ) ,
( minor isomer , partial ) 4.92 ( d , j = 10.8 hz , 0.6
h ) , 2.89 ( s , 1.5 h ) , 0.91 ( d , j = 6.1 hz , 1.5 h ) ; c nmr ( 75 mhz , cdcl3 , mixture of rotational isomers )
175.5 , 174.8 , 173.5 , 77.8 , 77.2 , 48.9 , 46.3 , 40.4 , 40.0 , 38.3 ,
37.2 , 35.2 , 35.0 , 33.6 , 33.2 , 32.98 , 32.96 , 31.2 , 39.7 , 27.9 , 27.6 ,
27.0 , 26.0 , 25.9 , 25.4 , 23.5 , 23.2 , 22.2 , 20.4 , 20.1 , 18.0 , 16.5 ;
hresims m / z 362.2660 [ m + na ] ( calcd for c20h37no3na ,
362.2665 ) .
the crude product was purified via column
chromatography ( 8:2 hexanes / etoac ) to afford compound 6 ( 0.006 g , 96% ) as a mixture of rotational isomers ( 2:1 ratio ) :
[ ]d23 + 42.1 ( c 0.5 , chcl3 ) ; ir ( neat , thin
film ) 2956 , 2930 , 2872 , 1727 , 1647 , 1459 , 1192 , 1156 cm ; h nmr ( 300 mhz , cdcl3 , mixture
of rotational isomers with mostly overlapping peaks , making the assignment
of major and minor components difficult ) ( major isomer ) 4.86
( d , j = 10.5 hz , 1 h ) , 3.513.42 ( m , 1 h ) ,
3.233.14 ( m , 1 hz ) , 2.88 ( s , 3 h ) , 2.312.48 ( m , 2
h ) , 2.182.31 ( m , 2 h ) , 1.971.79 ( m , 2 h ) , 1.231.66
( m , 15 h ) , 1.19 ( d , j = 7.0 hz , 3 h ) , 0.92 ( d , j = 5.8 hz , 3 h ) , 0.86 ( s , 11 h ) , ( minor isomer , partial )
4.83 ( d , j = 11.6 hz , 0.6 h ) , 2.91 ( s , 1 h ) , 1.15
( d , j = 7.2 hz , 1.5 h ) , 0.96 ( d , j = 5.7 hz , 1.5 h ) ; c nmr ( 75 mhz , cdcl3 ,
mixture of rotational isomers ) 176.8 , 176.6 , 173.3 , 173.0 ,
77.2 , 49.1 , 46.0 , 40.6 , 40.1 , 38.0 , 37.3 , 35.2 , 34.0 , 33.9 , 33.2 ,
33.1 , 33.0 , 31.3 , 29.7 , 28.9 , 27.2 , 25.9 , 24.8 , 23.9 , 22.8 , 19.9 ,
19.8 , 19.2 , 19.1 ; hresims m / z 362.2659
[ m + na ]
( calcd for c20h37no3na , 362.2665 ) . to a solution
of 1 ( 0.007 g , 0.021 mmol ) in chcl3 ( 2 ml )
was added a 6 n aqueous hcl solution dropwise until tlc analysis showed
complete consumption of the starting material .
the reaction mixture
was then quenched with a saturated aqueous solution of nahco3 , followed by extraction using etoac .
the
resultant crude aldehyde was dissolved in anhydrous meoh ( 1 ml ) and
treated with nabh4 ( 0.015 g , 0.39 mmol ) .
the reaction mixture
was allowed to stir at rt for 1 h before being quenched with h2o , extracted using etoac , dried over mgso4 , filtered ,
and concentrated .
the crude alcohol was purified by column chromatography
( 100% etoac ) to afford alcohol 9 ( 0.006 g , 73% over two
steps ) as a colorless oil : [ ]d24.3 + 26.8 ( c 0.20 , chcl3 ) ; ir ( neat , thin film ) 3296 , 2956 , 1728 , 1651 , 1562 ,
1462 , 1366 , 1164 cm ; h nmr ( 300 mhz ,
cdcl3 ) 6.10 ( bs , 1 h ) , 4.76 ( dd , j = 2.2 , 9.5 hz , 1 h ) , 3.63 ( t , j = 6.4 hz , 2 h ) ,
2.79 ( d , j = 4.8 hz , 3 h ) , 2.392.51 ( m , 1
h ) , 2.002.23 ( m , 2 h ) , 1.91 ( bs , 1 h ) , 1.631.80 ( m ,
2 h ) , 1.221.60 ( m , 10 h ) , 1.16 ( d , j = 7.0
hz , 3 h ) , 0.87 ( d , overlapped , 3 h ) , 0.86 ( s , 9 h ) ; c
nmr ( 75 mhz , cdcl3 ) 176.8 , 174.0 , 78.6 , 62.5 , 40.2 ,
37.6 , 36.2 , 34.5 , 33.3 , 32.5 , 28.8 , 26.2 , 25.9 , 25.9 , 23.5 , 23.0 ,
20.8 , 17.4 ; hresims m / z 380.2764
[ m + na ] ( calcd for c20h39no4na , 380.2771 ) . approximately 0.3
ml of an oxidant solution prepared via the mixing of naclo2 ( 0.060 g ) and nahpo4 ( 0.040 g ) in h2o ( 1 ml )
was added to a solution of palmyrolide aldehyde ( prepared as above ,
0.006 g , 0.016 mmol ) in tert - butanol ( 0.25 ml ) and
2-methyl-2-butene ( 0.1 ml ) . the reaction mixture was allowed to stir
for 30 min before being quenched with water , extracted with etoac ,
dried over mgso4 , filtered , and concentrated .
the crude
acid was purified by column chromatography ( 100% etoac ) to afford
acid 10 ( 0.002 g , 25% ) as a colorless oil : [ ]d23 + 10.8 ( c 0.10 , chcl3 ) ; ir ( neat , thin film )
3306 , 2960 , 1727 , 1633 , 1164 cm ; h nmr ( 300 mhz , cdcl3 ) 6.04 ( bs , 1 h ) , 4.78 ( dd , j = 10.2 , 1.9 hz , 1 h ) , 2.82 ( d , j = 4.8
hz , 3 h ) , 2.48 ( q , j = 6.9 hz , 1 h ) , 2.312.41
( m , 2 h ) , 2.022.23 ( m , 3 h ) , 1.601.82 ( m , 4 h ) , 1.211.59
( m , 6 h ) , 1.19 ( d , j = 7.0 hz , 3 h ) , 0.921.05
( m , 1 h ) , 0.88 ( d , overlapped , 3 h ) , 0.87 ( s , 9 h ) ; c
nmr ( 75 mhz , cdcl3 ) 176.4 , 78.5 , 40.1 , 37.6 , 36.2 ,
34.6 , 34.4 , 33.0 , 29.7 , 28.7 , 26.3 , 26.0 , 23.0 , 22.7 , 20.7 , 17.2 ;
hresims m / z 394.2558 [ m + na ] ( calcd for c20h37no5na ,
394.2563 ) .
the crude product was purified via column chromatography
( 100% etoac ) to afford compound 12 ( 0.004 g , 94% ) : [ ]d25.5 + 30.8 ( c 0.24 , chcl3 ) ; ir ( neat , thin film )
3304 , 2950 , 1729 , 1649 , 1560 , 1462 , 1366 , 1163 , 1074 cm ; h nmr ( 300 mhz , cdcl3 ) 6.25 ( bs ,
1 h ) , 4.76 ( dd , j = 9.9 , 1.6 hz , 1 h ) , 3.553.70
( m , 2 h ) , 2.77 ( d , j = 4.8 hz , 3h ) , 2.372.50
( m , 1 h ) , 2.27 ( bs , 1 h ) , 2.002.20 ( m , 2 h ) , 1.631.83
( m , 2 h ) , 1.211.60 ( m , 10 h ) , 1.16 ( d , j =
7.0 hz , 3 h ) , 0.921.04 ( m , 1 h ) , 0.86 ( d , overlapped , 3 h ) ,
0.85 ( s , 9h ) ; c nmr ( 75 mhz , cdcl3 )
177.0 , 174.1 , 78.4 , 62.2 , 40.0 , 37.5 , 36.2 , 34.6 , 34.5 , 33.1 , 32.4 ,
28.8 , 26.1 , 25.9 , 23.6 , 23.1 , 20.7 , 17.7 ; hresims m / z 380.2764 [ m + na ] ( calcd for c20h39no4na , 380.2771 ) .
primary cultures of
cerebrocortical neurons were obtained from embryonic day 17 swiss - webster
mice as described elsewhere .
briefly ,
pregnant mice were euthanized by co2 asphyxiation , and
embryos were removed under sterile conditions .
cerebral cortices were
collected , stripped of meninges , minced by trituration with a pasteur
pipet , and treated with trypsin for 25 min at 37 c .
the cells
were then dissociated by two successive trituration and sedimentation
steps in soybean trypsin inhibitor and dnase - containing isolation
buffer , centrifuged , and resuspended in eagle s minimal essential
medium with earle s salt ( mem ) supplemented with 1 mm l - glutamine , 10% fetal bovine serum , 10% horse serum , 100 iu / ml penicillin ,
and 0.10 mg / ml streptomycin ( ph 7.4 ) .
cells were plated onto poly - l - lysine - coated 96-well ( 9 mm ) , clear - bottomed , black - well culture
plates ( midsci ) at a density of 1.5 10 cells / well .
cells were then incubated at 37 c in a 5% co2 and
95% humidity atmosphere .
the culture media was changed every other
day , starting from day 5 in vitro using a serum - free
growth medium containing neurobasal medium supplemented with b-27 ,
100 iu / ml penicillin , 0.10 mg / ml streptomycin , and 0.2 mm l - glutamine .
all animal use protocols were
approved by the institutional animal care and use committee ( iacuc )
of creighton university .
[ na]i measurement and full in situ calibration of sodium - binding benzofuran isophthalate fluorescence
ratio were performed as described previously .
cells were grown in 96-well plates and washed four times with locke s
buffer ( 8.6 mm hepes , 5.6 mm kcl , 154 mm nacl , 5.6 mm glucose , 1.0
mm mgcl2 , 2.3 mm cacl2 , 0.1 mm glycine , ph 7.4 )
using an automated microplate washer ( biotek instruments ) . after measuring
the background fluorescence of each well , cells were incubated for
1 h at 37 c with dye - loading buffer ( 100 l / well ) containing
10 m sbfi - am ( invitrogen ) and 0.02% pluronic f-127 ( invitrogen ) .
cells were washed five times with locke s buffer , leaving a
final volume of 100 l in each well .
the plate was then transferred
back to the incubator for 15 min to allow the cells to equilibrate
after washing and placed in a flexstation ii ( molecular devices ) chamber
to detect na - bound sbfi emission at 505 nm ( cells were
excited at 340 and 380 nm ) .
fluorescence readings were taken once
every 5 s for 60 s to establish the baseline , and then 50 l
of different concentrations of ( )-palmyrolide a and/or veratridine
was added to each well from the compound plate at a rate of 26 l / s ,
yielding a final volume of 200 l / well . after correcting for
background fluorescence , sbfi fluorescence ratios ( 340/380 ) and concentration
each palmyrolide a analogue was dissolved in
dmso at a stock concentration of 50 mm and stored at 20 c
until used . on the day of an experiment ,
this dilution in locke s
buffer resulted in palmyrolide a analogue solutions that were 4 times
the desired final concentration .
( )-palmyrolide
a ( 1 ) and analogues ( 2 , 7 ,
and 8) were dissolved in cdcl3 at a concentration
of 2.5 mg in 50 l and transferred to a 1.7 mm nmr tube .
the noesy mixing time was 500 ms , the hmbc delay was set to 6.25 ms
( 8 hz ) , and the hetloc used a dispi-2 spinlock of 60 ms duration .
chemical shift assignments were based on analysis of the dqf - cosy ,
hsqc , and hmbc 2d experiments using ucsf sparky .
topspin ( bruker biospin ) was used to analyze coupling constants
in the h nmr and hetloc spectra .
noesy peak heights were
converted to upper distance restraints of 3 , 4 , 5 , or 6 using
in - house perl scripts . where restraints involved a group of atoms ,
pseudoatom corrections were applied by the amber restraint generation
tools .
force field parameters were estimated using
the gaff force field and the antechamber
program in the amber 14 package .
molecular
dynamics simulations were nonperiodic , vacuum simulations with a distance - dependent
dielectric term .
using sander in the amber 14 package each starting
structure was energy minimized , then subjected to 20 ps of simulated
annealing with the nmr - derived restraints in which the temperature
was raised from 10 k to 600 k then cooled to near 0 k. the simulated
annealing was run 20 times for each structure with a different random
number seed each time .
each collection of 20 structures was sorted
by restraint violation energy , and outliers were discarded . to align
structures ,
the selected ensemble was superimposed on c17 ,
o12 , c1318 , and n19 using cpptraj . to stereospecifically assign pairs of diastereotopic hydrogens ,
pairs of noes that involved the diastereotopic atoms and had differential
intensities
were identified , then compared with the distances between
these atoms in the ensemble of selected structures .
if the ensemble
mean of one distance was more than one standard deviation from the
alternative , then the diastereotopic hydrogens were stereospecifically
assigned .
|
a small library of synthetic ( )-palmyrolide
a diastereomers ,
analogues , and acyclic precursors have been examined with respect
to their interaction with voltage - gated sodium channels ( vgscs ) . toward
this goal ,
the ability of ( )-palmyrolide a and analogues to
antagonize veratridine - stimulated na+ influx in primary
cultures of mouse cerebrocortical neurons was assessed .
we found that
synthetic ( )-palmyrolide a and its enantiomer functioned as
vgsc antagonists to block veratridine - induced sodium influx . a detailed
nmr and computational analysis of four diastereomers revealed that
none had the same combination of shape and electrostatic potential
as exhibited by natural ( )-palmyrolide a. these data indicate
that the relative configuration about the tert - butyl
and methyl substituents appears to be a prerequisite for biological
function . additional testing revealed that the enamide double bond
was not necessary for blocking veratridine - induced sodium influx ,
whereas the acyclic analogues and other macrolide diastereomers tested
were inactive as inhibitors of vgscs , suggesting that the intact macrolide
was required .
|
Results and Discussion
Conclusion
Experimental Section
|
trigeminal neuralgia ( tn ) is a disorder of cranial nerve ( cn ) v that results in severe episodes of shock - like or lancinating pain in one or more of its three divisions ( v1v3 ) .
idiopathic tn and cases due to vascular compression of cn v are categorized as classical tn .
patients diagnosed with symptomatic tn experience trigeminal - related facial pain secondary to a brain tumor , skull deformity , or multiple sclerosis ( ms ) .
evidence suggests that the majority of cases of tn are the consequence of focal compression of the entry zone of the root of the trigeminal nerve , while only 2% of cases are observed in patients diagnosed with ms .
other than excruciating facial pain , there are no other direct medical symptoms associated with tn , and the condition does not decrease life expectancy .
however , many patients with tn struggle with accomplishing tasks that affect quality of life , which is how this disorder elicits a negative impact on the social and mental wellness of the patients who suffer from this illness . following the diagnosis of tn
however , many patients experience only limited relief from medication or are unable to endure the side effects of the prescribed drugs , and in turn seek neurosurgical intervention .
currently , surgical approaches include microvascular decompression ( mvd ) or a number of techniques that target the trigeminal ganglion or root which involve the destruction or blockage of portions of those anatomical structures [ 1 , 2 ] .
although the neurosurgical modalities are preferred in many clinical situations and have proven to be effective in achieving initial pain control , they are known to come with a variety of complications , and facial pain recurrence is likely .
stereotactic radiosurgery ( srs ) has proven to be an effective management approach for patients with medically and surgically refractory tn as a primary and repeat treatment modality .
the use of radiosurgery in the treatment of tn dates back to sweden in the 1950 's , where professor lars leksell performed radiogangliotomies directed at the gasserian ganglion .
since the time of leksell , advancements in radiosurgery and imaging technologies has led to the increasing popularity of srs as a treatment option for patients with tn .
one form of srs that can be delivered to a patient is through a machine called the gamma knife ( gk ) .
the gk device is a cobalt-60-based machine , with 201 separate 4 to 18 mm collimator openings , that emits multiple gamma rays that converge on a target specified by computer planning . for specific medication intolerable patient subsets , gamma knife radiosurgery ( gkrs )
can be used as an initial management approach , or as a secondary management approach following radiosurgery or one or more of the various surgical modalities . as the evidence examining the role of gkrs in the management of patients with tn is increasing ,
it is of utmost importance for physicians to understand the criteria associated with gkrs , so that the optimal course of treatment for their patients can be prescribed . an evidence - based review on the evaluation and treatment of tn by gronseth et al . found level c evidence indicating that gasserian ganglion percutaneous techniques , gkrs , and mvd may be considered for facial pain management for medically refractory patients .
however , questions remain regarding optimal treatment modalities in specific patient subsets . for this reason ,
the goal of this paper is to provide a modern review of the literature thoroughly analyzing the efficacy of gkrs in the treatment of patients with tn , as well as evaluating the treatment planning and methods associated with this evolving modality .
to identify contemporary studies assessing the clinical outcomes of patients treated with gkrs for tn , a pubmed search from 2006 to april 2011 was performed .
keywords for search included gamma knife or gamma knife radiosurgery or stereotactic radiosurgery trigeminal neuralgia or tic douloureux .
studies analyzed in this review included retrospective cohort studies and prospective cohort studies with 5 evaluated patients .
studies published only in abstract form and studies published in a language other than english were excluded from our analysis . due to our broad search strategy and the vast amount of world literature ,
we reviewed a total of 19 studies [ 4 , 5 , 925 ] analyzing the efficacy of patients with tn who were treated once with gkrs ( table 1 ) .
thirteen of the 19 evaluated studies [ 4 , 5 , 919 ] utilized the barrow neurological institute ( bni ) pain intensity scale as a measurement of response to treatment ( see section 3 ) .
of these 13 studies , only two [ 9 , 13 ] analyzed patients treated with gkrs as an initial management approach . with a median followup of 31 months , sheehan et al .
classified 87% of patients in bni class i - iiib , while chen et al .
classified 91% of patients in bni class i - iiib ( median followup = 15 months ) .
chen et al . also reported that five of the 44 patients ( 11% ) treated with gkrs developed hypoesthesia following the procedure .
the other 11 bni pain intensity scale studies we reviewed included patients where previous surgical procedures were performed in a fraction of patients [ 4 , 5 , 1012 , 1519 ] or all patients . of the 10 studies where previous surgical procedures were performed in a fraction of patients , nine reported outcomes in terms of categorizing patients in bni class i - iiib [ 4 , 5 , 1012 , 1518 ] .
classified 58.6% of patients in bni class i - iiib ( median followup = 48 months ) , kondziolka et al .
classified 71% of patients in bni class i - iiib at three years , dhople et al .
classified 72% of patients in bni class i - iiib ( median followup = 29 months ) , han et al
. classified 76.7% of patients in bni class i - iiib ( mean followup = 58 months ) , dhople et al .
classified 81% of patients in bni class i - iiib ( median followup = 5.6 years ) , matsuda et al .
classified 82% of patients in bni class i - iiib ( median followup = 37 months ) , little et al .
classified 83% of patients in bni class i - iiib ( median followup = 6.3 years ) , dellaretti et al .
classified 89.5% of patients in bni class i - iiib ( mean followup = 20.3 months ) , and park and hwang classified 94% of patients in bni class i - iiib with a minimum followup of 3 years .
reported clinical outcomes with respect to bni class i , which contained only 5.7% of patients .
the study that evaluated gkrs where previous surgical procedures were performed in 100% of patients classified 85% of patients in bni class i - iiib , with a median followup of 36 months .
we also reviewed two studies that used the excellent - good - fair - poor ( egfp ) categorical scale to assess patient outcomes [ 20 , 21 ] ( see section 3 ) .
treated 30 patients with tn with gkrs at iran gamma knife center between 2006 and 2007 .
the authors reported that 40% of patients had an excellent outcome , 10% of patients had a good outcome , 33% of patients had a fair outcome , and 17% of patients had a poor outcome following the procedure .
after surgery , 15 patients ( 54% ) reported an excellent outcome , one patient ( 4% ) reported a good outcome , two patients ( 7% ) reported a fair outcome , and 10 patients ( 36% ) reported a poor outcome .
the complications from mvd included facial numbness in six patients ( 21% ) , dysesthesias in three patients ( 11% ) , and delayed facial palsy in one patient ( 4% ) .
four of the 19 studies we evaluated [ 2225 ] used other methodologies in determining the effectiveness of gkrs . in a prospective controlled trial ,
rgis et al . analyzed 100 patients with tn treated with gkrs and reported that 83 patients ( 83% ) were completely pain free , 58 of which ( 58% ) discontinued all medication following the procedure ( minimum followup = 12 months ) .
ten patients ( 10% ) experienced radiation - induced complications , which included facial paresthesia or hypesthesia .
performed a study investigating the short - term efficacy of gkrs in 67 patients with medically refractory tn .
overall , 77.6% of patients witnessed some degree of pain relief , with 32.6% of those patients becoming completely pain free .
of the 67 patients , 10 ( 14.9% ) experienced complications from the procedure , which included hypoesthesia and paresthesia .
sixty eight patients ( 42.5% ) underwent prior invasive treatments . in clinical analysis , it was found that 61% of patients were pain free without medication , 29% of patients were pain free with medication , and 10% of patients did not respond to gkrs .
thirty eight patients ( 49.4% ) exhibited some level of pain improvement following gk treatment , with 23 of those patients ( 29.9% ) reporting a pain - free outcome .
twelve patients ( 15.6% ) experienced complications , which were reported to be mild facial sensory changes and mild facial nerve dysfunction .
as gkrs has proven to be an effective initial treatment for tn , numerous reports have been published analyzing patients treated on multiple occasions ( > 1 ) with gkrs .
we reviewed six studies evaluating patients treated more than once with gkrs [ 2732 ] ( table 2 ) .
of these six articles , two [ 29 , 32 ] utilized the bni pain intensity scale .
sixteen patients ( 76.2% ) exhibited compelling improvements and were placed in bni class i - ii .
huang et al . analyzed 65 medically refractory patients with tn who were treated with gkrs as a second treatment modality .
of these 65 patients , 30 ( 46% ) had undergone gkrs as an initial management approach .
the authors placed 22 patients ( 34% ) in bni class i , 11 patients ( 17% ) in bni class ii , four patients ( 6% ) in bni class iiia , and five patients ( 8% ) in bni class iiib .
overall , with a median followup of 64 months , 65% of patients reported successful results in terms of pain control rates . a total of three of the six reviewed studies evaluated patients using the egfp categorical scale [ 28 , 30 , 31 ] .
analyzed 37 patients treated a second time with gkrs for recurrent tn and reported that 17 patients ( 46% ) achieved excellent pain relief , nine patients ( 24% ) achieved good pain relief , five patients ( 14% ) achieved fair pain relief , and six patients ( 16% ) achieved poor pain relief .
however , the authors concluded that 57% of patients experienced some form of trigeminal dysfunction following repeat radiosurgery .
, huang et al . treated 28 patients with repeat gkrs and reported that 12 patients ( 43% ) exhibited excellent pain relief , five patients ( 18% ) exhibited good pain relief , and two patients ( 7% ) exhibited fair pain relief .
in addition , the authors found a statistically significant ( p = 0.047 ) correlation between cumulative radiation doses > 115 gy and facial numbness . in a separate study , huang et al .
specifically , a total of eight patients underwent mvd a mean of 7.6 months following repeat gkrs .
of the eight patients , seven ( 87.5% ) were completely pain free at a mean of 21 months following neurosurgery .
this data supports the use of mvd if multiple gk procedures are deemed ineffective .
kimball et al . treated 53 patients with repeat gkrs and analyzed the patients not lost during followup using the marseille scale , which categorizes patients into one of five classes , with a higher class statistically indicating a worse prognosis for the patient . with a mean
followup of 42 months , 20 patients ( 43.5% ) were categorized in marseille class i - ii , six patients ( 13% ) were categorized in marseille class iii - iv , and 20 patients ( 43.5% ) were categorized in marseille class v. the authors also reported a statistically significant ( p = 0.047 ) correlation between facial numbness and superior long - term pain relief .
a total of 22 patients ( 48% ) experienced trigeminal dysfunction of any kind , while 21 patients ( 46% ) experienced numbness in the face .
since gkrs can be performed as both initial and salvage treatment options for patients who suffer from tn , its efficacy has been compared in patients who undergo one versus multiple radiosurgery procedures .
we reviewed eight studies to further examine this matter [ 3 , 3339 ] ( table 3 ) .
four of the eight studies utilized the bni pain intensity scale to evaluate patient outcomes [ 3 , 3335 ] .
, it was reported that 75% , 60% , and 58% of patients with idiopathic tn had bni scores of i iiib at 1 , 3 , and 5 years , respectively .
the 1- , 3- , and 5-year - bni scores of i iiib in patients with ms - related tn were 56% , 30% , and 20% , respectively .
the authors concluded that repeat gkrs exhibited similar success rates when compared to the initial procedure .
similar to verheul et al . , park et al . did not find differences in terms of time to initial response , time to pain recurrence , and overall pain relief when comparing patients who underwent one versus two gk treatments .
however , it was observed that patients who received two gk treatments were more likely to have facial sensory changes when compared to patients treated a single time with radiosurgery .
little et al . performed a study where 79 patients with typical tn were treated with gkrs as a salvage procedure .
approximately five years following salvage gkrs , the authors reported that 50% of patients experienced pain relief and 20% of those patients were completely pain free .
in addition , a statistically significant ( p = 0.029 ) correlation between gkrs failure and prior mvd was found .
treated 37 patients ( 78% had failed prior surgery ) with ms - related tn with gkrs .
nine patients ( 24% ) underwent gkrs as their first procedure . the reported 1 , 3 , and 5 year bni scores of i iiib were 82.6% , 73.9% , and 54% , respectively . the other four studies we reviewed utilized the egfp categorical scale as a measurement of response to treatment [ 3639 ] .
two of the evaluated studies [ 36 , 37 ] were conducted by fountas et al . and analyzed patients treated with gkrs for idiopathic tn based on whether or not they had undergone previous surgical or radiosurgical procedures for facial pain control .
one of the studies evaluated 106 patients ( 19 previous radiosurgery procedures ) and concluded that the treatment group without a previous history of surgical or radiosurgical procedures exhibited superior clinical outcomes , with 1-year and 2-year complete pain relief rates of 82.5% and 78% , respectively .
the 1-year and 2-year complete pain relief rate in the patient group with a history of surgical or radiosurgical procedures was 69.4% and 63.5% , respectively .
as expected , similar results were found in the other study by fountas et al .
; however , no prior radiosurgical procedures were performed in the patient group with a history of prior procedures .
huang et al . conducted a study where 89 patients with idiopathic tn were treated with gkrs as an initial management approach , 20 of which underwent a subsequent gkrs procedure for facial pain recurrence . following the initial radiosurgical procedure ,
50 patients ( 56% ) had an excellent response , 12 patients ( 13.5% ) had a good response , and 7 patients ( 7.9% ) had a fair response .
following the second radiosurgical procedure , 11 patients ( 55% ) had an excellent response and one patient ( 5% ) had a good response . in a separate study , huang et al .
assessed 21 patients with benign tumor - related tn who were treated with gkrs as an initial or repeat procedure .
following the initial gk procedure to the tumor , 12 patients ( 57% ) had an excellent response and 1 patient ( 5% ) had a good response .
a total of eight patients were treated with a subsequent gkrs procedure targeted at the ipsilateral trigeminal root or ganglion due to facial pain recurrence . following the second radiosurgical procedure , the authors reported four patients ( 50% ) with an excellent response .
we identified six studies comparing patients treated with gkrs with patients treated with one of the various surgical modalities [ 2 , 4044 ] ( table 4 ) .
the authors of this review acknowledge the importance of percutaneous techniques in the management of tn ; however , our modern literature search predominantly yielded comparison studies analyzing the efficacy of mvd when compared to gkrs .
specifically , four of the six studies [ 2 , 4042 ] analyzed patients treated with gkrs against patients treated with mvd .
specifically , 36 were treated with mvd ( 45% ) , while 44 were treated with gkrs ( 55% ) .
the mvd treatment arm statistically differed from the gkrs treatment arm with respect to age ( median of 54 versus 74 years ) , preoperative symptom duration ( median of 2.6 versus 7.5 years ) , and the presence of major comorbidities ( 2.8 versus 58.3% ) .
the authors reported that patients treated with mvd exhibited superior levels of initial ( 100% ) , 2 year ( 88% ) , and 5 year ( 80% ) actuarial pain - free rates when compared to the patients treated with gkrs ( 78 , 50 , and 33% , resp . ) , with a p value of 0.0002 .
in addition to increased levels of patient satisfaction , as reported by required patient surveys , the mvd treatment group also had a decreased level of permanent mild ( 5.6% ) and severe sensory loss ( 0% ) when compared to the gkrs treatment group ( 6.8% and 2.3% , resp . ) .
two patients ( 5.6% ) in the mvd group experienced permanent mild paresthesias or numbness , one ( 2.8% ) patient experienced a cerebrospinal fluid leak from the wound , and one patient ( 2.8% ) experienced hearing loss and diplopia .
three patients ( 6.8% ) in the gkrs group experienced permanent mild paresthesias or numbness , one patient ( 2.3% ) experienced a more permanent sensory numbness , and one patient ( 2.3% ) experienced a transient headache and nausea following the gk procedure .
all patients were diagnosed with typical tn and did not undergo previous gk or mvd procedures . it was reported that patients treated with mvd exhibited superior levels of complete pain relief at 12 ( 68% ) and 18 months ( 68% ) when compared to the gkrs group , who 's complete pain relief rate was 58% at 12 months and 24% at 18 months ( p = 0.089 ) .
the treatment arms did not statistically differ in terms of 90% pain relief at 12 and 18 months .
this study could be criticized due to the large difference in the number of patients constituting the two treatment arms .
oh et al . evaluated a total of 45 elderly patients ( > 65 years of age ) diagnosed with idiopathic tn who were treated with either mvd ( 27 patients ) or gkrs ( 18 patients ) .
it was reported that three mvd patients ( 11% ) and three gk patients ( 17% ) underwent previous percutaneous procedures .
the mean followup period was reported to be 35.9 months for the mvd group and 33.1 months for the gkrs group . according to the bni pain intensity scale ,
the mvd group had a superior prognosis , with 17 patients ( 63% ) classified in bni class i - ii compared with the 10 patients ( 56% ) in the gkrs group classified in bni class i - ii .
the observed complications following mvd included constant headache in 11 patients ( 40.7% ) , facial paresthesia in five patients ( 18.5% ) , paresthesia of the tongue in two patients ( 7.4% ) , infection at the site of incision in one patient ( 3.7% ) , an acute subdural hemorrhage in one patient ( 3.7% ) , temporary hearing loss in one patient ( 3.7% ) , and otitis media with cerebrospinal leakage in one patient ( 3.7% ) . two patients ( 11% ) in the gkrs group experienced paresthesia .
compared the clinical outcomes of 19 patients treated with mvd with 15 patients treated with gkrs .
nine gk patients ( 60% ) and four mvd patients ( 21% ) underwent previous surgical procedures . the treatment
arms statistically differed ( p = 0.0005 ) with respect to mean patient age , with the mean age of the gkrs group exceeding the mvd group by 13 years ( 74 versus 61 years ) .
in addition , patient satisfaction was graded on a scale of 1 ( unsatisfied ) to 10 ( completely satisfied ) .
it was reported that the mean tn complexity grade was statistically different ( p < 0.001 ) between the treatment arms ( gk = 5.8 ; mvd = 3 ) .
the average response following the procedure was determined to be 3.4 for the mvd group and 2.4 for the gkrs group ( p = 0.017 ) . also , it was found that the satisfaction score for the mvd group was superior to the gkrs group ( 8.7 versus 6.4 ) , with a p value of 0.02 .
the authors reported a statistically significant correlation between tn complexity grade and clinical response ( p < 0.001 ) , as well as tn complexity grade and patient satisfaction ( p < 0.001 ) . to date
, no randomized trials have been conducted analyzing the outcomes of patients with tn who are treated with mvd compared to gkrs . in a large review on tn management ,
zakrzewska and linskey found evidence that mvd is an effective treatment for long - term facial pain relief but comes with an increased risk of ipsilateral hearing loss .
in addition , the authors concluded that single - dose srs is an effective treatment for long - term facial pain relief but puts patients at risk for facial numbness or facial paresthesias .
investigation into this matter in the form of a randomized controlled trial would provide the best evidence in terms of facial pain relief and procedure - related complications .
in addition , we reviewed two studies comparing patients treated with gkrs with patients treated with posterior fossa exploration ( pfe ) [ 43 , 44 ] , both of which were conducted by pollock and colleagues at the mayo clinic college of medicine .
one of the studies was a specific prospective comparison of 91 patients treated with pfe and 49 patients treated with gkrs for idiopathic tn as an initial management approach .
the treatment arms statistically differed in terms of age ( gkrs = 67.1 years ; pfe = 58.2 years ) , with a p value < 0.001 .
it was reported that patients treated with pfe were more likely to be pain free and off medications at 1 year ( 84% ) and 4 years ( 77% ) when compared to the gkrs group ( 66 and 56% , resp . )
retreatment for recurrent facial pain was performed in 15% of the patients in the pfe treatment arm and 35% of patients in the gkrs treatment arm ( p = 0.009 ) .
also , it was found that nonbothersome facial numbness occurred more frequently in the gkrs group ( p = 0.04 ) .
an additional study from the mayo clinic evaluated patients with recurrent tn who underwent 3 or more surgical procedures .
the authors reported that patients treated with pfe exhibited superior levels of complete pain relief at 3 years of followup when compared to patients treated with gkrs , balloon compression , and glycerol rhizotomy ( p < 0.01 ) and underwent additional surgery for recurrent facial pain less often when compared to patients treated with the other modalities ( p = 0.02 ) .
clinical 006futcomes did not differ between patients treated with gkrs and patients treated with the percutaneous techniques .
srs can be performed by a variety of tools , which include gkrs , cyberknife technology , and linear accelerator ( linac)-based treatment .
our analysis yielded one study whose primary endpoint was to devise a method using cyberknife treatment planning that would mimic the dosimetric characteristics of the gk treatment plan in five patients undergoing radiosurgery for tn .
both the isodose lines and critical structures were identified using the gkrs treatment plan and were transferred to the cyberknife treatment planning system .
it was reported that the average length of the trigeminal nerve receiving a dose of 60 gy was 4.5 mm for the gk , 4.5 mm for the nonisocentric cyberknife , and 4.4 mm for the isocentric cyberknife .
the authors found it more difficult to minimize the dose to critical structures when using cyberknife technology .
also , the dose falloff of gkrs was found to be steeper when compared to cyberknife technology due to , what the authors hypothesized , the large number of gamma rays produced which converge on the focal point with precision .
as previously mentioned , the gk machine 's primary functional unit is cobalt-60 , which is used to emit photon energy through 201 separate 4 to 18 mm collimator openings that converge on a target specified by a treatment planning system .
balamucki et al . performed a study examining if the half life of cobalt ( 5.26 years ) relates to the outcomes for patients being treated for tn with gkrs .
the authors collected data on 239 gkrs procedures performed at their institution between 1999 and 2004 .
patient surveys were used to measure responses to radiosurgery . with the followup time ranging from one to six months , it was reported that 80% of patients experienced some degree of pain relief and that 56% of those patients were pain free .
the authors concluded that clinical outcomes remained consistent during the first half life of cobalt-60 .
an area of controversy in the treatment of patients with tn is defining the optimal maximum radiosurgery dose that can be delivered to specific patient subsets .
we analyzed five studies whose primary endpoint was to assess gkrs - dosing efficacy [ 4852 ] .
kim et al . utilized the bni pain intensity scale to assess 66 tn patients treated with a gk maximum dose of 80 gy and 44 tn patients treated with a gk dose of 85 gy . although the two groups did not statistically differ in terms of facial rain relief and procedure - related complications , the authors did report that patients treated with a gk dose of 85 gy experienced a more rapid response to treatment when compared to the patients treated with a gk dose of 80 gy .
arai et al . analyzed 165 patients with tn treated with a gkrs dose of 80 gy .
specifically , the authors divided the patients into two groups , which differed in the radiation dose rate received ( low - dose rate = 1.212.05 gy / min ; high - dose rate = 2.063.74 gy / min ) . using the bni pain intensity scale as a clinical evaluation method
, it was reported that the low - dose - rate group and the high - dose - rate group did not statistically differ in terms of initial pain relief , maintenance of pain relief , and clinical complications .
patients in group one were treated with a gk dose < 90 gy with no beam channel plugging , patients in group two were treated with a gk dose equal to 90 gy with no beam channel plugging , and patients in group three were treated with a gk dose equal to 90 gy with beam channel plugging . although the trend did not reach full statistical significance ( p = 0.054 ) , patients in group three exhibited the highest level of pain relief , while patients in group one exhibited the lowest level of pain relief .
the authors also observed that the three groups statistically differed ( p < 0.0001 ) in terms of trigeminal nerve dysfunction , with patients in group three experiencing the highest rate of mild and bothersome complications and patients in group one experiencing the lowest rate of mild and bothersome complications .
similar to the results of massager et al . , morbidini - gaffney et al . reported positive outcomes in patients treated with gk doses > 85 gy .
the authors also found that patients treated with two isocenters were more likely to have superior bni pain intensity scale scores during their course of followup when compared to patients treated with a single isocenter .
the median initial dose was 80 gy , and the median retreatment dose was 45 gy . although the authors did not report any predictors in terms of facial pain control and patient morbidity , they did compare the results of their study with seven published retreatment articles and found that successful levels of pain control ( > 50% ) were significantly correlated with cumulative gkrs doses > 130 gy , as well as new trigeminal nerve dysfunction ( > 20% ) . in addition to dose selection efficacy in select patient cohorts , the radiosurgical target of cn v is another subject matter that requires further clinical investigation .
we reviewed three studies [ 5355 ] analyzing specific gkrs targeting methods in the treatment of tn and one study that examined the accuracy of gkrs to its image - guided target .
compared patients treated with gkrs targeted at the dorsal root entry zone ( 59 patients ) with patients whose radiosurgical target was the retrogasserian zone of the trigeminal nerve ( 41 patients ) . with a median followup of 30 months , the dorsal root entry target group exhibited statistically superior levels of initial complete pain remission ( p = 0.003 ) and experienced less complications than the retrogasserian zone group ( p = 0.009 ) .
chen et al . also reported positive results with the dorsal root entry zone - targeting approach , with a success rate of 82.8% and a complication rate of 15% .
park et al . compared the dorsal root entry zone and retrogasserian zone - targeting methods in the treatment of 39 patients with medically refractory tn .
the authors reported that the two treatment arms did not statistically differ in treatment success ( bni class i - iiib ) with respect to the bni pain intensity scale .
however , patients treated with the retrogasserian zone - targeting method experienced a substantially shorter time of response to gkrs than patients treated with the dorsal root entry zone - targeting method ( p = 0.044 ) .
although the two groups did not statistically differ with regard to treatment - related morbidities , it was found that the patients whose targeting approach was the dorsal root entry zone experienced a greater amount of bothersome complications than the retrogasserian zone group .
massager et al . analyzed the targeting accuracy of gkrs in 65 patients treated for tn whose six month followup mri showed focal contrast enhancement of the trigeminal nerve .
the authors found that the median deviation of the coordinates between the intended radiosurgical target and the center of contrast enhancement was 0.91 mm in euclidean space .
the median radiation dose fitting into the contrast enhancement region was determined to be 77 8.7 gy .
this small deviation from the gkrs target explains the high accuracy and precise nature of the machine .
the two most common methods of measuring patient outcomes from gkrs in the management of tn are the barrow neurological institute pain intensity scale ( table 5 ) and the excellent - good - fair - poor ( egfp ) categorical scale ( table 6 ) .
the bni pain intensity scale divides patients into one of five classes , with a higher class indicating a worse prognosis for the patient .
patients in bni class iiia do not experience trigeminal pain but require the use of medication .
patients in bni class iiib experience some trigeminal pain that can be satisfactorily managed with medication . patients in bni class iv experience some trigeminal pain that is not satisfactorily managed with medication . patients in bni class
excellent outcomes are defined as complete pain relief without the need of medication .
good outcomes are defined as complete pain relief with the need of medication .
poor outcomes are defined as a < 50% pain relief rate or treatment failure .
for patients with medically refractory forms of tn , gkrs has proven to be an effective initial and repeat treatment option .
cumulative research suggests that patients treated a single time with gkrs exhibit similar levels of facial pain control when compared to patients treated multiple times with gkrs .
however , patients treated on multiple occasions with gkrs are more likely to experience facial numbness and other facial sensory changes when compared to patients treated once with gkrs .
although numerous articles have reported mvd to be superior to gkrs in achieving facial pain relief , the findings of these comparison studies are weakened by the vast differences in patient age and comorbidities between the two studied groups and can not be considered conclusive .
further evidence in the form of a phase iii - randomized trial is needed to confirm the clinical outcomes of patients treated with either modality .
questions remain regarding optimal gkrs dosing and targeting strategies , which warrants further investigation into this controversial matter .
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since its introduction by leksell , gamma knife radiosurgery ( gkrs ) has become increasingly popular as a management approach for patients diagnosed with trigeminal neuralgia ( tn ) .
for this reason , we performed a modern review of the literature analyzing the efficacy of gkrs in the treatment of patients who suffer from tn . for patients with medically refractory forms of the condition ,
gkrs has proven to be an effective initial and repeat treatment option .
cumulative research suggests that patients treated a single time with gkrs exhibit similar levels of facial pain control when compared to patients treated multiple times with gkrs .
however , patients treated on multiple occasions with gkrs are more likely to experience facial numbness and other facial sensory changes when compared to patients treated once with gkrs .
although numerous articles have reported mvd to be superior to gkrs in achieving facial pain relief , the findings of these comparison studies are weakened by the vast differences in patient age and comorbidities between the two studied groups and can not be considered conclusive .
questions remain regarding optimal gkrs dosing and targeting strategies , which warrants further investigation into this controversial matter .
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1. Introduction
2. Review of Gamma Knife Radiosurgery for Trigeminal Neuralgia
3. Treatment Planning and Methods
4. Conclusions
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drywood termites cause significant damage to wood in structures in the united states , with two species , incisitermes minor ( hagen ) and cryptotermes brevis ( walker ) , being responsible for the majority of damage ( su and scheffrahn , 1990 ; grace , 2009 ) .
the economic cost of control and repair of damage is second only to that for subterranean termites ( su and scheffrahn , 1990 ) .
remedial control of drywood termites in the united states relies primarily on either ( 1 ) fumigation of the entire structure with a toxic gas or heated air , or ( 2 ) localized chemical or physical treatments designed to eradicate small , localized colonies ( lewis and haverty , 1996 ; lewis , 2008 ; lewis and rust , 2009 ) .
fumigation treatments are likely to kill all of the termites in a structure , while localized treatments can destroy all termites in a colony , provided the treatment is delivered to the entire gallery system .
the size , number , and dispersion of drywood termite colonies in a structure can vary greatly , depending on the age of the infestation and the success of preventative or remedial treatments .
drywood termite colonies also are considered single piece nesters , i.e. , they nest within their food source and do not forage from the nesting site to a food source ( abe , 1987 ) .
in fact , large , dispersed colonies ( or even small colonies ) can live in a single piece of wood or in multiple pieces of wood that are joined together ( grace et al . ,
drywood termites are cryptic , seldom leaving obvious or visible external signs of their presence in wood . a commonly used sign for determining the presence of drywood termites is the occurrence of fecal pellets , ejected through a kick - out hole in the external surface of wood from the internal galleries ( ebeling , 1975 ) .
these pellets often are found as conical piles or are scattered on horizontal surfaces below infested wood .
these hexagonally sided pellets are diagnostic for drywood termites , and can be used to distinguish damage from that by other wood - destroying insects ( ebeling , 1975 ; moore , 1992 ) .
grace and yamamoto ( 2009 ) demonstrated the relationship between the cellulose and lignin content of the food utilized by small groups of c. brevis and incisitermes immigrans ( light ) , and the quantity of fecal pellets produced over time .
they also discussed the use of the size and number of fecal pellets for estimating both size and age of drywood termite colonies .
drywood termites excrete feces in the form of hard , even - shaped fecal pellets .
these fecal pellets contain the same mixture of hydrocarbons as the insects that produced them , albeit in slightly different proportions ( haverty et al .
, 2005 ) . because cuticular hydrocarbons are species specific in termites ( page et al . , 2002 )
rather than simply signaling the general presence of termites or providing a diagnosis of the species of termite inhabiting the wood , we postulated that these pellets could be chemically characterized so as to determine the status of a colony as active ( alive ) or inactive ( dead ) . here , we report quantification of the hydrocarbons in pellets of i. minor aged for up to 1 year after they were produced .
we document the changes in proportions of selected hydrocarbons as an indication of the length of time since the pellets were excreted .
collection of termites and preparation of termite containment unit termites , i. minor , were removed from one naturally infested board ( 98.9 13.3 271.8 cm ) collected on 19 july 2006 from lakeview , california , and stored at the university of california richmond field station .
the board was cut across the grain into pieces , 58 cm thick , and stored at room temperature .
a wood chisel was used to separate pieces of wood into smaller pieces , 0.51.0 cm thick .
all remaining live termites , including soldiers and primary reproductives ( alates ) , were placed in a termite containment unit ( tcu ) .
termites thus collected were likely from a large mixed colony ( booth et al . , 2010 ) or from multiple colonies .
we were not able to assign the various galleries in the wood to any particular colony.fifteen birch tongue depressors were bundled together , and three equally spaced holes drilled across the length of the bundle using a 2.8-mm bit .
bamboo skewers , 1013 cm in length , were inserted into each of the three holes in the bundle of tongue depressors .
spaces , 36 mm , were left between each tongue depressor allowing termites access to all tongue depressors .
two sets of skewered tongue depressors were placed into a clean plastic container ( 17.5 12.5 6 cm ) , one on top of the other , such that the tongue depressors on the top layer fit into the gaps created by the tongue depressors on the bottom layer .
tongue depressors were lightly misted with water , and then 6,662 drywood termites ( a mixture of pseudergates or workers , alates , and soldiers ) were placed in the tcu .
tcus , with termites , were maintained in a dark cabinet in the laboratory under ambient conditions prior to collection of fecal pellets .
a single colony or a mixture of two or more colonies , representing a single location within california , was prepared in this manner . pellet collection and aging process
after all the tcus had acclimated to laboratory conditions over several weeks , new holding chambers were prepared .
all wood debris , pellets , dead termites , and damaged tongue depressors were discarded .
the termites in the new holding chambers were maintained in the laboratory under ambient conditions for 2 weeks . at the end of this period
, all pellets were removed , and sub - samples collected and readied for the aging study .
concurrently , three samples of 20 workers , three samples of 20 alates , and one sample of 20 soldiers were also collected for hydrocarbon analysis .
live termites were placed in a 20-ml scintillation vial and frozen until extraction.fecal pellets were separated from debris by sequentially sifting them through successively smaller sieves ( haverty et al . , 2005 ) .
thus , all substances such as hand lotion , lip balm , parafilm , and waxed paper were kept away from the work area .
gloves were worn when handling pellets , and implements that came in contact with pellets were wiped with a paper towel dampened with absolute ethanol to remove any oils or waxes .
pellets were separated from fine debris by removing individual pellets with forceps or with an aspirator , until a sample weighing approximately 200 mg was obtained.in developing this protocol we made one estimate and one critical assumption .
the estimate was that at least 1,000 pellets were needed for each hydrocarbon analysis ( based on preliminary analyses of pellets collected from drywood termites maintained in our laboratory ) .
( 1997 ) using c. brevis and i. snyderi ( light ) from southern florida . in that study , 40 i. snyderi ( pseudergates or workers ) produced 573 pellets over 4 weeks , slightly less than 150 pellets / wk or about 3.5 pellets / individual / wk . in a study with small groups of c. brevis and i. immigrans , grace and yamamoto ( 2009 ) found that these species produced 4.9 to 7.0 pellets / individual / wk . for our study , we assumed that i. minor would produce pellets at roughly the same rate and , therefore , we needed about 3,000 to 5,000 individuals in a tcu for this study .
we prepared one tcu to collect the requisite quantity of fecal pellets.four sub - sampling intervals , each replicated three times , were used : 0 , 30 , 90 , and 365 days from the initial collection date .
sub - samples were stored in clean , 20-ml scintillation vials , sealed with 1.5 1.5 mm mesh screen .
vials were stored in a dark cabinet at the university of california richmond field station at ambient temperature.voucher samples of i. minor pseudergates or workers and soldiers from this study ( fresh , not dried ) were preserved in 85% ethanol and deposited in the essig museum , university of california at berkeley ( haverty et al . ,
extraction procedure and characterization of hydrocarbons hydrocarbons from workers , alates , soldiers , and fecal pellets of i. minor were extracted , characterized , and quantified as previously reported ( haverty et al . , 2005 ) .
frozen termite samples were thawed and dried at 70c for approximately 1 h before extraction . each sub - sample of fecal pellets or termites
was placed in a 20-ml scintillation vial , and immersed in 10 ml of n - hexane for 10 min . after extraction
, hydrocarbons were separated from other compounds through 4 cm of activated sigma silica gel ( 70230 mesh ) in pasteur pipette mini - columns .
the resulting hydrocarbon fractions were evaporated to dryness under a stream of nitrogen and redissolved in 60 l of n - hexane for gas chromatography - mass spectrometry ( gc - ms ) analysis .
a 3-l aliquot was injected into the gc-ms.gc-ms analyses were performed on an agilent 6890 gas chromatograph interfaced with an agilent 5973 mass selective detector , using agilent chemstation data analysis software ( g1701ca version c.00.00 ) .
the gc - ms was equipped with an hp-1ms , fused silica capillary column ( 30 m 0.25 mm i.d , 0.25 m film thickness ) , and was operated in split mode ( split ratio of 30:1 ) , using helium as carrier gas .
the column oven was programmed from 200320c at 3c min , with a final hold of 11 min .
electron impact ( ei ) mass spectra were obtained at 70 ev.all chemicals of interest were identified by their retention times and mass spectra .
mass spectra of methyl - branched alkanes were interpreted as described by blomquist et al .
olefins were identified by their mass spectra , although double bond positions were not determined ( haverty et al . , 2005).in the text , tables , and figures , we use shorthand nomenclature to identify individual hydrocarbons or mixtures of hydrocarbons .
this shorthand uses a descriptor for the location of methyl groups ( x - me ) , the total number of carbons ( cxx ) in the hydrocarbon component excluding the methyl branch(es ) , and the number of double bonds following a colon ( cxx : y ) .
thus , pentacosane is represented as n - c25 , 11-methylnonacosane as 11-mec29 , 13,17-dimethylhentriacontane as 13,17-dimec31 , and heptatriacontatriene as c37:3 .
hydrocarbons are presented in the tables for each caste and worker fecal pellets in the order of elution from our gc - ms system .
statistical analysis gc - ms peak ( some peaks contained more than one compound or a mixture of positional isomers ) areas were converted to percentages of total hydrocarbon fraction , enabling mean ( sd ) relative amounts of each hydrocarbon peak for workers and alates , and overall mean ( sd ) of each hydrocarbon peak for the fecal pellets , to be calculated.the percentage of each hydrocarbon peak for pellets of each aging period was transformed to the log of the percentage ( dependent variable y ) and regressed against the days of aging ( independent variable x ) .
hydrocarbons with slopes different from 0 ( = 0.05 ) were separated into two groups : those with a significant positive slope and those with a significant negative slope . for each aging period ( 0 , 30 , 90 , and 365 day ) , an index of age ( iage ) was created by subtracting the sum of the percentages of the hydrocarbons with a negative slope over time from the sum of the percentages of the hydrocarbons with a positive slope over time [ iage = ( peakpositive ) minus ( peaknegative ) , for each aging period ] .
the iage for each aging period then was regressed against the aging period ( r development core team , 2004 ) .
the cuticular hydrocarbons for i. minor pseudergates and fecal pellets were characterized previously ( haverty et al . , 2000 , 2005 ) .
the composition of the hydrocarbon mixture for this species and the hydrocarbons from their fecal pellets in the present study are displayed in fig .
1total ion chromatogram of cuticular hydrocarbons from a workers of incisitermes minor ( hagen ) and b from fecal pellets from the same collection .
acronyms for hydrocarbons are derived as follows : x - me location of methyl groups , the total number of carbons cxx in the hydrocarbon component excluding the methyl branch(es ) , and cxx : y the number of double bonds following a colontable 1relative abundance of hydrocarbons from pseudergates , alates , soldiers , and fecal pellets of incisitermes minor ( hagen)hydrocarbonspseudergates n = 3alates n = 3soldiers n = 1fecal pellets n = 122-mec220.06 ( 0.02)0.08 ( 0.02)0.090.11 ( 0.02)n - c231.30 ( 0.06)1.34 ( 0.05)1.481.35 ( 0.18)2-mec231.45 ( 0.40)1.72 ( 0.35)2.112.13 ( 0.28)3-mec231.31 ( 0.38)1.38 ( 0.30)1.712.06 ( 0.29)n - c240.46 ( 0.01)0.43 ( 0.02)0.480.32 ( 0.05)2-mec240.13 ( 0.03)0.19 ( 0.03)0.190.15 ( 0.02)3-mec240.08 ( 0.02)0.10 ( 0.02)0.120.11 ( 0.02)n - c2516.35 ( 0.16)11.75 ( 0.40)12.648.65 ( 1.05)2-mec250.18 ( 0.01)0.27 ( 0.01)0.240.14 ( 0.02)3-mec250.23 ( 0.04)0.36 ( 0.04)0.340.26 ( 0.03)n - c262.25 ( 0.15)1.17 ( 0.07)1.510.87 ( 0.12)2-mec260.14 ( 0.01)0.13 ( 0.01)0.140.07 ( 0.01)n - c2714.04 ( 0.55)6.81 ( 0.31)10.055.91 ( 0.65)2-mec270.13 ( 0.01)0.09 ( 0.01)0.130.06 ( 0.01)3-mec270.20 ( 0.01)0.19 ( 0.01)0.250.12 ( 0.01)n - c280.43 ( 0.05)0.17 ( 0.01)0.350.15 ( 0.03)n - c293.38 ( 0.33)1.27 ( 0.07)2.971.38 ( 0.18)11-mec290.06 ( 0.01)0.15 ( 0.01)0.100.09 ( 0.01)9,13-dimec290.70 ( 0.02)0.82 ( 0.01)0.910.63 ( 0.06)9,13,17-trimec290.11 ( 0.02)0.27 ( 0.00)0.160.22 ( 0.03)n - c300.04 ( 0.00)0.07 ( 0.00)0.100.0013-mec300.01 ( 0.02)0.08 ( 0.00)0.040.0010,14-dimec300.64 ( 0.06)1.45 ( 0.02)0.880.90 ( 0.03)10,14,18-trimec300.18 ( 0.02)0.43 ( 0.01)0.240.28 ( 0.02)n - c310.14 ( 0.01)0.10 ( 0.01)0.200.09 ( 0.01)15- ; 13- ; 11- ; 9-mec310.36 ( 0.03)0.82 ( 0.01)0.420.49 ( 0.02)13,17- ; 11,15-dimec317.45 ( 0.68)13.74 ( 0.52)8.5510.67 ( 1.80)9,13,17-trimec310.57 ( 0.05)1.19 ( 0.02)0.720.89 ( 0.06)7,11,15-trimec310.54 ( 0.04)0.99 ( 0.01)0.720.81 ( 0.09)14- ; 12-mec320.17 ( 0.01)0.33 ( 0.02)0.190.27 ( 0.03)12,16-dimec322.01 ( 0.14)3.57 ( 0.05)2.222.97 ( 0.28)10,14,18- ; 8,12,16-trimec320.46 ( 0.03)0.80 ( 0.02)0.610.82 ( 0.06)15- ; 13-mec330.93 ( 0.05)1.57 ( 0.03)0.921.30 ( 0.08)13,17- ; 11,15- ; 9,13-dimec334.63 ( 0.31)6.87 ( 0.11)5.117.41 ( 1.03)9,13,17-trimec331.30 ( 0.08)2.02 ( 0.02)1.863.47 ( 0.33)7,13,17-trimec330.21 ( 0.02)0.32 ( 0.01)0.320.47 ( 0.07)c35:31.01 ( 0.03)2.93 ( 0.17)1.641.76 ( 0.17)c35:22.31 ( 0.06)4.46 ( 0.14)2.713.55 ( 0.15)12,16-dimec34 ; c35:10.68 ( 0.02)1.16 ( 0.03)0.851.40 ( 0.10)10,14,18- ; 8,12,16-trimec340.28 ( 0.00)0.50 ( 0.04)0.450.89 ( 0.14)15- ; 13- ; 11-mec350.56 ( 0.02)0.85 ( 0.05)0.671.11 ( 0.09)13,17-dimec351.27 ( 0.09)1.96 ( 0.02)1.833.95 ( 0.46)9,13,17-trimec350.14 ( 0.01)0.28 ( 0.03)0.270.93 ( 0.14)c37:31.74 ( 0.06)3.33 ( 0.16)2.723.28 ( 0.34)c37:21.67 ( 0.06)2.55 ( 0.08)2.182.90 ( 0.10)12,16-dimec360.17 ( 0.01)0.34 ( 0.02)0.280.71 ( 0.08)c37:10.78 ( 0.02)0.76 ( 0.03)0.810.83 ( 0.06)10,14,18-trimec36 ; c37:10.44 ( 0.03)0.35 ( 0.05)0.430.58 ( 0.10)13 ; 11-mec370.71 ( 0.01)0.60 ( 0.04)0.770.89 ( 0.14)13,17- ; 11,15-dimec370.87 ( 0.05)1.27 ( 0.01)1.252.13 ( 0.22)9,13,17-trimec370.09 ( 0.01)0.13 ( 0.01)0.130.30 ( 0.06)c39:30.09 ( 0.02)0.18 ( 0.02)0.290.49 ( 0.16)c39:3 ; 12-mec380.49 ( 0.04)0.56 ( 0.03)0.761.03 ( 0.18)14,18 ; 12,16-dimec380.02 ( 0.03)0.14 ( 0.01)0.140.24 ( 0.04)c39:1 ; 10,14,18-trimec382.73 ( 0.19)1.73 ( 0.04)2.531.74 ( 0.23)13- ; 11-mec391.75 ( 0.09)1.03 ( 0.03)1.701.26 ( 0.20)13,17- ; 11,15-dimec390.90 ( 0.01)1.04 ( 0.14)1.361.44 ( 0.08)9,13,17-trimec390.41 ( 0.03)0.17 ( 0.09)0.110.20 ( 0.03)12- ; 10-mec400.38 ( 0.05)0.20 ( 0.01)0.350.26 ( 0.07)12,16-dimec400.27 ( 0.00)0.27 ( 0.02)0.340.32 ( 0.05)c41:13.31 ( 0.27)1.70 ( 0.04)2.791.58 ( 0.25)13- ; 11-mec412.78 ( 0.20)1.45 ( 0.06)2.591.74 ( 0.31)13,17- ; 11,15-dimec413.34 ( 0.13)2.90 ( 0.15)4.003.33 ( 0.19)9,13,17-trimec410.79 ( 0.02)0.30 ( 0.02)0.330.42 ( 0.19)12- ; 10-mec420.30 ( 0.03)0.15 ( 0.01)0.250.26 ( 0.27)12,16-dimec420.48 ( 0.04)0.37 ( 0.02)0.520.47 ( 0.12)c43:12.72 ( 0.28)1.25 ( 0.06)2.271.31 ( 0.26)15- ; 13-mec431.00 ( 0.14)0.46 ( 0.05)0.870.61 ( 0.16)13,17-dimec431.99 ( 0.16)1.18 ( 0.16)1.931.68 ( 0.28)14,18-dimec440.12 ( 0.02)0.07 ( 0.01)0.120.13 ( 0.06)c45:10.42 ( 0.09)0.16 ( 0.02)0.330.21 ( 0.07)13-mec450.000.000.000.04 ( 0.03)13,17-dimec450.37 ( 0.08)0.18 ( 0.02)0.340.43 ( 0.13)mean percent ( sd ) of total hydrocarbon composition
. compounds are listed in elution orderacronyms for hydrocarbons are derived as follows : location of methyl groups ( x - me ) , the total number of carbons ( cxx ) in the hydrocarbon component excluding the methyl branch(es ) , and the number of double bonds following a colon ( cxx : y)these hydrocarbons were not reported for i. minor pseudergates or fecal pellets in haverty et al .
( 2005)an isomeric mixture or two or more compounds co - eluted in this peak total ion chromatogram of cuticular hydrocarbons from a workers of incisitermes minor ( hagen ) and b from fecal pellets from the same collection .
acronyms for hydrocarbons are derived as follows : x - me location of methyl groups , the total number of carbons cxx in the hydrocarbon component excluding the methyl branch(es ) , and cxx : y the number of double bonds following a colon relative abundance of hydrocarbons from pseudergates , alates , soldiers , and fecal pellets of incisitermes minor ( hagen ) mean percent ( sd ) of total hydrocarbon composition .
compounds are listed in elution order acronyms for hydrocarbons are derived as follows : location of methyl groups ( x - me ) , the total number of carbons ( cxx ) in the hydrocarbon component excluding the methyl branch(es ) , and the number of double bonds following a colon ( cxx : y ) these hydrocarbons were not reported for i. minor pseudergates or fecal pellets in haverty et al .
( 2005 ) an isomeric mixture or two or more compounds co - eluted in this peak hydrocarbons from termites
n - alkanes and dimethylalkanes were the predominant classes of hydrocarbons in all castes and in fecal pellets ( table 2 ) , as previously reported by haverty et al .
n - alkanes comprised from 23.12% to 38.39% , unsaturated components from 15.21% to 19.03% , terminally branched monomethylalkanes from 3.90% to 5.32% , internally branched monomethylalkanes from 7.70% to 9.01% , and dimethylalkanes from 25.25% to 36.18% , of the total hydrocarbon content .
there was also a homologous series of trimethylalkanes , representing 8.26% to 9.48% of the total hydrocarbon content .
table 2relative abundance of hydrocarbon classes from pseudergates , alates , soldiers , and fecal pellets of incisitermes minor ( hagen)hydrocarbon classpseudergatesalatessoldiersfecal pelletsnormal alkanes38.3923.1229.7718.71olefins18.3821.1120.3120.66terminally branched methylalkanes3.904.495.325.19internally branched methylalkanes9.017.708.888.33dimethylalkanes25.2436.1829.7937.40trimethylalkanes5.097.405.929.71percent of total hydrocarbon compositionall peaks with co - eluting olefins and saturated compounds are summarized as olefinsthe hydrocarbons of i. minor characterized in this study included all of the compounds reported by haverty et al .
( 2005 ) , as well as 18 additional hydrocarbons ( table 1 ) .
these additional hydrocarbons were not abundant , never representing more than 0.85% of the total hydrocarbon ( as for the mixture of 12,16-dimec34 and c35:1 in soldiers ) content , and usually much less .
the detection of these additional hydrocarbons in this study are likely due to the greater concentration of the extracted hydrocarbons used or , possibly , colony to colony variation .
relative abundance of hydrocarbon classes from pseudergates , alates , soldiers , and fecal pellets of incisitermes minor ( hagen ) percent of total hydrocarbon composition all peaks with co - eluting olefins and saturated compounds are summarized as olefins hydrocarbons from termite fecal pellets in general , the hydrocarbons from whole - body extracts of i. minor were represented in extracts of fecal pellets . as with whole - body extracts of i. minor , dimethylalkanes were the predominant class of hydrocarbon , followed by olefins and normal alkanes ( table 2 ) .
1 ) were found in termites and not in fecal pellets , but these were present in small quantities , less than 0.1% of the total hydrocarbon content .
the lack of these two hydrocarbons in fecal pellets may be a function simply of the concentration of the extracts .
changes in hydrocarbons of fecal pellets over time we identified 73 hydrocarbon peaks in fecal pellets of i. minor ( table 1 ) . of these peaks
( data from non - significant slopes not reported ) , 19 of 73 ( 26% ) had a significant change , either positive ( five ) or negative ( 14 ) , over time ( fig . 2 ) .
this is a much higher number of statistically significant slopes ( different from 0 ) , than would be expected by chance alone ( 5% of 73 peaks is < 4 peaks ) .
2changes in percent content of individual hydrocarbons , over time , from fecal pellets of incisitermes minor workers . a hydrocarbons with significant ( = 0.05 ) negative slopes , and b hydrocarbons with significant positive slopes .
acronyms for hydrocarbons are derived as follows : x - me location of methyl groups , the total number of carbons , cxx in the hydrocarbon component excluding the methyl branch(es ) , and cxx : y the number of double bonds following a colonthe index , iage , for each of the three replications ( a , b , and c at 0 , 30 , 90 , and 365 day ) was plotted against the associated number of days ( fig .
the fitted line , with a 95% confidence interval for the means to show the variability of the replicates , had a high adjusted r (= 0.888 ) value , suggesting that it might be possible to predict fecal age by the index .
3fitted line and 95% confidence intervals for a plot of the mean of index of age ( iage ) against age .
values for 30 day are jittered a small amount so that all three data points can be seen changes in percent content of individual hydrocarbons , over time , from fecal pellets of incisitermes minor workers . a hydrocarbons with significant ( = 0.05 ) negative slopes , and b hydrocarbons with significant positive slopes .
acronyms for hydrocarbons are derived as follows : x - me location of methyl groups , the total number of carbons , cxx in the hydrocarbon component excluding the methyl branch(es ) , and cxx : y the number of double bonds following a colon fitted line and 95% confidence intervals for a plot of the mean of index of age ( iage ) against age .
values for 30 day are jittered a small amount so that all three data points can be seen we do not know the basis by which peaks increase or decrease over the 1-year period .
in general , those that increased in relative abundance tended to have higher initial relative abundances ; i.e. , mono- , di- , or tri - methylalkanes with carbon chains of 31 , 32 , or 33 .
those that decreased were represented in five of the six classes of hydrocarbons , ranging from c26 to c45 .
the composition of the hydrocarbon mixture of insects is genetically controlled ( toolson and kuper - simbrn , 1989 ; kaib et al . , 1991 ; page et al . , 1991 ; coyne et al . , 1994 ) .
this composition can be slightly affected by diet and environmental conditions ( hadley , 1977 ; espelie et al . , 1994 ; chapman et al . , 1995
( 1996 ) demonstrated significant seasonal variation in quantities of some hydrocarbons of coptotermes formosanus shiraki from hawaii ; these differences were small and associated with the production of alates .
however , the qualitative mix of hydrocarbons has been shown to be stable over decades among species of cone beetles in the genus conophthorus ( page et al . , 1990 ) .
the motivation for our study was to devise a method for the evaluation of the success or failure of drywood termite treatments , including fumigation and local application methods .
with the recent classification of sulfuryl fluoride ( the only fumigant active ingredient registered in california ) as a greenhouse gas ( mhle et al . , 2009 ) and
the anticipated increase in local treatments , we felt that there would be considerable interest by the industry , regulatory agencies , and consumers for a simple and accurate means to determine whether or not a targeted colony / infestation is still in a structure .
our method , based on changes in hydrocarbon chemistry over time , shows promise to this end .
future research includes : ( 1 ) validating these findings for additional species of drywood termites and geographic populations of the same species , ( 2 ) validating these findings at different times of the year , and ( 3 ) exploring seasonality in pellet production and hydrocarbon quality in pellets .
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hydrocarbon mixtures extracted from fecal pellets of drywood termites are species - specific and can be characterized to identify the termites responsible for damage , even when termites are no longer present or are unable to be recovered easily . in structures infested by drywood termites , it is common to find fecal pellets , but difficult to sample termites from the wood . when fecal pellets appear after remedial treatment of a structure , it is difficult to determine whether this indicates that termites in the structure are still alive and active or not .
we examined the hydrocarbon composition of workers , alates , and soldiers of incisitermes minor ( hagen ) ( family kalotermitidae ) and of fecal pellets of workers .
hydrocarbons were qualitatively similar among castes and pellets .
fecal pellets that were aged for periods of 0 , 30 , 90 , and 365 days after collection were qualitatively similar across all time periods , however , the relative quantities of certain individual hydrocarbons changed over time , with 19 of the 73 hydrocarbon peaks relatively increasing or decreasing .
when the sums of the positive and negative slopes of these 19 hydrocarbons were indexed , they produced a highly significant linear correlation ( r2 = 0.89 ) .
consequently , the quantitative differences of these hydrocarbons peaks can be used to determine the age of worker fecal pellets , and thus help determine whether the colony that produced them is alive or dead .
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Introduction
Methods and Materials
Results and Discussion
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there
is a need for computer methods that can calculate the aqueous solvation
free energies of solute molecules accurately and efficiently .
explicit - solvent models provide a physically
accurate and atomistically detailed model of solvent , but they can
be computationally expensive .
boltzmann
( pb ) , generalized born ( gb ) , and
weighted surface area ( wsa ) approaches , are computationally efficient because they treat
water as a continuum .
however , they are sometimes inaccurate , smearing
out the particulate nature of water molecules , and they may
have limited transferability to situations for which they have not
been optimized .
solvation modeling has
often been improved by combining the advantages of implicit- and explicit - solvent
models .
one such approach is the semi - explicit assembly ( sea ) water model . in the sea approach ,
water s solvation behavior is precomputed
in explicit - solvent simulations of water around model spheres that
are then combined together as building blocks to represent any given
solute structure at run time .
the solvation free energy gsolv for a target solute molecule is calculated
from a sum over solvating waters .
sea has been shown to predict efficiently
the air - to - water transfer free energies ( ghyd ) of small - molecule solutes in both prospective and
retrospective studies with reasonable accuracy .
even
so , the sea model does not give accurate solvation free energies of
ions or solutes having high charge density .
here , we describe
field - sea , a considerable improvement over sea , which gives accurate
free energies of transfer of ions and charged solutes , at no additional
computational cost and with no degradation of the predictions for
nonpolar and uncharged polar solutes . in overview , we studied full
md simulations of ionic and neutral solutes in tip3p water , described
below , and found that the results could be captured by using a solvation
free - energy field surface ( in field - sea ) , instead of using precomputed
waters ( as in sea ) .
moreover ,
we found that a weakly charged solute atom that is adjacent to a strongly
charged solute atom retains some of the restrictions of its solvating
water molecules that its neighboring charge has ; see figure 1 .
field - sea captures this effect using an adaptive
boundary method . a solvent water molecule around a solute molecule .
on the left , each solute atom ( weakly charged ) has a predefined radius ,
irrespective of its neighboring solute atoms , leading to the locus
of water centers shown by the black dashed curve . on the right ,
one
solute atom is strongly charged , leading to two consequences : its
own solvating water molecule is pulled in tightly , and neighboring
solute atoms have tighter water interactions too .
such effects may not be captured in simplified solvation
models that treat atoms as having fixed radii , independent of neighboring
atoms .
below , we describe the field - sea approach to computing solvation
free energies . the original sea model is described elsewhere , and our related explicit - solvent and linearized poisson
boltzmann
equation modeling ( lpbe ) are described
in the supporting information ( si ) . in both the original sea and the new
field - sea described here ,
first , in a precomputation step , various
model spheres are solvated in an explicit - solvent model , such as tip3p .
this provides a database of component free energies that are used
in the second step . in the second step , at the run time ,
any given
solute molecule of interest is assembled from an appropriate concatenation
of these spheres .
the solute s solvation free energy is computed
by summing the component sphere free energies . on the one hand , this
approach provides the speed advantages of simple additivity - based
models as we only need to perform the first step once for a given
solvent model . on the other hand , this procedure is more accurate
than additivity - based approaches because ( 1 ) sea sums are regional ,
not local , and ( 2 ) the database encodes the microscopic response observed
in explicit - solvent simulations .
the new field - sea instead captures
the solvation free energy using a continuous solvation field . to do this ,
the charging free energies for a series of lennard - jones
( lj ) spheres solvated in tip3p water were calculated with thermodynamics
integration ( ti ) in the set of precomputations ( see explicit solvent
free energy calculations in the si ) .
we
construct a free - energy contour ( see si figure s1 ) as a combination of the electric field at the first solvation
shell boundary ( e = q / rw , where e is the signed
magnitude of the electric field , q is the sphere s
charge , and rw is the distance from the
sphere center to the first peak of the water oxygen s radial
distribution function , rdf , around the given sphere ) , the curvature c = 1/rw at this boundary , and
the charging free energy of the spheres . within each charge step ( q ) , all of the data of free energy , electric field , and
curvature were fitted to a formula , which can be taken as an expansion
of the born model1 equation 1 will
be used to calculate the free energy associated with any surface spot
on an arbitrary solute solvent boundary ( esub ) .
this free energy could also be calculated from interpolation
between data points on the free - energy contour .
in addition
to a charging free - energy contour , we also need a contour for estimating
the explicit - solvent - accessible surface of a given solute molecule .
to generate this
, we calculated the rdf of water oxygen around each
charged sphere and picked the first peak in the rdf , denoted as the
boundary - sphere distance rw .
all of the rw values from these spheres and their charge
and lj parameters ( lj and lj )
were used to build an rw contour . to generate a solvent - accessible
surface for a given solute
, an rw for
each atom is first calculated from interpolation with its partial
charge , lj , and lj on the rw contour .
a lee richards surface is then constructed from the nonoverlapping
sections of these rw spheres centered
on their associated atom sites .
the fixed rw boundary is dependent upon only the individual parameters
of the surface atoms .
for solute molecules with multiple partial charge
sites ( especially molecular ions , where strong electric fields are
involved ) , a more physical representation of the explicit - solvent - accessible
surface would be one that responds to the whole solute s electric
field .
in other words , the surface atom distance , rw , is determined not only by the surface atom s
partial charge , but also by neighboring atoms charges .
taking
into account such collective electrostatic effects , we adjust the
fixed rw boundary in an adaptive manner ,
described by the following three - step procedure : ( 1)we cull
all surface segments within 1.4 of any solute atom . as we are
starting with a lee richards solvent - accessible surface , the
minimum rw possible in the surface construction
is half of a water molecule diameter ( corresponding to a solute atom
with a 0 radius ) .
this partial - culling process simply removes
potential numerical instabilities from surface sites overlapping solute
atom centers while providing some adjustable starting surface sites
that penetrate within the fixed rw boundary .
we calculate
the electric field at a given dot a about atom a2where n is the number
of solute atoms , ria represents a vector from atom i to surface
dot a , and ria is its length ; signa = 1 when raai=1n ( qi / ria3)ria 0 , and signa = 1 when raai=1n ( qi / ria3)ria < 0 , and raa is the vector from atom a to dot a. from the electric field , we
calculate the corresponding charge by3where raa is the length of vector raa . we interpolate with this new charge and
atom a s lj parameters to get a new rw , rw , new .
in addition ,
we assume that rw can not expand ( if rw , new > rw , original , we let rw , new = rw , original ) . this nonexpansion assumption is supported by solute
solvent
boundary plots of model diatomic solutes ( see si figure s2 ) . finally , the rw , new s of all atom s potential surface dots are averaged
to yield an rw for this atom , used in
the later culling process . ( 3 ) we cull the buried dots adaptively .
because each dot atom distance ( e.g. , daa ) is adjusted independently in the above
procedure , the shell of potential surface dots about an atom will
no longer be spherical .
thus , culling buried dots is no longer a simple
process of eliminating dots within a neighboring atom s rw , and an adaptive culling process is needed .
to adaptively cull
buried surface dots , when we check
whether a given dot a is buried by a neighboring
atom b , we first determine a corresponding charge
at dot a from the electric field at this dot .
this
is used along with atom b s lj parameters
to get a new rw , rw , badp , following the above rw adjustment procedure .
atom
distance , dab , with rw , badp , to determine if the
dot is buried in atom b. if dab is less than rw , badp , it is removed from the set of potential surface
dots .
we call the new dot surface resulting from the above culling
procedure the adaptive boundary .
our term field - sea refers both to
the field and to the adaptive boundary .
the adaptive nature of the
boundary only pertains to multiatom solutes ; the adaptive and fixed rw boundaries are identical for single - atom solutes ,
like monatomic ions . while our adaptation procedure could , in principle ,
be applied iteratively , we found no further improvement after a single
calculation .
the charging
free energy can be calculated for any given field - sea surface via4where ndot is the total number of surface - exposed dots , mi is the total number of dots
( exposed + occluded ) for corresponding atom i ( each
surface dot , j , of atom i , only
corresponds to 1/mi of
this atom sphere s total surface area or total solvation free
energy ) , and esub , j is
the subenergy associated with exposed surface dot j , calculated from the free - energy contour . here ,
esub , j is calculated from eq 1 using the signed magnitude of the electric field , ej , and the curvature at dot j , 1/rij .
ej is defined as negative when ejrij < 0 , where ej is the electric
field ( vector ) at dot j , rij is the vector from atom i to its surface dot j , and rij is this vector s length .
now ,
we take the total solvation free energy to be the sum of polar and
nonpolar components5 assuming the polar component of the free energy of transfer
( gpol ) can be uniquely described
by both the surface electric field and geometry of the solute , this
molecular gpol can be accurately
calculated from the simple summation of the surface dots energies
process described above .
we consider the gsolv of monatomic ions as an initial test . for molecular
solutes ,
to account for the role of solute conformation in the solvation
free energy , we average gsolv results
from calculations on 50 conformations ( though 10 conformations are
usually enough ; see the si ) . these solute
conformations were generated from 5 ns tip3p water md simulations
with a 100 ps snapshot interval .
we developed field - sea because of the errors that we
observed in simpler methods in solvating ions ; see figure 2 .
the explicit - solvent curve shows the hydration
asymmetry discussed by others , where positively charged solutes are less favored than negatively
charged solutes of similar size .
gsolv as the function of a model lj sphere (
= 0.22 nm , = 0.06538 kj / mol ) charge for tip3p , lpbe , and field - sea .
for comparison at infinite - dilution conditions ,
an ewald correction
is applied to the tip3p and field - sea results . here , we tested field - sea on the solvation free energies
of monatomic ions using ion parameters developed by aqvist or joung and cheatham ( table s2 , si ) .
these ions have different
sizes and charges ; therefore , they have different charge densities .
table 1 and figure s3 ( si ) show the results of field - sea calculations , compared with
lpbe and tip3p .
lpbe results using the lj surface do not capture quantitatively
the gsolv of ions with high charge
densities .
in contrast , field - sea reproduces the tip3p gsolv values regardless of the specific lj parameters
and charges .
these results indicate that field - sea is accurately reproducing
explicit - solvent free energies of solvation of monatomic ions , provided
that their charge and lj parameters are near or encompassed within
the range of the rw and free - energy contours .
a common approximation in simplified
solvation models is to suppose that atoms have fixed
atom
types , where any particular solute atom has a given value
of charge and radius , independent of its atomic neighbors . however ,
our tip3p explicit - solvent simulations described below show that this
approximation is a source of error . how the solvation surface is constructed
our explicit - solvent simulations show that the solvation shell is
not just a simple union of the spherical surfaces of all of the atoms
making up the solute molecule .
imagine a diatomic solute with partially
charged atom a covalently connected to partially
charged atom b. our tip3p simulations show that when
atom a attracts water molecules , it also shrinks
the solvation shell of waters around atom b. in this
way , the solvation surface around the diatomic molecule a b can be more complex than the simple union
of independent spherical solvation shells around atoms a and b. to address such situations , we developed
an adaptive method that gives a more explicit - like solvation boundary . to test our adaptive boundary method , we made up 22 fictitious
diatomic solutes ( see si table s4 ) , computed
their solvation free energies , and compared to their solvation in
tip3p .
we constructed these solutes by taking pairs of ordinary simulation
atom types , placing them at fixed covalent bond distance apart , and
giving each atom a charge and radius that we could vary systematically
over the series . because of the fictitious charges that we give them ,
these are not molecules observed in nature
however , they are physically
plausible molecules that provide us with a systematic series for learning
about how explicit - solvent models handle solvated charges .
figure 3 shows the computed free energies of these diatomic
solutes from field - sea when using the fixed rw and adaptive boundaries in comparison to explicit - solvent
tip3p simulations .
lpbe and original sea results are shown in the si instead of here for the sake of simplicity .
in summary
, we find that when the tip3p gsolv is weak , for example , when the charge density of solute
atoms is low , the fixed rw boundary works
fairly well .
when the gsolv grows
to 100 kcal / mol and larger ( more negative ) , it becomes increasingly
important to use an adaptive boundary .
these strongly solvated model
molecules often have large , unbalanced partial charges on the solute
atoms , and these situations lead to exaggerated distortions of the
explicit solvation shell .
field - sea gsolv for model diatomic solutes ( triangles : fixed rw ; circles : adaptive boundary ) compared to tip3p simulations .
figure 4 shows how a neighboring
solute atom can affect the solvation shell about another atom .
the
fixed rw and adaptive boundaries are identical
in figure 3a as both carbon atoms are weakly
charged and there is only minor collective electrostatic perturbation
on the boundary . however ,
when the solutes are more highly charged
and large charging energies are involved , as in figure 3b , the weakly charged carbon atom s solvation shell
will be distorted by its neighboring highly charged oxygen atom . in
this case ,
water molecules penetrate more deeply into the carbon s
solvent - accessible surface to better solvate the oxygen charge ( the
right part of the white curve in the dark blue region ) .
also , water
molecules pack more tightly around the carbon atom and reduce its
apparent rw ( the left part of the white
curve in the blue and light blue regions ) .
both of these effects are
captured well by adaptive field - sea boundaries , leading to field - sea
charging energies that are in excellent agreement with explicit simulation
results .
oxygen density around ( a ) weakly charged
and ( b ) strongly charged diatomic solutes .
the blue contours indicate
water density greater than the bulk value , with the darker blue regions
indicating the enhanced water probability density .
the black line
shows the nonadaptive fixed rw boundary .
for the weakly charged diatomic , the adaptive and nonadaptive boundaries
coincide .
the adaptive and nonadaptive boundaries differ . in order to establish that the accuracy of field - sea is not degraded
relative to sea on nonionic solutes , we applied field - sea with both
fixed rw and adaptive boundaries to a
standard set of 504 neutral small molecules .
this set contains an alchemically diverse
range of functional groups , for which solvation free energies are
available from experiments and tip3p simulations .
figure 5 shows 504 neutral solutes solvation free
energy from tip3p simulation , from the lpbe ( white diamonds ) and field - sea
( white circles ) ( see si figure s4 for nonadaptive
fixed rw field - sea results ) .
field - sea
shows a rmse ( root - mean - square error ) of 1kt with a negligible mse ( mean - signed error ) , comparable to the original
sea and considerably better than the
lpbe , which bears a systematically negative error .
while the fixed rw boundary field - sea results are comparable
to the lpbe ( table 1 ) , it overestimates the
free energy when weakly charged c atoms are neighbored by highly charged
o or n atoms , as in the cases of alcohols , amines , ethers , and esters
( si table s5 ) .
these errors arise because
the fixed rw boundary can not capture water
molecule penetration into the c atom s solvent - accessible surface
( see figure 4b ) , situations the adaptive boundary
handles directly . therefore , while these are simply neutral solutes ,
collective solute interactions still play a clear role in their overall
hydration .
figure s5 ( si ) compares
the total solvation free energy from field - sea with experimental results .
the mse / rmse of field - sea to experimental solvation free energy ( 0.67/1.45
kcal / mol , table s6 ( si ) ) is comparable
to that of the much more computationally expensive tip3p water model
( 0.66/1.22 kcal / mol ) .
these results indicate
that field - sea can accurately compute solvation free energies over
the full range from charge - dense ions to neutral polar and nonpolar
molecular solutes . here , we test field - sea on an expanded set of molecular
ions and biomolecules ( e.g. , acetate , butylammonium , etc . ; see si table s7 for the complete list and results ) .
these are ions that are larger and more complex than the simple
atomic and diatomic ions described above and should better test the
need for adaptive boundary considerations .
figure 5 compares solvation energies from lpbe ( orange diamond ) and
field - sea ( orange circle ; see si figure
s6 for field - sea results with a fixed rw boundary ) with tip3p results for 35 molecular ions .
the errors for
both lpbe and field - sea with a fixed rw boundary are around 10 kcal / mol ( table 1 ) .
while this might be regarded as acceptable in light of the nearly
100 kcal / mol span of free energies covered in the tip3p simulations ,
using an adaptive boundary with field - sea has half of this rmse .
field - sea
also performs well in multivalent molecular ion solvation ( si figure s7 ) and is as accurate as explicit - solvent
calculations compared to experimental values for ionic solute solvation
( table s6 and figure s8 , si ) .
md simulations
of gsolv for 504 neutral solutes
( white ) , 35 molecular ions ( orange ) , and 22 capped amino acid dipeptides
( cyan ) in tip3p water , compared to ( a ) lpbe and ( b ) field - sea .
we also tested our methods on 22 capped amino
acids ( n - acetyl - x - methylamide , x = glu , arg , leu ,
etc . ;
si table s8 ) , which are widely used
biological models in both theoretical studies and experiments .
these are useful precursor
structures for the foundation of hydrophobicity scales , used in estimating
the solvation of larger biomolecular structures . here
, we investigate the solvation
free energies of a full series of capped amino acids with field - sea .
as the size of solute increases , computing the total solvation
show , lpbe
( cyan diamond ) and field - sea ( cyan circle ) both yield solvation free
energies that agree well with tip3p calculations .
again , the adaptive
boundary helps field - sea considerably , cutting the rmse to half of
that seen from the lpbe or fixed rw cases .
these results indicate that the accuracy of field - sea does not degrade
as the solute size further increases .
the solvation boundaries
in field - sea are made from a set of surface dots .
above , we used 80 dots / atom ,
the same as was used in previous sea studies .
what is the minimum number of grid dots that we need to properly
represent the first - shell boundary ? to investigate this , we performed
an analysis of accuracy versus relative computational time as a function
of the granularity of the boundary .
this analysis indicates that the
rmse for field - sea results on the 504 neutral molecule set is essentially
uncompromised even down to a granularity of 5 dots / atom , without increasing
the rmse above 1 kcal / mol ( figure s9 and s10 ( si ) , the granularity does not affect the accuracy for charged
solutes either ) . at 5 dots / atom ,
field - sea is roughly 5-fold faster
than dipolar sea at 80 dots / atom , the minimum surface granularity
recommended for this method .
these results
indicate that while field - sea is sensitive to the physicality of the
solvation boundary , it is less sensitive to the granularity of its
depiction .
we have
described field - sea , a method for computing solvation free energies
of solutes in water .
it improves upon an earlier method called sea
( semi - explicit assembly ) .
sea captured the physics of solvation by
presimulating toy spheres in explicit water , collecting a database
of structural and energetic properties of those waters and then assembling
at run time the solvation physics as sums over appropriate toy spheres
to properly represent a given solute . in field - sea , this procedure
differs in using a solvation free - energy field , rather than explicit
waters .
furthermore , field - sea uses an adaptive boundary , allowing
solvating waters to approach a solute atom to different degrees depending
on neighboring atoms .
relative to sea , field - sea captures the solvation
free energies of ions and charged solutes accurately , is faster to
compute , and has no degradation of performance on nonpolar and polar
solutes .
both sea and field - sea offer advantages over implicit - solvent
modeling in that they entail no adjustable solute atom radii parameters .
sea and field - sea are built upon a corresponding force field and explicit - solvation
model . here
one of the
key observations that arises from our md simulations of charged solutes
in tip3p water , which is captured by field - sea , is that atoms that
are adjacent to charged atoms in solutes acquire partial characteristics
of those charged atoms .
for example , a weakly charged atom s
solvation shell is shrunk by its neighboring big charges .
an implication
of this for implicit - solvent modeling is that atomic radii should
not be treated as fixed for solvation in water ; an atom s radius
in implicit - solvent modeling can depend on the nature of its neighboring
atom .
|
previous
work describes a computational solvation model called semi - explicit
assembly ( sea ) .
the sea water model computes the free energies of
solvation of nonpolar and polar solutes in water with good efficiency
and accuracy .
however , sea gives systematic errors in the solvation
free energies of ions and charged solutes .
here , we describe field - sea ,
an improved treatment that gives accurate solvation free energies
of charged solutes , including monatomic and polyatomic ions and model
dipeptides , as well as nonpolar and polar molecules . field - sea is
computationally inexpensive for a given solute because explicit - solvent
model simulations are relegated to a precomputation step and because
it represents solvating waters in terms of a solute s free - energy
field .
in essence , field - sea approximates the physics of explicit - model
simulations within a computationally efficient framework .
a key finding
is that an atom s solvation shell inherits characteristics
of a neighboring atom , especially strongly charged neighbors .
field - sea
may be useful where there is a need for solvation free - energy computations
that are faster than explicit - solvent simulations and more accurate
than traditional implicit - solvent simulations for a wide range of
solutes .
|
Introduction
Theory and Methods
Results and Discussion
Conclusions
|
selective serotonin reuptake inhibitors ( ssris ) , serotonin and norepinephrine reuptake inhibitors , and noradrenergic and specific serotonergic antidepressants are categorized as first - line treatments in many countries , including japan.1,2 thus , health care providers have the opportunity to choose an antidepressant from a variety of first - line treatment options . to select the most appropriate treatment for each patient , it is important for clinicians to be aware of the differences in efficacy between comparable antidepressants at the doses approved in their countries .
one long - standing approach to comparing efficacy has been meta - analysis of several randomized controlled trials that compare widely used treatments.310 however , results have been inconsistent , perhaps partly due to the use of different methodologies .
recent meta - analyses have used mixed treatment comparisons in which both direct and indirect comparisons were used.11,12 duloxetine , a second - generation norepinephrine reuptake inhibitor that has demonstrated efficacy , safety , and tolerability in patients with mdd,1316 has been the subject of several recent mixed treatment comparisons . a recent analysis by cipriani et al12 compared the effects of 12 new - generation antidepressants in adults with mdd , and gartlehner et al11 conducted an analysis of 234 studies that included placebo as a comparator . however , these results are not applicable to clinical practice in japan , because they included data from patients taking duloxetine up to 120 mg / day , which is twice the maximum dosage ( 60 mg / day ) approved in japan .
another drawback was the inclusion of patients who took antidepressants not approved or not even available for use in japan .
therefore , it is uncertain whether these results can be applied to daily clinical practice in countries ( eg , japan ) where the approved dosage for duloxetine is 4060 mg / day and ssris are limited to four compounds , ie , paroxetine , sertraline , escitalopram , and fluvoxamine . to the best of our knowledge , there are no meta - analyses that compare the efficacy of duloxetine 60 mg / day with these ssris .
the primary purpose of the current study was to provide clinicians with efficacy results that would allow a comparison of duloxetine 60 mg / day with four approved ssris in japan .
the eli lilly clinical trial database contains all clinical trials of duloxetine for patients with mdd that were conducted by eli lilly or its partners outside of japan .
we reviewed this database and selected randomized controlled trials that included duloxetine and ssri for the acute treatment of mdd .
for the ssri selection , four ssris that were approved in japan for the treatment of mdd ( eg , paroxetine , sertraline , escitalopram , and fluvoxamine as of july 2014 ) were included . as a result ,
a total of four studies ( three studies on duloxetine 40 or 80 mg / day versus paroxetine 20 mg / day and one study on duloxetine 60 mg / day versus escitalopram 10 mg / day ) were selected ( table 1 ) . as a next step ,
patients who received more than the approved daily dose ranges in japan ( duloxetine 80 mg / day ) were excluded from the analysis . no studies meeting the criterion for comparison were excluded .
common inclusion criteria for the included studies ( hmat a , hmat b , hmcv , hmcr ) were as follows : male or female outpatients aged at least 18 years who met dsm - iv ( diagnostic and statistical manual for mental disorders , 4th edition)17 criteria for nonpsychotic mdd and had a clinical global impression score 4 , a 17-item hamilton rating scale for depression ( hamd17 ) total score 15 , or a montgomery sberg depression rating scale score 22 .
the hmat study ( groups a and b ) was a multicenter , parallel , double - blind , randomized , placebo - controlled and active comparator - controlled study with a blinded placebo lead - in and placebo lead - out .
the primary objective was to assess mean changes in hamd17 total score from baseline to endpoint ( week 8) .
the treatments were duloxetine 40 or 80 mg / day , paroxetine 20 mg / day , or placebo .
the hmcv study was a multicenter , randomized , double - blind , double - dummy , parallel , active treatment - controlled phase iii trial comparing the efficacy and safety of duloxetine 60 mg / day and paroxetine 20 mg / day for inpatients and outpatients with mdd .
the study included 8 weeks of active treatment followed by a one - week dose - tapering period .
the primary objective of the study was to determine whether duloxetine 60 mg / day was noninferior to paroxetine 20 mg / day in the acute treatment of patients with mdd , as assessed by the baseline - to - endpoint change in hamd17 total score over an 8-week period.13 the hmcr study was a multicenter , randomized , 8-month , placebo - controlled , double - blind study that evaluated the comparative efficacy of duloxetine and escitalopram for patients with mdd.14,15 the primary objective was to compare the onset of antidepressant efficacy for patients taking duloxetine 60 mg / day or escitalopram 10 mg / day .
this objective was evaluated by testing the hypothesis that the percentage of patients taking duloxetine who met onset criteria at week 2 was not inferior to ( or at least as good as ) the percentage of patients taking escitalopram .
the primary endpoint was mean change from baseline in hamd17 total score at week 8 .
secondary endpoints were mean change in hamd17 subscale score , response rate , remission rate , and mean change in hamilton anxiety rating scale ( hama ) total score from baseline to week 8 .
response was defined as a 50% reduction in hamd17 total score from baseline at week 8 .
our analyses included two groups : all randomized patients who had a hamd17 total score 15 and a subgroup of more severe patients who had a hamd17 total score 19 .
this division was included because some studies suggest that patients with severe symptoms may have different treatment needs.4,18 hamd17 items include ( 1 ) depressive mood ; ( 2 ) feelings of guilt ; ( 3 ) suicide ; ( 4 ) early insomnia ; ( 5 ) middle insomnia ; ( 6 ) late insomnia ; ( 7 ) work and activities ; ( 8) psychomotor retardation ; ( 9 ) agitation ; ( 10 ) psychic anxiety ; ( 11 ) somatic anxiety ; ( 12 ) somatic symptoms gastrointestinal ; ( 13 ) general somatic symptoms ; ( 14 ) genital symptoms ; ( 15 ) hypochondriasis ; ( 16 ) weight loss ; and ( 17 ) insight.19 five hamd17 subscales were used for the subgroup analysis : bech ( items 1 , 2 , 7 , 8 , 10 , 13 ) and maier ( 1 , 2 , 7 , 8 , 9 , 10 ) , comprising items generally considered to be the core symptoms of depression ; retardation ( items 1 , 7 , 8 , 14 ) , evaluating the degree of energy and interest levels ; sleep ( items 4 , 5 , 6 ) assessing the degree of insomnia ; and anxiety / somatization ( items 10 , 11 , 12 , 13 , 15 , 17 ) and hama total score , assessing anxiety.1923 for the analysis of primary and secondary endpoints , missing data were imputed using the last observation carried forward approach .
continuous variables were calculated using analysis of covariance based on last observation carried forward values as follows : one analysis of covariance model was calculated for each study ( counting the groups a and b of the hmat study as two studies ) , with a fixed effect for investigator , treatment , and baseline score as covariates .
effect sizes in each model were calculated using least squares mean differences divided by the standard deviation of the residuals provided by the model of this study .
overall least squares mean estimates and effect sizes were calculated as a weighted mean of the corresponding estimates in all studies , with weights based on within - study variance , assuming a fixed study effect .
the binary outcomes were analyzed using the cochran - mantel - haenszel test adjusted for study .
relative risk is presented with 95% confidence intervals ( ci ) and p - values .
the odds ratio ( or ) was adjusted for the study by calculating the ors within each study and then weighting these ors across studies .
absolute numbers and averages were not adjusted for study and therefore can not be directly compared with the ors .
safety and tolerability measures included patient - reported treatment - emergent adverse events and overall rates of study discontinuation due to adverse events .
reasons for discontinuation from the study were listed and treatment - emergent adverse events were reported using the medical dictionary for regulatory activities terminology .
safety was analyzed by treatment descriptive statistics , and categorical safety measures were evaluated using fisher s exact test .
the eli lilly clinical trial database contains all clinical trials of duloxetine for patients with mdd that were conducted by eli lilly or its partners outside of japan .
we reviewed this database and selected randomized controlled trials that included duloxetine and ssri for the acute treatment of mdd .
for the ssri selection , four ssris that were approved in japan for the treatment of mdd ( eg , paroxetine , sertraline , escitalopram , and fluvoxamine as of july 2014 ) were included . as a result ,
a total of four studies ( three studies on duloxetine 40 or 80 mg / day versus paroxetine 20 mg / day and one study on duloxetine 60 mg / day versus escitalopram 10 mg / day ) were selected ( table 1 ) . as a next step ,
patients who received more than the approved daily dose ranges in japan ( duloxetine 80 mg / day ) were excluded from the analysis . no studies meeting the criterion for comparison were excluded .
common inclusion criteria for the included studies ( hmat a , hmat b , hmcv , hmcr ) were as follows : male or female outpatients aged at least 18 years who met dsm - iv ( diagnostic and statistical manual for mental disorders , 4th edition)17 criteria for nonpsychotic mdd and had a clinical global impression score 4 , a 17-item hamilton rating scale for depression ( hamd17 ) total score 15 , or a montgomery sberg depression rating scale score 22 .
the hmat study ( groups a and b ) was a multicenter , parallel , double - blind , randomized , placebo - controlled and active comparator - controlled study with a blinded placebo lead - in and placebo lead - out .
the primary objective was to assess mean changes in hamd17 total score from baseline to endpoint ( week 8) .
the treatments were duloxetine 40 or 80 mg / day , paroxetine 20 mg / day , or placebo .
the hmcv study was a multicenter , randomized , double - blind , double - dummy , parallel , active treatment - controlled phase iii trial comparing the efficacy and safety of duloxetine 60 mg / day and paroxetine 20 mg / day for inpatients and outpatients with mdd .
the study included 8 weeks of active treatment followed by a one - week dose - tapering period .
the primary objective of the study was to determine whether duloxetine 60 mg / day was noninferior to paroxetine 20 mg / day in the acute treatment of patients with mdd , as assessed by the baseline - to - endpoint change in hamd17 total score over an 8-week period.13 the hmcr study was a multicenter , randomized , 8-month , placebo - controlled , double - blind study that evaluated the comparative efficacy of duloxetine and escitalopram for patients with mdd.14,15 the primary objective was to compare the onset of antidepressant efficacy for patients taking duloxetine 60 mg / day or escitalopram 10 mg / day .
this objective was evaluated by testing the hypothesis that the percentage of patients taking duloxetine who met onset criteria at week 2 was not inferior to ( or at least as good as ) the percentage of patients taking escitalopram .
the primary endpoint was mean change from baseline in hamd17 total score at week 8 .
secondary endpoints were mean change in hamd17 subscale score , response rate , remission rate , and mean change in hamilton anxiety rating scale ( hama ) total score from baseline to week 8 .
response was defined as a 50% reduction in hamd17 total score from baseline at week 8 .
our analyses included two groups : all randomized patients who had a hamd17 total score 15 and a subgroup of more severe patients who had a hamd17 total score 19 .
this division was included because some studies suggest that patients with severe symptoms may have different treatment needs.4,18 hamd17 items include ( 1 ) depressive mood ; ( 2 ) feelings of guilt ; ( 3 ) suicide ; ( 4 ) early insomnia ; ( 5 ) middle insomnia ; ( 6 ) late insomnia ; ( 7 ) work and activities ; ( 8) psychomotor retardation ; ( 9 ) agitation ; ( 10 ) psychic anxiety ; ( 11 ) somatic anxiety ; ( 12 ) somatic symptoms gastrointestinal ; ( 13 ) general somatic symptoms ; ( 14 ) genital symptoms ; ( 15 ) hypochondriasis ; ( 16 ) weight loss ; and ( 17 ) insight.19 five hamd17 subscales were used for the subgroup analysis : bech ( items 1 , 2 , 7 , 8 , 10 , 13 ) and maier ( 1 , 2 , 7 , 8 , 9 , 10 ) , comprising items generally considered to be the core symptoms of depression ; retardation ( items 1 , 7 , 8 , 14 ) , evaluating the degree of energy and interest levels ; sleep ( items 4 , 5 , 6 ) assessing the degree of insomnia ; and anxiety / somatization ( items 10 , 11 , 12 , 13 , 15 , 17 ) and hama total score , assessing anxiety.1923 for the analysis of primary and secondary endpoints , missing data were imputed using the last observation carried forward approach .
continuous variables were calculated using analysis of covariance based on last observation carried forward values as follows : one analysis of covariance model was calculated for each study ( counting the groups a and b of the hmat study as two studies ) , with a fixed effect for investigator , treatment , and baseline score as covariates .
effect sizes in each model were calculated using least squares mean differences divided by the standard deviation of the residuals provided by the model of this study .
overall least squares mean estimates and effect sizes were calculated as a weighted mean of the corresponding estimates in all studies , with weights based on within - study variance , assuming a fixed study effect .
the binary outcomes were analyzed using the cochran - mantel - haenszel test adjusted for study .
relative risk is presented with 95% confidence intervals ( ci ) and p - values .
the odds ratio ( or ) was adjusted for the study by calculating the ors within each study and then weighting these ors across studies .
absolute numbers and averages were not adjusted for study and therefore can not be directly compared with the ors .
safety and tolerability measures included patient - reported treatment - emergent adverse events and overall rates of study discontinuation due to adverse events .
reasons for discontinuation from the study were listed and treatment - emergent adverse events were reported using the medical dictionary for regulatory activities terminology .
safety was analyzed by treatment descriptive statistics , and categorical safety measures were evaluated using fisher s exact test .
at baseline , patients were mostly middle - aged ( mean age 40.842.0 years ) , female ( range 63.8%67.4% ) , with mean hamd17 total scores ranging from 17.6 to 19.0 and mean hama total scores ranging from 14.4 to 15.8 .
the majority of patients were caucasian ; patients from east asia accounted for approximately 30% of duloxetine - treated and ssri - treated patients ( table 2 ) .
completion rates were approximately 70% ( 68.6% for duloxetine - treated patients , 72.3% for ssri - treated patients , and 66.8% for placebo - treated patients ) .
adverse events were common reasons for discontinuation ( duloxetine - treated patients , 8.9% ; ssri - treated patients , 7.1% ; placebo - treated patients , 6.0% ; see table 3 ) .
discontinuation due to lack of efficacy was more likely in placebo - treated patients than in duloxetine - treated or ssri - treated patients ( 13.3% versus 3.2% and 3.2% , respectively ) .
a comparison of the mean changes in hamd17 total , subscale scores and hama total score from baseline at week 8 ( last observation carried forward ) is shown in table 4 ; corresponding effect sizes are shown in figure 1 . in the total population
, no statistically significant differences were found in the mean change in hamd17 total score at week 8 between duloxetine - treated and ssri - treated patients ( 10.08 and 9.69 , respectively ) . however , the differences between duloxetine versus placebo and ssri versus placebo were statistically significant ( p<0.01 ) .
duloxetine and ssri were comparable on most other efficacy measures ; however , duloxetine - treated patients showed a greater mean change from baseline in hamd17 retardation subscale score compared with ssri - treated patients ( least squares mean difference [ 95% ci ] ) ( 0.33 [ 0.60 , 0.07 ] ) .
for the more severe subgroup with hamd17 total scores 19 at baseline , again statistically significant differences were found in mean change in hamd17 total scores at week 8 , but greater efficacy for duloxetine was seen with the bech , maier , and retardation subscale scores compared with ssris : hamd17 bech ( 0.62 [ 1.16 , 0.08 ] ) , hamd17 maier ( 0.65 [ 1.18 , 0.12 ] ) , and hamd17 retardation ( 0.45 [ 0.83 , 0.07 ] ) . in the total population , hamd17 total score effect sizes for duloxetine and ssri versus placebo were comparable ( 0.260 and 0.256 , respectively ) .
these were seen on the retardation subscales for the total population ( 0.337 versus 0.191 , respectively ) in the more severe population ( 0.409 versus 0.154 , respectively ) , and on the bech and maier subscales for the more severe population ( figure 1 ) .
the response rates at week 8 were 42.0% for the duloxetine - treated patients , 44.5% ( n=307 ) for the ssri - treated patients , and 24.4% ( n=77 ) for the placebo - treated patients .
no statistically significant difference was found between the duloxetine - treated and ssri - treated patients ( or 1.05 ; 95% ci 0.801.37 ) .
statistically significant differences were shown for the duloxetine - treated versus placebo - treated patients ( or 1.61 ; 95% ci 1.112.33 ) and the ssri - treated versus placebo - treated patients ( or 1.83 ; 95% ci 1.272.64 ) .
the remission rates at week 8 were 35.2% for the duloxetine - treated patients , 36.1% ( n=249 ) for the ssri - treated patients , and 21.2% ( n=67 ) for the placebo - treated patients .
no statistically significant differences were found at week 8 for the duloxetine - treated versus ssri - treated patients ( or 1.11 ; 95% ci 0.641.92 ) , the duloxetine - treated versus placebo - treated patients ( or 1.53 ; 95% ci 0.743.18 ) , or the ssri - treated versus placebo - treated patients ( or 1.60 ; 95% ci 0.773.33 ) .
the most common treatment - emergent adverse events are summarized in table 5 . among duloxetine - treated patients ,
nausea ( 25.7% ) , dry mouth ( 17.3% ) , dizziness ( 12.6% ) , constipation ( 11.0% ) , and decreased appetite ( 11.9% ) had a higher incidence than in placebo - treated patients , but the rates of these adverse events were similar between duloxetine - treated and ssri - treated patients except for nausea and dry mouth .
at baseline , patients were mostly middle - aged ( mean age 40.842.0 years ) , female ( range 63.8%67.4% ) , with mean hamd17 total scores ranging from 17.6 to 19.0 and mean hama total scores ranging from 14.4 to 15.8 .
the majority of patients were caucasian ; patients from east asia accounted for approximately 30% of duloxetine - treated and ssri - treated patients ( table 2 ) .
completion rates were approximately 70% ( 68.6% for duloxetine - treated patients , 72.3% for ssri - treated patients , and 66.8% for placebo - treated patients ) .
adverse events were common reasons for discontinuation ( duloxetine - treated patients , 8.9% ; ssri - treated patients , 7.1% ; placebo - treated patients , 6.0% ; see table 3 ) .
discontinuation due to lack of efficacy was more likely in placebo - treated patients than in duloxetine - treated or ssri - treated patients ( 13.3% versus 3.2% and 3.2% , respectively ) .
a comparison of the mean changes in hamd17 total , subscale scores and hama total score from baseline at week 8 ( last observation carried forward ) is shown in table 4 ; corresponding effect sizes are shown in figure 1 . in the total population
, no statistically significant differences were found in the mean change in hamd17 total score at week 8 between duloxetine - treated and ssri - treated patients ( 10.08 and 9.69 , respectively ) . however , the differences between duloxetine versus placebo and ssri versus placebo were statistically significant ( p<0.01 ) .
duloxetine and ssri were comparable on most other efficacy measures ; however , duloxetine - treated patients showed a greater mean change from baseline in hamd17 retardation subscale score compared with ssri - treated patients ( least squares mean difference [ 95% ci ] ) ( 0.33 [ 0.60 , 0.07 ] ) .
for the more severe subgroup with hamd17 total scores 19 at baseline , again statistically significant differences were found in mean change in hamd17 total scores at week 8 , but greater efficacy for duloxetine was seen with the bech , maier , and retardation subscale scores compared with ssris : hamd17 bech ( 0.62 [ 1.16 , 0.08 ] ) , hamd17 maier ( 0.65 [ 1.18 , 0.12 ] ) , and hamd17 retardation ( 0.45 [ 0.83 , 0.07 ] ) . in the total population , hamd17 total score effect sizes for duloxetine and ssri versus placebo were comparable ( 0.260 and 0.256 , respectively ) .
these were seen on the retardation subscales for the total population ( 0.337 versus 0.191 , respectively ) in the more severe population ( 0.409 versus 0.154 , respectively ) , and on the bech and maier subscales for the more severe population ( figure 1 ) .
the response rates at week 8 were 42.0% for the duloxetine - treated patients , 44.5% ( n=307 ) for the ssri - treated patients , and 24.4% ( n=77 ) for the placebo - treated patients .
no statistically significant difference was found between the duloxetine - treated and ssri - treated patients ( or 1.05 ; 95% ci 0.801.37 ) .
statistically significant differences were shown for the duloxetine - treated versus placebo - treated patients ( or 1.61 ; 95% ci 1.112.33 ) and the ssri - treated versus placebo - treated patients ( or 1.83 ; 95% ci 1.272.64 ) .
the remission rates at week 8 were 35.2% for the duloxetine - treated patients , 36.1% ( n=249 ) for the ssri - treated patients , and 21.2% ( n=67 ) for the placebo - treated patients .
no statistically significant differences were found at week 8 for the duloxetine - treated versus ssri - treated patients ( or 1.11 ; 95% ci 0.641.92 ) , the duloxetine - treated versus placebo - treated patients ( or 1.53 ; 95% ci 0.743.18 ) , or the ssri - treated versus placebo - treated patients ( or 1.60 ; 95% ci 0.773.33 ) .
the most common treatment - emergent adverse events are summarized in table 5 . among duloxetine - treated patients , nausea ( 25.7% ) , dry mouth ( 17.3% ) , dizziness ( 12.6% ) , constipation ( 11.0% ) , and decreased appetite ( 11.9% ) had a higher incidence than in placebo - treated patients , but the rates of these adverse events were similar between duloxetine - treated and ssri - treated patients except for nausea and dry mouth .
our results show that duloxetine and ssri treatment within their respective approved dosage in japan demonstrated comparable efficacy with regard to overall depressive symptoms
. however , certain hamd17 subscale scores responded differently to duloxetine compared with ssris , and this may clarify the difference in clinical attributes among these antidepressants .
first , when assessing overall efficacy using the mean changes in hamd17 total scores from baseline , both duloxetine and ssri demonstrated greater efficacy than placebo , but did not indicate any significant difference between each other for either the total population ( hamd17 total score 15 ) or the more severe population ( hamd17 total score 19 ) . on the one hand , this is consistent with a prior report by thase et al,4 where all randomized patients across any severity were analyzed and no significant difference in efficacy between duloxetine and ssri was found .
however , on the other hand , this is inconsistent with prior reports that duloxetine 120 mg / day showed greater efficacy than ssris in patients with at least moderately severe depression.4,16 given this and the finding of a modest dose response trend for duloxetine efficacy by mallinckrodt et al16 this inconsistency may be explained by the upper limit on the dosage of duloxetine ( 60 mg / day ) in this study .
second , when assessing energy and interest using retardation subscale scores , duloxetine showed greater efficacy regardless of baseline depression severity ( hamd17 total score 15 or 19 ) .
this is consistent with previous reports by mallinckrodt et al16 and martinez et al.24 furthermore , katz et al25 reported that desipramine ( a norepinephrine reuptake inhibitor ) demonstrated better efficacy than paroxetine ( an ssri ) on psychomotor retardation symptoms .
these results may be explained by the effect of duloxetine on norepinephrine , which has been shown to be associated with arousal and activity.26 the current results show some inconsistency with a report by cipriani et al in which some ssris were significantly more efficacious than duloxetine .
this is partly due to the different methodologies used in that study and in our current study . in cipriani
et al,12 approximately one third of the comparisons were direct comparisons from randomized controlled trials and many of the pairwise comparisons were based on only one study .
degree of heterogeneity and statistical incoherence ( estimates based on the direct comparisons are not contained in the 95% ci for indirect comparisons ) in the study network were detected .
study conclusions based on direct comparisons were much more conservative than those based on indirect ones . on the other hand ,
the strengths of this database analysis are that it analyzed individual patient data , used the same analysis method and endpoint across studies , and included similar patients .
the adopted database analysis design allowed for a homogeneous patient population , with almost the same inclusion criteria and the exact same endpoint . although our analysis resulted in a comparatively smaller number of studies and patients , it achieved high internal validity and reproducibility .
in addition , direct comparisons of head - to - head data enabled us to avoid the uncertainty of how much the two different evidence levels are weighted when pooling direct and indirect comparisons .
the integrated database only included studies that were conducted by eli lilly or its partners outside japan ; therefore , sponsorship bias can not be fully mitigated .
using this database resulted in inclusion of fewer studies than publication - based meta - analyses ; however , this methodology enabled us to analyze individual patient data that are not available in many publications .
similarly , using this global database resulted in inclusion of caucasians as the majority ( 58.3% ) of the patient population .
another limitation of this meta - analysis is that it only consisted of two ssris ( paroxetine and escitalopram ) although four ssris are available in japan .
furthermore , ssri doses used in this analysis ( paroxetine 20 mg / day and escitalopram 10 mg / day ) were at the lower approved dosage in japan ( 2040 mg / day for paroxetine , and 1020 mg / day for escitalopram ) , whereas the duloxetine doses used in this analysis ( 40 mg / day or 60 mg / day ) were the dosages approved in japan .
further research that includes all available ssris using flexible dosing within their full therapeutic ranges will make it possible to generalize conclusions to the ssri group as a whole .
in addition , the exclusion of patients randomized to duloxetine > 60 mg / day limits the ability to generalize conclusions in some countries with approved dosages above a maximum of 60 mg / day , but this was not within the scope of our analysis .
lastly , in these analyses , no correction for multiplicity was performed , so superiority based on p - values can not be assessed .
the integrated database only included studies that were conducted by eli lilly or its partners outside japan ; therefore , sponsorship bias can not be fully mitigated . using
this database resulted in inclusion of fewer studies than publication - based meta - analyses ; however , this methodology enabled us to analyze individual patient data that are not available in many publications .
similarly , using this global database resulted in inclusion of caucasians as the majority ( 58.3% ) of the patient population .
another limitation of this meta - analysis is that it only consisted of two ssris ( paroxetine and escitalopram ) although four ssris are available in japan .
furthermore , ssri doses used in this analysis ( paroxetine 20 mg / day and escitalopram 10 mg / day ) were at the lower approved dosage in japan ( 2040 mg / day for paroxetine , and 1020 mg / day for escitalopram ) , whereas the duloxetine doses used in this analysis ( 40 mg / day or 60 mg / day ) were the dosages approved in japan .
further research that includes all available ssris using flexible dosing within their full therapeutic ranges will make it possible to generalize conclusions to the ssri group as a whole .
in addition , the exclusion of patients randomized to duloxetine > 60 mg / day limits the ability to generalize conclusions in some countries with approved dosages above a maximum of 60 mg / day , but this was not within the scope of our analysis .
lastly , in these analyses , no correction for multiplicity was performed , so superiority based on p - values can not be assessed .
comparison between duloxetine and ssri demonstrated comparable efficacy within the japan - approved dose range in patients with mdd .
results of the hamd17 subscale analysis indicated that duloxetine might be superior to ssris in improving loss of energy and interest .
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backgroundapproved doses of antidepressants in japan are usually lower than those in the usa and european union , but to date meta - analyses comparing antidepressants have all used the higher doses approved in the usa and european union and often have used indirect comparisons .
the purpose of this study was to conduct an integrated database analysis of patient level data to compare the effects of duloxetine with those of selective serotonin reuptake inhibitors ( ssris ) at the doses approved in japan.methodspooled data were analyzed from four randomized , double - blind , placebo - controlled studies that compared duloxetine at the dose range approved in japan ( 4060 mg / day ) with other ssris ( paroxetine 20 mg / day or escitalopram 10 mg / day ) and placebo in patients with major depressive disorder . in total , 1,694 patients were included in the analysis ( duloxetine , n=688 ; selective serotonin reuptake inhibitors , n=690 ; placebo , n=316 ) .
the primary outcome measure was the mean change from baseline at week 8 in 17-item hamilton rating scale for depression ( hamd17 ) total and subscale scores.resultsduloxetine and both selective serotonin reuptake inhibitors were superior to placebo in hamd17 total score at week 8 in both the all - randomized group and the more severe subgroup ( hamd17 total scores 19 ) .
duloxetine was superior to ssris in improving the hamd17 retardation subscale score ( least squares mean difference [ 95% confidence interval ] ) : all - randomized group , 0.33 [ 0.60 , 0.07 ] , p=0.015 ; severe subgroup , 0.45 [ 0.83 , 0.07 ] , p=0.020).conclusionwithin the dose range approved in japan for patients with major depressive disorder , duloxetine and selective serotonin reuptake inhibitors demonstrated comparable overall efficacy , with a possible advantage for duloxetine in improving loss of energy and interest . to the best of our knowledge , this analysis is unique not only in evaluating dosages specific to japan , but also in using individual patient data and the same endpoint across studies to allow for strictly direct head - to - head data comparisons as opposed to pooling direct and indirect comparisons .
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Introduction
Materials and methods
Study selection and data collection
Statistical analysis
Results
Patient demographics and disposition
Efficacy comparison using HAMD
Response
Remission
Safety
Discussion
Limitations
Conclusion
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we all have a very different understanding of how we define a need for help .
this is most clearly felt when encountering individuals who , despite outside temperatures below zero , prefer sleeping rough in the street over the warm place in an overnight homeless shelter that a social worker or other care worker has offered them .
if they additionally show no response to medical diagnoses such as open ulcers , flu infections or severe breathing difficulties , we can safely say that this person s behaviour deviates fundamentally from the expectations and understanding that health professionals would normally associate with need for action .
being exposed to people who reject such support services , which seem well - motivated and necessary from a medical point of view , is only one of several fruitful challenges that are deliberately provoked as part of the new teaching concept in the institute for the history of medicine at giessen university .
the objectives are as follows : to get students to reflect systematically and critically upon conventional and thus familiar ways of thinking and acting , expectations and beliefs in medicine ; to get students to reflect upon central concepts such as bedarf / bedrfnis ( needs in objective vs. subjective terms ) that are normative in character and have a structuring and guiding effect on medical care services ; to advance and refine students soft skills , including the ability to put themselves in the position of other people , in particular those using medical services ; to employ the method of participant observation in order to gain awareness of patients concrete social and environmental living conditions , in this case people on the fringe of society ; and to raise students awareness of what can be deemed suitable support services for people whose behaviour is not consistent with the conventional expectations of health professionals . to get students to reflect systematically and critically upon conventional and thus familiar ways of thinking and acting , expectations and beliefs in medicine ; to get students to reflect upon central concepts such as bedarf / bedrfnis ( needs in objective vs. subjective terms ) that are normative in character and have a structuring and guiding effect on medical care services ; to advance and refine students soft skills , including the ability to put themselves in the position of other people , in particular those using medical services ; to employ the method of participant observation in order to gain awareness of patients concrete social and environmental living conditions , in this case people on the fringe of society ; and to raise students awareness of what can be deemed suitable support services for people whose behaviour is not consistent with the conventional expectations of health professionals .
these teaching objectives are implemented and pursued , among others , in the interdisciplinary field querschnittsbereich 2 / q 2 history , ethics and theory of medicine ,
that is part of the approbationsordnung fr rzte/(ao : regulations for licensing doctors ) ( ao , 2002 ) . as a matter of fact , some of the objectives
are also presented and discussed in some medical school - curricula in medical sociology and psychology , as well as in community medicine / public health , however , in these contexts , the primary emphasis here is on the methodological and conceptual repertoire from their related disciplines , sociology and psychology . -
similar teaching concepts as those presented in q 2 can also be found at an international level , where they tend to be associated with the concept of medical humanities ( " kulturwissenschaften in der medizin ) , .
medical faculties vary in their interpretation and weighting of these teaching concepts ; however , in the context of research and debate in medical education , the main focus is on the relationship between the three approaches ( history , ethics , theory : additive or integrative ; weighting of individual components ) , which are specified explicitly in the ao and which intend to introduce a reflective perspective on current medicine , .
this paper sets out to discuss the possibility of supplementing the above - described methods with methodological approaches and concepts from another discipline based in cultural studies , namely medical anthropology ( or medical ethnology ) , and to apply these in the context of a specifically designed form of experience to which students are exposed . outside of germany
, medical anthropology is an established discipline in university departments , degree courses and associations .
the seminar medical care on the fringes of society : participant observation and change in perspectives has been designed with these approaches and experiences in mind .
it has been offered as a seminar option in q 2 at the medical school of giessen university since the 2010 - 11 winter semester .
the new teaching concept was developed on the basis of the following findings and considerations : in the standard degree courses required by the regulations for licensing doctors medical students will get to experience doctor - patient interaction almost exclusively in a hospital setting or in doctors surgeries , in other words , in institutional environments which in terms of their organisation and management of internal procedures are driven by the rationality of medical thinking ( in addition to political and economic requirements ) . as a result ,
the focus is on problems as defined by physicians as well as related activities ( diagnosis , therapy , consulting ) .
the doctors and other medical staff working in such contexts are naturally familiar with the environment , but those who come to these institutions complaining of being unwell or suffering from acute illness ( patients from a doctor s point of view ) will often be new to this environment .
this in itself can indeed become a source of additional stress , leading to feelings of , among others , uncertainty , anxiety and reluctance in doctor / patient interaction .
the asymmetrical relationship between doctors and patients that is predefined by polar concepts such as ailing / healthy , laypeople / experts , those seeking help / those providing help is further exacerbated by the lack of familiarity / familiarity in the actual setting.as has been repeatedly demonstrated , the problem definitions employed in medical settings revolve around specific illnesses , perceptions of the body und self - images that have been defined by doctors and through medical research .
however , they do not necessarily reflect how those attending medical care institutions would themselves define their problems and concerns .
the priorities for action that result from the individual illness of the patient not seldom deviate from the priorities that are deduced from a scientifically based perception of disease in medicine , . in recent years
, the debate has become increasingly systematic , for instance in relation to the treatment of patients suffering from chronic illness , e.g. , acute pain syndromes , or advanced stages of cancer : it became clear that procedures that are primarily rooted in medical rationality ( maximum diagnosis and therapy including all available possibilities afforded by science and technology ) not seldom contradict the need of the patient , which is to live their remaining life as fully and comfortably as possibly given the circumstances according to their own subjective criteria , .
however , the issue of systematically considering and ultimately prioritising the patient s perspective still continues to be on the periphery in medical training .
the same applies to medico - ethical considerations and evaluations , which too will yield different conclusions if , for example , the risks of therapeutic or research interventions are systematically understood not as predefined medical entities ( e.g. , by way of morbidity and mortality rates for specific medical complications ) , but viewed and evaluated instead from the point of view of the patient .
the above - outlined problem becomes clear if we consider the different aspects of meaning inherent in the conceptual pair
bedarf/bedrfnis , terms referring to needs in the perspective of the physician , or the patient respectively : while the use of the term bedarf presupposes ( scientific ) objectivity and is therefore widely employed in the sense of measurable and essentially quantifiable entities in medical and political debates on medical care , the term
bedrfnis is far more closely linked to the subjective perception of an individual or a particular predefined group.empirical research on medical care for people with migratory backgrounds in a number of different health care systems strongly points to the fact that the issues and problems as regards medical or health care services that become apparent with socially marginalised groups are by no means exclusively limited to these . on the contrary
, these problems tend to be phenomena of a ubiquitous nature that apply universally in the context of medical care and doctor / patient interaction , which become strikingly apparent when dealing with socially marginalised groups .
if looked upon closely , in the german health care system such phenomena , including , for example , that patients do not fully understand the medical information that is given them , or that patients perceptions of their body , and the way in which they describe illness or pain is considered inadequate by medical staff , are by no means limited to people of exotic or migratory backgrounds , but can equally be observed with native users , .
moreover , there is evidence that medical staff tends to de - individualise , standardise and in part stigmatise behaviour of people with a migrant background .
such behaviour is frequently perceived as exotic and therefore attributed to the culture or supposed biological disposition . upon closer inspection , such stereotyping and stigmatising perceptions and evaluation
may also be observed in relation to certain patients from within the own society : ample evidence in support of this is provided by patients suffering from chronic pain or obesity .
another striking example are socially marginalised groups such as black americans in the united states ( e.g. , , , ) ; but this too happens in the german health care system , , .
stereotypical ways of thinking and acting as well as implicit normative assumptions , which act as a potential source of misunderstandings , conflict or problems in medicine , can therefore be illustrated and reflected upon using the example of socially marginalised groups . in the standard degree courses required by the regulations for licensing doctors
medical students will get to experience doctor - patient interaction almost exclusively in a hospital setting or in doctors surgeries , in other words , in institutional environments which in terms of their organisation and management of internal procedures are driven by the rationality of medical thinking ( in addition to political and economic requirements ) . as a result , the focus is on problems as defined by physicians as well as related activities ( diagnosis , therapy , consulting ) .
the doctors and other medical staff working in such contexts are naturally familiar with the environment , but those who come to these institutions complaining of being unwell or suffering from acute illness ( patients from a doctor s point of view ) will often be new to this environment .
this in itself can indeed become a source of additional stress , leading to feelings of , among others , uncertainty , anxiety and reluctance in doctor / patient interaction .
the asymmetrical relationship between doctors and patients that is predefined by polar concepts such as ailing / healthy , laypeople / experts , those seeking help / those providing help is further exacerbated by the lack of familiarity / familiarity in the actual setting . as has been repeatedly demonstrated , the problem definitions employed in medical settings revolve around specific illnesses , perceptions of the body und self - images that have been defined by doctors and through medical research .
however , they do not necessarily reflect how those attending medical care institutions would themselves define their problems and concerns .
the priorities for action that result from the individual illness of the patient not seldom deviate from the priorities that are deduced from a scientifically based perception of disease in medicine , . in recent years
, the debate has become increasingly systematic , for instance in relation to the treatment of patients suffering from chronic illness , e.g. , acute pain syndromes , or advanced stages of cancer : it became clear that procedures that are primarily rooted in medical rationality ( maximum diagnosis and therapy including all available possibilities afforded by science and technology ) not seldom contradict the need of the patient , which is to live their remaining life as fully and comfortably as possibly given the circumstances according to their own subjective criteria , .
however , the issue of systematically considering and ultimately prioritising the patient s perspective still continues to be on the periphery in medical training . the same applies to medico - ethical considerations and evaluations , which too will yield different conclusions if , for example , the risks of therapeutic or research interventions are systematically understood not as predefined medical entities ( e.g. , by way of morbidity and mortality rates for specific medical complications ) , but viewed and evaluated instead from the point of view of the patient .
the above - outlined problem becomes clear if we consider the different aspects of meaning inherent in the conceptual pair
bedarf/bedrfnis , terms referring to needs in the perspective of the physician , or the patient respectively : while the use of the term bedarf presupposes ( scientific ) objectivity and is therefore widely employed in the sense of measurable and essentially quantifiable entities in medical and political debates on medical care , the term
bedrfnis is far more closely linked to the subjective perception of an individual or a particular predefined group .
empirical research on medical care for people with migratory backgrounds in a number of different health care systems strongly points to the fact that the issues and problems as regards medical or health care services that become apparent with socially marginalised groups are by no means exclusively limited to these . on the contrary ,
these problems tend to be phenomena of a ubiquitous nature that apply universally in the context of medical care and doctor / patient interaction , which become strikingly apparent when dealing with socially marginalised groups .
if looked upon closely , in the german health care system such phenomena , including , for example , that patients do not fully understand the medical information that is given them , or that patients perceptions of their body , and the way in which they describe illness or pain is considered inadequate by medical staff , are by no means limited to people of exotic or migratory backgrounds , but can equally be observed with native users , .
moreover , there is evidence that medical staff tends to de - individualise , standardise and in part stigmatise behaviour of people with a migrant background .
such behaviour is frequently perceived as exotic and therefore attributed to the culture or supposed biological disposition . upon closer inspection , such stereotyping and stigmatising perceptions and evaluation
may also be observed in relation to certain patients from within the own society : ample evidence in support of this is provided by patients suffering from chronic pain or obesity .
another striking example are socially marginalised groups such as black americans in the united states ( e.g. , , , ) ; but this too happens in the german health care system , , .
stereotypical ways of thinking and acting as well as implicit normative assumptions , which act as a potential source of misunderstandings , conflict or problems in medicine , can therefore be illustrated and reflected upon using the example of socially marginalised groups .
the three findings outlined above provided the basis for a teaching concept that incorporates the educational potential provided by concepts and procedures used in medical anthropology .
moreover , it draws on the specific experience that students gathered working with people from socially marginalised groups , or more precisely : without a permanent address .
the aim was to systematically draw students attention to the following aspects and dimensions : the significance of each particular life situation , experience of suffering and particular ways of patients interaction in order to design ( medical ) support services that will pick up on their specific needs ; in this context the concept and method of field research and participant observation from ( medical ) anthropology is taught;the unsettling impact an unfamiliar setting can have in the context of doctor / patient interaction , and as a consequence gaining awareness of the importance of one s own subjectivity ( i.e. , of the medical staff ) in one s dealing with persons requiring help ; in this context the modified concept of counter - transference from ethno - psychoanalysis ( following devereux ) is addressed;getting to know and immersing themselves in the living environments , health problems and specific needs of people living on the fringe of society following the model of cultural immersion approaches , : aside from more general phenomena such as poverty and physical neglect , we are dealing here with precarious conditions in terms of hygiene and nutrition , multimorbidity , often coinciding with psychiatric comorbidity , along with the effect of stigmatisation and shame over the ( non-)use of available ( medical ) support services .
the aim is to look closely at that which is ( supposedly ) exotic and bewildering , and to overcome the fear of entering unfamiliar territory;exposing the problems of the concept need for help in the context of the concrete state of a concrete individual ( using the conceptual pair bedarf / bedrfnis ) : on the one hand , the focus here is on analysing central guiding concepts in medicine that bear relevance in relation to medical theory ; on the other , the intention is to draw students attention to the fact that the medical perspective on the discomfort presented ( on scientific grounds , objective ) needs to be meaningfully and systematically complemented by including the patient s perspective ( subjective , in the context of their biography and life situation , partly ambivalence / rejection of support services ) .
the significance of each particular life situation , experience of suffering and particular ways of patients interaction in order to design ( medical ) support services that will pick up on their specific needs ; in this context the concept and method of field research and participant observation from ( medical ) anthropology is taught ; the unsettling impact an unfamiliar setting can have in the context of doctor / patient interaction , and as a consequence gaining awareness of the importance of one s own subjectivity ( i.e. , of the medical staff ) in one s dealing with persons requiring help ; in this context the modified concept of counter - transference from ethno - psychoanalysis ( following devereux ) is addressed ; getting to know and immersing themselves in the living environments , health problems and specific needs of people living on the fringe of society following the model of cultural immersion approaches , : aside from more general phenomena such as poverty and physical neglect , we are dealing here with precarious conditions in terms of hygiene and nutrition , multimorbidity , often coinciding with psychiatric comorbidity , along with the effect of stigmatisation and shame over the ( non-)use of available ( medical ) support services . the aim is to
look closely at that which is ( supposedly ) exotic and bewildering , and to overcome the fear of entering unfamiliar territory ; exposing the problems of the concept need for help in the context of the concrete state of a concrete individual ( using the conceptual pair bedarf / bedrfnis ) : on the one hand , the focus here is on analysing central guiding concepts in medicine that bear relevance in relation to medical theory ; on the other , the intention is to draw students attention to the fact that the medical perspective on the discomfort presented ( on scientific grounds , objective ) needs to be meaningfully and systematically complemented by including the patient s perspective ( subjective , in the context of their biography and life situation , partly ambivalence / rejection of support services ) . in achieving these objectives , as briefly outlined above , in addition to the medical body of knowledge , concepts and methods from ( medical ) anthropology were introduced as central components of the seminar .
this essentially involves the method of field research including participant observation , : central to this method is that those at the focus of medical attention will be visited in their everyday living settings .
an additional effort is made to find out about their physical and social environment and their daily routines in order to comprehend the importance of these factors in relation to the patients perceptions of their body and state of mind , and in relation their help - seeking behaviour .
in addition to visiting the living environment of those affected , systematic monitoring and as far as possible participating in their everyday lives , interviewing informers from the group in focus , together with the systematic documentation of experiences and reflections , form essential elements of this method , .
this will enable students to get a better grasp , for example , of the behaviour displayed by the strangers they encounter ( which at first glance may often seem obscure and inadequate ) within the logic and conditionality of the particular contextual setting .
students will also be introduced to the central concept and methodological approach of counter - transference ( as modified by the ethnologist and psychoanalyst george devereux ) : according to this concept contrary to a widely shared assumption researchers and other professionals working with people too respond emotionally to these persons and the conditions or behaviours observed in connection with them .
such emotional responses range from sympathy to various feelings of ambivalence to fear or aggression , which can impact upon professional interaction as well as prolong or shorten it , or even lead to selective perception and specific interpretation of observed behaviours and even persons . following devereux , researchers or doctors
are asked to systematically observe and reflect upon their own subjectivity and employ their findings , as it were , as an examination tool in its own right that will in addition to other methods yield a more holistic and rounded image of the other .
encounters with homeless people can as a result of their often radically different way of living and occasionally extreme physical neglect etc . trigger particularly striking responses from those providing help ; a situation that serves well as an introduction to systematic self - reflection , among other things , on behavioural patterns that are likely to result from such emotional responses .
in addition to concepts and methods based in anthropology / ethnology and ethno - psychoanalysis , the seminar also teaches the basic principles about the general social , economic and legal conditions affecting the life of people without a permanent address .
cooperation with relevant institutions plays a major role in this : our main partner is frankfurter verein fr soziale heimsttten e.v . ( fv ) ,
in the summer semester we additionally have elisabeth strassenambulanz ( esa ) ( backed by : caritasverband frankfurt / m . ) on board .
the fv is an institution that has close ties with the city and offers an extensive range of services to different groups of homeless in frankfurt / main . during the winter months fv social workers drive the klte - bus to those spots where the homeless
are known to congregate and , if required , supply them with tea , blankets and sleeping pads .
the social workers are the first points of contact for the homeless and , if required , will arrange placements offering follow - on support services , including transport organisation .
the esa provides services aimed at improving the health of women and men in housing difficulties , bearing in mind the unusual living conditions of these people . that a profound knowledge of the particular needs of homeless people as well as of institutional and legal possibilities in dealing with homeless people plays a central role in designing and implementing meaningful teaching concepts
however , the focus there was on teaching concepts that concentrate mainly on providing medical treatment for socially marginalised groups , and less on the meta - level of systematic self - reflection and the ability to take on a different perspective , which is the focal point of the project presented here .
the seminar is structured into a preparatory theoretical - methodological module , a practical module , namely the field work ( in the winter semester with the klte - bus , in the summer semester visiting social work in the context of the esa ) , and a follow - up module .
the seminar groups consist of around 10 students ; this figure is based upon the limited number of individuals when accompanying the klte - bus and in the context of the esa ( one student per appointment ) .
the first part of the preparatory sessions ( 2 hours ) takes place in the classrooms at giessen university . here
students are informed about the legal , social and medical situation of people on the fringe of society ; previous experience and expectations from students are likewise discussed .
in addition , students are familiarised with the experience of patients rejecting their offer of support , which can often be disconcerting ; this includes introducing them to the different perceptions people have of illness , and to the concept of counter - transference .
the structure of the responsible social institutions , particularly those based in frankfurt ( fv , esa ; among others ) is presented and practical information on the planned field trip given .
people on the fringe of society : the field work starts now . the second part of the preparatory session ( 2 hours ) takes place on site in the rooms of the fv in frankfurt a preliminary stage of the field trip : the students leave their familiar environment in order to gain new and unfamiliar impressions . in the rooms of the overnight shelter , amidst the homeless persons staying there , fv social workers provide in - depth information interspersed with specialist and personal contributions .
they also shed light on the medical and social situation of the individuals in the shelter and discuss expectations and particularities as regards the organisation of this part of the field work with the students .
the field work proper does not take place in the group setting , but students go out invidudually : one student at a time accompanies the fv or esa social workers on their on - site visits to homeless people .
a 6-hour period is scheduled for this hands - on experience ; in the winter months it includes a trip on the klte - bus between 8 p.m. and 2 a.m. ; in summer the field trip lasts just as long but takes place during the day or in the evening .
students accompany the social workers on their visits on foot or by bike or in the car ; some students get to attend call - outs on the esa s medical bus . during the feed - back sessions ( 2x2 hours )
students relate the different impressions they have had , contextualise them and explain how they felt , thus creating a wide spectrum of different impressions , evaluations and responses , which provide ample opportunities to reflect upon ways of action and potential changes in perception when dealing with patients .
possible essay topics ( the seminar is assessed by essay ) begin to take shape ; these are discussed in - depth and in relation to how individual students approach their work , as well as formal specifications . in the essay students
are asked to present their own personal observations and experiences during the field trip ( e.g. , feeling insecure in the unfamiliar environment , personal responses to coming face to face with the living environment , the appearance and behaviour of homeless people ) ; in a second step the aim is to reflect upon these observations and their significance for medical thinking and acting .
given the limited number of participants ( ten students per semester ) and the relative short period for which the seminar has been available ( since the 2010 - 11 winter semester ; only three seminar groups have fully completed the class ) we are thus far only able to qualitatively comment on the course ; the total number of participants is still too low to allow for any meaningful quantitative - statistical analysis . to date
, 31 students overall have participated in the seminar : 16 women and 15 men .
the anonymous written evaluation contained three open questions ( positive aspects include ; negative aspects include ; what do you take out of this experience for your further studies ? ) and nine questions to be answered by way of a graded scale ( school grades a to f ) . among these ,
three questions were of a general nature ( please assess how much you have learned ; relevance for training as a medical doctor ; overall school grade ) , six related to specific aspects of the seminar , such as design and structure of the teaching concept , preparation of the lecturer , availability and comprehensibility of relevant information / literature or learning environment . the overall feedback on the structure of the course , on the preparation of the lecturer ( b.m . ) and on the learning environment was between positive and very positive ( grade b or a in analogy to the school grading system ) .
the questions was the seminar interesting ? and the seminar encouraged reflection received the top grade ( a ) .
somewhat in contrast likewise an unanimous perception the relevance for hands - on practical application ( relevant for my training as a doctor ) was recognised but felt to be less positive ( grades b and c ) . in answering the open questions , the on - site experience with social workers and ( in a different response ) the
individual tuition as a result of this was perceived to be particularly worthwhile .
another positive comment was that attention was drawn to how a person s concrete living environment affected their health .
finally , the opportunity to look outside the box was felt to be a profound experience that would shape the students further course of studies .
the essays too contained statements that reflected the intensity of the seminar and the potential inherent in the anthropological approach complemented by field work and participant observation .
the following statement has been taken from the discussion section of one of the essays :
never before did i look at frankfurt s bahnhofsviertel in this way [ ] .
another participant in the seminar felt that the field research method opened up an exotic world [ for him ] .
the human being that crosses our daily horizon as a penniless and neglected figure on the periphery suddenly gets to be at the heart of our attention .
both in the evaluation and occasionally also in the essays the wish was expressed to actually extend the practical part of the seminar . arrangements for receiving written feedback from seminar participants at predefined intervals ( one , two and five years ) have been made , and interviews about the long - term effects of learning and reflection processes are planned with focus groups .
the preliminary experiences we gained with the teaching concept providing medical care on the fringe of society : participant observation and change in perspective illustrate that the spectrum of methodological approaches in q 2 ( ao ) can indeed be complemented by concepts from medical anthropology , in combination with exposing students to people in extreme social situations .
the objective of getting students to reflect systematically upon historical , ethical - normative and theoretical foundations in medicine can therefore be extended and intensified in particular as regards the following three aspects : making students aware of the importance of the specific local setting in medical thinking and acting ( aided by the concept of participant observation and field research from ethnology ) , the importance of the subjectivity of all those involved in doctor / patient interaction , including medical staff ( aided by the modified concept of counter - transference from ethno - psychoanalysis ) , and the fact that key medical terms ( such as medically defined need vs. individual needs of the patient ) are essentially context - dependent . making students aware of the importance of the specific local setting in medical thinking and acting ( aided by the concept of participant observation and field research from ethnology ) , the importance of the subjectivity of all those involved in doctor / patient interaction , including medical staff ( aided by the modified concept of counter - transference from ethno - psychoanalysis ) , and the fact that key medical terms ( such as medically defined need vs. individual needs of the patient ) are essentially context - dependent . at a more general level students
will learn how to put themselves in the position of different protagonists in a range of medical settings , and practice the skill of reflecting critically upon putative conceptual / theoretical and normative - ethical assumptions in medicine .
the teaching project therefore ties in with international models that likewise seek to integrate these teaching objectives in their medical training .
the method of field research ( or inspired by this in the context of medical training that of cultural immersion ) has proven to be very helpful for understanding the perceptions and needs of socially or ethically marginalised groups , and in principle all other groups in health care , , , , [ . in relation to this in the context of the new teaching concept the attention on social determinants of health and how they impact on the pathogenesis and course of health conditions
yield synergies with other teaching concepts , for example in medical sociology , community medicine / public health or general medicine , , which focus on similar matters .
collaborations with community medicine practitioners working for example at technical colleges / fachhochschulen or other institutions outside universities could likewise bear relevance .
, it will be necessary to know and familiarise oneself with the central ( medical ) anthropological concepts and methods .
further , opportunities will have to be created that will enable cooperation with such institutions that are willing to set up the organisational framework and make available their expertise to enable the direct contact of students with people on the fringe of society in their everyday living environments .
the new curricular option described here could only be realized through the co - operation and engagement of many members of staff at the frankfurter verein fr soziale heimsttten e.v . as well as the elisabeth - straen - ambulanz frankfurt / m .
( esa ) . we want to express special thanks to heiko ewald and christine heinrichs , together with the staff of the kltebus-project , the ostpark - project , the beratungszentrum fr aufsuchende sozialarbeit , as well as dr .
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this paper examines the new teaching concept providing medical care on the fringe of society : participant observation and change in perspectives in the context of the interdisciplinary field of querschnittsbereich 2/q 2 ( the transdisciplinary section under ao , the german regulations for licensed physicians ) that explores the history , theory and ethics of medicine .
the disciplinary approach usually adopted in q 2 is supplemented with concepts from medical anthropology ; in addition students will be exposed to people in extreme social situations . the aim is to make students aware of and invite them to reflect upon : the importance of participant observation in the specific on - site setting of medical thinking and acting ; the importance of the subjectivity of all those involved in doctor / patient interaction ; and the fact that key medical terms ( such as the need as seen by the physician vs. the need as seen by the patient ) are essentially context - dependent in their interpretation . at a more
general level students will learn how to put themselves in the position of different protagonists in a range of medical settings , and practice the skill of reflecting critically upon putative conceptual / theoretical and normative - ethical assumptions in medicine .
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Introduction
Project description
Implementation
Evaluation
Discussion
Acknowledgement
Competing interests
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in the recent millennium , the constant trend of change in the demands of the community as well as transforming the trend of knowledge production has highlighted the necessity for researchers to adopt a more comprehensive approach .
increasingly , many academic disciplines are utilizing qualitative research ( qr ) as the qualitative method investigating the why and how of the process of a developed concept ( 1 , 2 ) .
qualitative research is sometimes defined as interpretive research , and as interpretations can be incorrect or biased , the findings may be controversial ( 3 ) . however , qualitative research is not only useful as the first stage of quantitative research , but can also play a key role in validating it or in providing a different viewpoint on the same social phenomena ( 4 ) .
qualitative studies tend to use methods that result in text production rather than numerical outputs .
given that the researcher is considered to be the research instrument , and the plan of inquiry needs to be developed and altered as the study progresses , a qualitative researcher can not depend upon traditional approaches to address certain concerns such as bias and credibility .
therefore , learning from a series of mistakes is often considered an integral part of qualitative research ( 5 , 6 ) . in this study ,
a literature review was carried out in international electronic databases including pubmed , web of sciences , cumulative index to nursing and allied health literature ( cinahl ) , scopus , ebsco , embase and science direct without any time limitation , using the search terms qualitative research ,
these keywords were also searched on national electronic databases including scientific information database ( sid ) , iran medex and medical articles library ( medlib ) using the same strategy .
authors of the present article endeavor to shine a light on the ethical issues affecting researchers and propose strategies to face the ethical challenges of qualitative studies , so as to provide applicable and trustworthy outcomes
. this could be the basis for the formulation of specific ethical guidelines in this regard .
up to the 1970s , qualitative research was solely employed by anthropologists and sociologists . during the 1970s and 1980s ,
however , it was favored by various disciplines and experts of different branches of science and humanity such as health care , psychology , nursing , management , political science , education , and communication studies ( 2 , 7 ) .
qualitative research has been conducted in the field of nursing in order to identify , describe and explain related concepts , experiences and phenomena and to develop the nursing knowledge .
, qualitative research has been performed to achieve the concepts of patient care and other main perceptions in the nursing profession .
qualitative studies provide nurses with sensitivity to the lived experiences of individuals from different nursing care aspects ( 4 , 8) .
in the case of nurses who perform qualitative research , ethical issues are raised when the nurse - patient relationship in the research area leads to some degree of therapeutic communication for the participants ( 9 ) .
thus , nurse researchers must be aware of the impact of the questioning on the participants , and in order to decrease such harmful effects on human subjects , the reflexive approach is recommended ( 10 ) . in qualitative studies researchers
are often required to clarify their role in the research process ( 11 ) . in the qr procedure
the researcher is involved in all stages of the study from defining a concept to design , interview , transcription , analysis , verification and reporting the concepts and themes .
therefore , whenever instruments are involved in qualitative research , a human being will be an integral part of the process ( 12 ) .
it is argued that humans have increasingly become the instrument of choice for naturalistic research due to certain characteristics : they are highly responsive to environmental stimuli , have the ability to interact with the situation , pull together different pieces of information at multiple levels simultaneously , and perceive situations holistically ; moreover , they are able to process findings the instant they become available , can present immediate feedback , and feel unusual responses .
nevertheless , researchers need to improve the abilities that make them appropriate human instruments and consequently , their interpersonal skills are of major importance in natural settings and study processes ( table 1 ) ( 13 , 14 ) .
the relationship and intimacy that is established between the researchers and participants in qualitative studies can raise a range of different ethical concerns , and qualitative researchers face dilemmas such as respect for privacy , establishment of honest and open interactions , and avoiding misrepresentations ( 19 ) .
ethically challenging situations may emerge if researchers have to deal with contradicting issues and choose between different methodological strategies in conflict arises .
in such cases , disagreements among different components such as participants , researchers , researchers discipline , the funding body and the society may be inevitable ( 20 , 21 ) . some important ethical concerns that should be taken into account while carrying out qualitative research are : anonymity , confidentiality and informed consent ( 22 ) . according to richards and schwartz findings ( 22 ) , the term
confidentiality conveys different meanings for health care practitioners and researchers . for health care practitioners , confidentiality means that no personal information is to be revealed except in certain situations . for researchers , however , the duty of confidentiality is less clear and involves elaboration of the form of outcome that might be expected from the study ( 22 , 23 ) .
the researcher must endeavor to minimize the possibility of intrusion into the autonomy of study participants by all means .
when highly sensitive issues are concerned , children and other vulnerable individuals should have access to an advocate who is present during initial phases of the study , and ideally , during data gathering sessions .
it is sometimes even necessary that the researcher clarify in writing which persons can have access to the initial data and how the data might be used ( 24 , 25 ) .
informed consent has been recognized as an integral part of ethics in research carried out in different fields . for qualitative researchers , it is of the utmost importance to specify in advance which data will be collected and how they are to be used ( 26 ) .
the principle of informed consent stresses the researcher s responsibility to completely inform participants of different aspects of the research in comprehensible language .
clarifications need to include the following issues : the nature of the study , the participants potential role , the identity of the researcher and the financing body , the objective of the research , and how the results will be published and used ( 27 ) .
informed consent naturally requires ongoing negotiation of the terms of agreement as the study progresses ( 26 ) .
many people consider it necessary to participate in research that their peers , community and/or society may benefit from .
therefore , qualitative health researchers need to clarify that the research they carry out will benefit science and can contribute to the improvement of health policy ( 5 ) .
the qualitative method is utilized to explain , clarify and elaborate the meanings of different aspects of the human life experience .
therefore , researchers can interpret people s experiences because they are involved in human activities .
the principle of no harm to participants ought to be considered by researchers , who should be aware of the potential harms that might be inflicted upon study subjects .
obviously , sometimes a conflict between the right to know ( defended on the basis of benefits to the society ) and the right of privacy ( advocated based on the rights of the individual ) may happen ( 27 , 28 ) .
there are several effective strategies to protect personal information , for instance secure data storage methods , removal of identifier components , biographical details amendments and pseudonyms ( applicable to names of individuals , places and organizations ) ( 27 ) .
researchers have the responsibility of protecting all participants in a study from potentially harmful consequences that might affect them as a result of their participation .
it is getting increasingly common for research ethics committees to seek documented proof of consent in a written , signed , and ideally , witnessed form .
researchers can only do their best to protect their respondent s identity and hold the information strictly confidential as there would be no guarantee for it otherwise ( 29 ) .
furthermore , in investigations of sensitive topics where written consent puts the informants at risk , audio recorded oral consent would be more appropriate ( 30 ) .
therefore , researchers should seriously consider the potential impact they may have on the participants and vice versa , and details of such interactions should be clearly mentioned in research proposals ( 23 ) .
overall , the role of the researcher as ( a ) stranger , ( b ) visitor , ( c ) initiator , ( d ) insider - expert or other should be well defined and explained ( 3 ) . as brenner quoted kvale state that , preparing an ethical protocol can cover issues in a qualitative research project from planning through reporting ( 30 ) . in qualitative research ,
data are collected with a focus on multifaceted interviews and narratives to produce a description of the experiences .
the researchers , therefore , play the role of a mediator between the experiences of the respondents and the community of concerned people ( 28 , 31 ) .
the post - interview comment sheet could assist the researcher to note the feelings of informants , as well as interpretations and comments that occurred during the interview ( 32 ) .
data collection needs to be as overt as possible , and findings should be recorded .
although there is no guarantee of absolute confidentiality , openly recording field notes assists participants to decide what they wish to have on the record . in health care research , the problem may be even more exaggerated as the researcher is sometimes the health provider as well ( 33 ) . in comparison with other research methods , ethnography has singular characteristics .
when a researcher aims to study the culture of certain people , living amongst them is inevitable .
this method of collecting data is a subject of debate from an ethical point of view .
participants should always be aware of the information that has been obtained and is being recorded , and consent to it .
sometimes this can not be achieved easily and conflicts may happen , as in studies of cultural and ethnic characteristics ( 18 ) .
the physical presence of the researchers within the culture requires them to be responsible for their role and potential consequences on the field .
for instance , when criminals or a group of war veterans suffering from a disease are the subject of a study , the risks involved in living amongst them should be considered .
ethnographers must be vigilant about any distractions stemming from close interactions that can be potentially harmful to participants in the long run ( 33 , 34 ) .
researchers can benefit from supervision sessions directed at learning , mentoring and skill development , all of which can foster their ability to carry out research without risking their health . adequate professional supervision ( which may be outside of the university ) can be of service to researchers in dealing with the potential stress associated with the study ( 35 37 ) . in order to gain explicit data ,
these steps include participant observation , ethnographic record , descriptive observation , taxonomic analysis , selected observation , componential analysis , discovering the cultural theme , cultural inventory , and finally writing ethnography ( 38 , 39 ) .
researchers should always be aware of the precise reason for involvement in a study in order to prevent undesirable personal issues .
the probability of exposure to vicarious trauma as a result of the interviews needs to be evaluated .
interviewers should be properly scheduled to provide the researcher with sufficient recovery time and reduce the risk of emotional exhaustion , while allowing ample time for analysis of the objective and emotional aspects of the research .
it is also necessary for the researcher to be familiar with signs of extreme fatigue and be prepared to take necessary measures before too much harm is done ( 40 42 ) .
the relationship and intimacy that is established between the researchers and participants in qualitative studies can raise a range of different ethical concerns , and qualitative researchers face dilemmas such as respect for privacy , establishment of honest and open interactions , and avoiding misrepresentations ( 19 ) .
ethically challenging situations may emerge if researchers have to deal with contradicting issues and choose between different methodological strategies in conflict arises .
in such cases , disagreements among different components such as participants , researchers , researchers discipline , the funding body and the society may be inevitable ( 20 , 21 ) . some important ethical concerns that should be taken into account while carrying out qualitative research are : anonymity , confidentiality and informed consent ( 22 ) . according to richards and schwartz findings ( 22 ) , the term
confidentiality conveys different meanings for health care practitioners and researchers . for health care practitioners , confidentiality means that no personal information is to be revealed except in certain situations . for researchers , however , the duty of confidentiality is less clear and involves elaboration of the form of outcome that might be expected from the study ( 22 , 23 ) .
the researcher must endeavor to minimize the possibility of intrusion into the autonomy of study participants by all means .
when highly sensitive issues are concerned , children and other vulnerable individuals should have access to an advocate who is present during initial phases of the study , and ideally , during data gathering sessions .
it is sometimes even necessary that the researcher clarify in writing which persons can have access to the initial data and how the data might be used ( 24 , 25 ) .
informed consent has been recognized as an integral part of ethics in research carried out in different fields . for qualitative researchers , it is of the utmost importance to specify in advance which data will be collected and how they are to be used ( 26 ) .
the principle of informed consent stresses the researcher s responsibility to completely inform participants of different aspects of the research in comprehensible language .
clarifications need to include the following issues : the nature of the study , the participants potential role , the identity of the researcher and the financing body , the objective of the research , and how the results will be published and used ( 27 ) .
informed consent naturally requires ongoing negotiation of the terms of agreement as the study progresses ( 26 ) .
many people consider it necessary to participate in research that their peers , community and/or society may benefit from .
therefore , qualitative health researchers need to clarify that the research they carry out will benefit science and can contribute to the improvement of health policy ( 5 ) .
the qualitative method is utilized to explain , clarify and elaborate the meanings of different aspects of the human life experience .
therefore , researchers can interpret people s experiences because they are involved in human activities .
the principle of no harm to participants ought to be considered by researchers , who should be aware of the potential harms that might be inflicted upon study subjects . obviously , sometimes a conflict between the right to know ( defended on the basis of benefits to the society ) and the right of privacy ( advocated based on the rights of the individual ) may happen ( 27 , 28 ) .
there are several effective strategies to protect personal information , for instance secure data storage methods , removal of identifier components , biographical details amendments and pseudonyms ( applicable to names of individuals , places and organizations ) ( 27 ) .
researchers have the responsibility of protecting all participants in a study from potentially harmful consequences that might affect them as a result of their participation .
it is getting increasingly common for research ethics committees to seek documented proof of consent in a written , signed , and ideally , witnessed form .
researchers can only do their best to protect their respondent s identity and hold the information strictly confidential as there would be no guarantee for it otherwise ( 29 ) .
furthermore , in investigations of sensitive topics where written consent puts the informants at risk , audio recorded oral consent would be more appropriate ( 30 ) .
therefore , researchers should seriously consider the potential impact they may have on the participants and vice versa , and details of such interactions should be clearly mentioned in research proposals ( 23 ) .
overall , the role of the researcher as ( a ) stranger , ( b ) visitor , ( c ) initiator , ( d ) insider - expert or other should be well defined and explained ( 3 ) . as brenner quoted kvale state that , preparing an ethical protocol can cover issues in a qualitative research project from planning through reporting ( 30 )
in qualitative research , data are collected with a focus on multifaceted interviews and narratives to produce a description of the experiences .
the researchers , therefore , play the role of a mediator between the experiences of the respondents and the community of concerned people ( 28 , 31 ) .
the post - interview comment sheet could assist the researcher to note the feelings of informants , as well as interpretations and comments that occurred during the interview ( 32 ) .
data collection needs to be as overt as possible , and findings should be recorded . although there is no guarantee of absolute confidentiality , openly recording field notes assists participants to decide what they wish to have on the record . in health care research
, the problem may be even more exaggerated as the researcher is sometimes the health provider as well ( 33 ) . in comparison with other research methods ,
ethnography has singular characteristics . when a researcher aims to study the culture of certain people , living amongst them is inevitable .
this method of collecting data is a subject of debate from an ethical point of view .
participants should always be aware of the information that has been obtained and is being recorded , and consent to it .
sometimes this can not be achieved easily and conflicts may happen , as in studies of cultural and ethnic characteristics ( 18 ) .
the physical presence of the researchers within the culture requires them to be responsible for their role and potential consequences on the field .
for instance , when criminals or a group of war veterans suffering from a disease are the subject of a study , the risks involved in living amongst them should be considered .
ethnographers must be vigilant about any distractions stemming from close interactions that can be potentially harmful to participants in the long run ( 33 , 34 ) .
researchers can benefit from supervision sessions directed at learning , mentoring and skill development , all of which can foster their ability to carry out research without risking their health . adequate professional supervision ( which may be outside of the university ) can be of service to researchers in dealing with the potential stress associated with the study ( 35 37 ) . in order to gain explicit data , ethnographers need to know the role of instrument details .
these steps include participant observation , ethnographic record , descriptive observation , taxonomic analysis , selected observation , componential analysis , discovering the cultural theme , cultural inventory , and finally writing ethnography ( 38 , 39 ) .
researchers should always be aware of the precise reason for involvement in a study in order to prevent undesirable personal issues .
the probability of exposure to vicarious trauma as a result of the interviews needs to be evaluated .
interviewers should be properly scheduled to provide the researcher with sufficient recovery time and reduce the risk of emotional exhaustion , while allowing ample time for analysis of the objective and emotional aspects of the research .
it is also necessary for the researcher to be familiar with signs of extreme fatigue and be prepared to take necessary measures before too much harm is done ( 40 42 ) .
it is argued that qualitative research that deals with sensitive topics in depth can pose emotional and other risks to both participants and researchers .
clear protocols for dealing with distress should be in place so that both parties involved in research can use them if necessary .
it is not usually easy to predict what topics are likely to lead to distress , and researchers should therefore receive sufficient training in predicting traumatic situations .
preventive measures for researchers who carry out sensitive qualitative studies should include official arrangements for a peer support program consisting of a list of researchers who are involved , or a constellation of researcher support activities aiming at improving psychological fitness in the form of a professional confidence building module .
other such measures include offering adequate supervision to provide opportunities for self - development and self - care , and facilitating the process of self - reflection and self - monitoring .
strategies for emotional distancing need to be considered and adopted if the research topic or participants have the potential to be emotionally challenging .
an appropriate planning should be in place before the commencement of the fieldwork , and it must be perfectly clear how the study should be conducted and what level of relationship development is necessary .
measures must also be taken so that levels of self - disclosure , objective displays of emotion during the interviews , and strategies to end the relationships are well defined and communicated .
one of the most prominent tasks of qualitative researchers is to minimize the flaws in observation and endeavor to gain truthful knowledge .
therefore , it is necessary for researchers to continuously update their investigation skills in terms of methodology and find novel techniques to better carry out studies in the field of health and sociology . as explained before ,
qualitative research is carried out in natural settings , which requires researchers to work in close collaboration with other members of the team and under direct supervision to discuss and resolve issues as they arise .
therefore , development of practical strategies and communicating them to researchers can be of great benefit and assist them in conducting more perceptive qualitative studies .
it is noteworthy that such research should be directed towards making a difference in people s lives , improving care delivery in different settings and at all levels , and providing a framework for health sciences without any ethical disturbances . as a result of the extensive body of research in the field of medical sciences ,
research ethics committees are formed to provide independent advice to participants , researchers , funders / sponsors and healthcare organizations on the extent to which research proposals comply with universally endorsed ethical standards . in the history of social and medical science , there have been a few research studies that seriously injured people , and many more in which their welfare was not sufficiently protected .
to return to the matter of privacy , the researcher should not rely solely on the informant to identify possible intrusion , but needs to work at anticipating it in advance .
confidentiality does not necessarily preclude intrusion , as anonymity by itself is not enough to protect a person s privacy or prevent disclosure of personal issues .
investigators should refrain from soliciting private information that is not closely related to the research question .
considering the aforementioned challenges , it is recommended to conduct further research in order to provide meticulous and explicit ethical protocols , guidelines and codes with respect to qualitative studies .
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considering the nature of qualitative studies , the interaction between researchers and participants can be ethically challenging for the former , as they are personally involved in different stages of the study .
therefore , formulation of specific ethical guidelines in this respect seems to be essential .
the present paper aimed to discuss the necessity to develop explicit guidelines for conducting qualitative studies with regard to the researchers role . for this purpose ,
a literature review was carried out in domestic and international databases by related keywords.health care providers who carry out qualitative research have an immense responsibility .
as there is no statistical analysis in qualitative studies , the researcher has to both evaluate what he or she observes and to interpret it .
providing researchers with the necessary skills and applying stringent supervision can lead to better extraction of reliable information from qualitative studies .
this article presents a debate in order to illustrate how researchers could cover the ethical challenges of qualitative studies and provide applicable and trustworthy outcomes.researchers face ethical challenges in all stages of the study , from designing to reporting .
these include anonymity , confidentiality , informed consent , researchers potential impact on the participants and vice versa .
it seems of paramount importance that health care providers , educators and clinicians be well informed of all the different aspects of their roles when acting as qualitative researchers .
hence , these adroit roles need to be well defined , and the use of practical guidelines and protocols in all stages of qualitative studies should be encouraged .
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Introduction
An overview on qualitative research in health care
Role of researchers in qualitative studies
Ethical challenges in qualitative studies:
The researcher-participant relationship
Research design
Data gathering and data analysis
Conclusion
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crh is a 41-amino - acid peptide hormone synthesized in the paraventricular nucleus of the hypothalamus .
its function in the central nervous system is to stimulate the hypothalamic - pituitary - adrenal ( hpa ) axis as part of the stress response .
crh is also produced in peripheral tissues including the placenta of humans and hominine primates [ 13 ] .
placental crh secretion results in an exponential rise of crh concentration in maternal plasma during the third trimester of gestation .
the rate of the increase is related to gestational length , since the rise of crh level is accelerated in pregnancies ending with preterm birth , while the increase is retarded in pregnancies that continue after term .
because of this relationship , it is believed that the regulation of placental crh production is linked to the mechanism that determines the length of pregnancy and triggers labour and delivery .
the mechanism regulating the exponential increase in placental crh expression remains unclear although positive feedback by glucocorticoids and increasing numbers of syncytial cell nuclei are suggested explanations [ 5 , 6 ] .
spontaneous and agonist - induced syncytium formation by cytotrophoblasts is associated with crh expression with camp being a strong stimulant of both syncytial differentiation and crh gene activity .
molecular studies using crh promoter - reporter constructs indicated that transcription factor complexes bound to a consensus cyclic - amp response element ( cre ) at 224 bp upstream of the major transcription initiation site mediated the camp - stimulation , although a nonconsensus second promoter site was also implicated in the cyclic nucleotide response [ 811 ] .
it has been suggested that these molecular interactions are involved in the gestational age dependent control of crh gene activity .
epigenetic chromatin modifications define cell - specific gene expression potential and alter gene expression patterns during cell differentiation and development .
methylation of cytosines at cpg dinucleotides in dna is a well - characterized epigenetic chromatin modification generally associated with closed chromatin structure and gene repression .
furthermore , methylation of cpg sites within particular transcription factor binding sequences may modify transcription factor binding affinity and alter regulatory changes in gene expression [ 1417 ] .
the human crh proximal promoter contains 9 cpg dinucleotides with one located within the methylation sensitive cre sequence .
in addition , the promoter is within 1000 bp distance from an intragenic cpg island , which corresponds to a cpg island shore
region , the methylation of which is related to tissue specific gene expression . therefore , in the present investigation we have explored the possibility that methylation of the promoter contributes to the control of crh expression in trophoblast cells .
we used the bewo choriocarcinoma cell line in the experiments , which is a well - characterized trophoblast model exhibiting dynamic dna methylation as well as ability to syncytialise and increase crh expression when stimulated with camp [ 1921 ] .
the bewo human choriocarcinoma - derived cell line was obtained from the american type culture collection ( manassas , va , usa ) ( atcc # ccl-98 ) .
the culture medium was dmem / f12 ( with hepes and l - glutamine , without phenol red ) supplemented with 10% ( v / v ) fetal bovine serum ( fbs ) and 1x antibiotic - antimycotic ( gibco / life technologies , mulgrave , vic , australia ) .
cells were cultured at 37c in a humidified atmosphere of 5% co2 in air . at approximately 80% confluence cells
were detached with trypsin / edta solution ( gibco ) , washed , counted , and transferred into six - well plates at a density of 0.8 10 cells / well .
cultures reaching 50% confluence were incubated with fresh medium with or without 8-br - camp ( 8-bromoadenosine-3,5-cyclic monophosphate , 2.5 10 mol l ) and/or 5-aza - dc ( 5-aza-2-deoxycytidine , 5
10mol l ) ( sigma - aldrich , sydney , nsw , australia ) followed by harvesting for rna and dna isolation .
drug concentrations were optimised in previous studies and showed no toxic effects in bewo cells at the concentrations and exposure times employed [ 19 , 22 , 23 ] .
rna was extracted from cells using the rneasy mini kit ( qiagen , chadstone centre , vic , australia ) according to the manufacturer 's protocol .
rna was eluted from the rneasy spin columns with 30 l of rnase - free water and quantified using a nanodrop 1000 spectrophotometer ( thermo fisher scientific australia , scoresby , vic , australia ) .
contaminating dna was removed by dnase treatment using the turbo dna - free kit ( ambion / life technologies ) following the routine protocol .
the total reaction volume was 20 l including 2 l of 10x dnase buffer , 1 l of dnase , and up to 2 g of rna .
rna integrity in all samples was assessed by agarose gel electrophoresis . prior to reverse transcription , the rna samples were spiked with 5 10 copies of alien rna transcript ( supplied with the alien qrt - pcr inhibitor alert kit , stratagene / integrated sciences , chatswood , nsw , australia ) per microgram rna .
the alien rna transcript served as a reference rna of equal abundance in all samples and pcr runs .
rna was reverse transcribed using the superscript iii first - strand synthesis system for rt - pcr ( invitrogen ) with random hexamer primers .
real - time pcr was performed using an applied biosystems 7500 real - time pcr system with reagents supplied by the manufacturer ( applied biosystems / life technologies ) .
the amplification reaction contained template cdna from 20 ng of reverse - transcribed rna , 6 10 moles l forward and 3 10 moles l reverse primer , sybr green master mix , and milliq water to a total volume of 25 l .
the crh cdna primers were designed and optimised by sehringer et al . and are listed in table 1 .
primer sequences for amplifying alien cdna are proprietary and were used according to the manufacturer 's instructions ( stratagen / integrated sciences ) .
the temperature sequence was 50c for 2 min , 95c for 10 min , 40 cycles of 95c for 15 s , and 60c for 1 min , followed by melting curve analysis .
no - template control and no - reverse transcriptase controls for all samples were included to detect residual genomic dna .
expression levels of the crh mrna were determined relative to alien rna according to the ct method .
cells grown in six - well plates were collected in 750 l pbs ( phosphate - buffered saline ; 137 10mol l nacl , 2.7 10 mol l kcl , 8 10mol l na2hpo4 , and 2 10 mol l kh2po4 , ph 7.4 ) using cell scrapers and centrifuged for 5 min at 300 g .
cell pellets were resuspended in 200 l of pbs and processed for dna isolation as per the manufacturer 's instructions .
genomic dna was eluted from the mini spin columns with 200 l milliq water , quantified using the nanodrop 1000 spectrophotometer , and stored at 4c .
up to 300 ng of genomic dna was bisulfite - converted and purified using the methylseqr bisulfite conversion kit ( applied biosystems ) .
the crh proximal promoter regions were pcr - amplified using the toptaq master mix kit ( qiagen ) and two sets of nested primers designed with the methyl primer express software v.1.0 ( applied biosystems ) .
pcr reactions contained purified bisulfite - converted dna template , 25 l of 2x toptaq master mix , 4 10 mol l of each primer , and milliq water up to 50 l final volume .
the conditions for the first pcr amplification included an initial step at 94c for 3 min , followed by 30 cycles of 94c for 30 s , 54c for 30 s , and 72c for 1 min and a final extension step at 72c for 10 min .
one microliter of a 20-fold diluted aliquot of the first pcr reaction was used as template for the second pcr amplification using the nested primer set .
pcr conditions were 94c 30 min , 30 cycles of 94c for 30 s , 50c for 30 s , and 65c for 1 min and an extension step at 65c for 10 min . following amplification , 20 l of the pcr reaction mixture
was separated by agarose gel electrophoresis and the amplification product was visualised with ethidium bromide .
the gel slice containing the amplified dna fragment was excised , extracted , and purified using the wizard sv gel and pcr clean - up system ( promega , auburn , vic , australia ) .
the dna was collected in 50 l milliq water by the centrifugation of the sv minicolumn .
the bisulfite - converted and pcr - amplified dna was ligated into the pgem - t easy vector using reagents provided by the manufacturer ( promega ) . the 10 l reaction mixtures contained ligation buffer , 3 weiss units of t4 dna ligase , 50 ng of pgem - t easy vector , and pcr product at 3 : 1 insert : vector molar ratio .
a positive control using the control dna provided and a negative control ( no pcr product ) were also included .
the ligation mixture was used subsequently to transform jm109 competent cells ( promega ) according to the manufacturer 's protocol .
fifty l of transformed cell suspension was spread onto duplicate lb / ampicillin / iptg / x - gal plates and incubated at 37c overnight .
the white streak colonies were picked the next day and cultured in 5 ml luria broth at 37c overnight .
plasmids were isolated from the minicultures using the genelute plasmid miniprep kit ( sigma - aldrich , castle hill , nsw , australia ) .
the presence of inserts was verified by digesting an aliquot from each plasmid preparation with ecor i ( promega ) followed by agarose gel electrophoresis .
plasmids containing the expected size inserts ( 300 bp ) were sequenced from both directions by the australian genome research facility ( agrf , brisbane , qld , australia ) .
the sequencing primers were designed by promega and produced by invitrogen / life technologies ( forward : 5-tatttaggtgacactatag-3 , reverse : 5-tatttaggtgacactatag-3 ) .
quality control was automatically performed and any sequence with an unacceptably low conversion rate or high number of sequencing errors was excluded .
ten randomly selected clones , representing individual gene copies , were processed for methylation frequency analyses from each dna sample using the chi - square test and fisher 's exact test as appropriate .
significant one - sided tests are reported in cases when the two - sided tests showed borderline significance . the stata ( college station , tx , usa ) software package was used for the statistical calculations .
as shown in figure 1 , abundance was not significantly different between 0 h and 24 h and between 24 h and 28 h of incubation
. between 48 h and 72 h , a 2.9-fold increase ( p = 0.029 ) was observed , which was coincident with the reported spontaneous syncytialisation of bewo cells . in the presence of 8-br - camp
( 2.5 10 mol l ) , a powerful stimulant of syncytial differentiation , significant increases of crh mrna level were detected between 0 h and 24 h ( p < 0.0001 ) , 24 h and 48 h ( p = 0.03 ) , and 48 h and 72 h ( p = 0.049 , one - sided t - test ) with a maximum at 72 h , which was 23.2-fold higher than the 0 h level ( figure 1 ) . in cultures treated with the dna methyltransferase inhibitor 5-aza - dc ( 5 10mol l ) a slight , but significant , increase of crh mrna abundance
was observed between 0 h and 24 h ( 1.43-fold , p = 0.028 , one - sided t - test ) , but there was no further change between 24 h and 48 h and between 48 h and 72 h. combined treatment with 8-br - camp and 5-aza - dc resulted in robust increases of crh mrna levels between 0 h and 24 h ( p < 0.0001 ) , 24 h and 48 h ( p = 0.0026 ) , and 48 h and 72 h ( p = 0.039 ) reaching a 86.4-fold rise at 72 h compared to 0 h ( figure 1 ) .
8-br - camp increased crh mrna abundance relative to vehicle at all time points ( 24 h , p = 0.0042 ; 48 h , p = 0.0066 ; 72 h , p = 0.0004 ; figure 1 ) , which confirmed previous findings of the stimulatory effects of 8-br - camp on crh gene expression in bewo cells .
5-aza - dc treatment had no effect at 24 h and 48 h and reduced crh mrna abundance relative to vehicle at 72 h , effectively blocking the increase seen between 48 h and 72 h in vehicle - treated cells ( p = 0.0021 , figure 1 ) . combined treatment with the cyclic nucleotide and the dna methyltransferase inhibitor upregulated crh mrna expression at all time points beyond the level reached in response to 8-br - camp alone ( 24 h , p < 0.0001 ; 48 h , p = 0.0002 ; 72 h , p = 0.0029 ; figure 1 ) .
the significant effects of 5-aza - dc on crh mrna expression suggested that dna methylation was involved in the control of crh gene activity . to explore this further
, we have determined the effects of 8-br - camp and 5-aza - dc on the methylation of the 9 cpg sites present in the crh proximal promoter .
bisulfite sequencing revealed partial methylation ( 38% of the 9 cpg sites combined ) at 0 h , before treatments commenced ( figure 2 ) , which increased spontaneously to 57% ( p = 0.001 ) by 72 h of culture .
treatment with 8-br - camp ( 250 m ) resulted in a similar increase of methylation ( to 61% ) , not significantly different from the vehicle - treated control .
treatment with 5-aza - dc for 72 h reduced promoter methylation to 23% , which was significantly less than the control ( p = 0.001 ) . combined treatment with 5-aza - dc and 8-br - camp increased the level of methylation compared to 5-aza - dc alone ( to 33% , p = 0.011 ) but did not reach the methylation level observed in cells treated with 8-br - camp only ( p = 0.001 , figure 2 ) .
clonal bisulfite sequencing determines cytosine methylation with single base resolution in individual alleles ( gene copies ) .
the technique enabled us to determine the particular cpg sites that undergo methylation changes under the treatment conditions that influence methylation levels overall , as presented in figure 2 .
the scheme in figure 3 shows the positions of the 9 methylatable cpg dinucleotides in the human crh proximal promoter .
the two major transcription initiation sites and the two sequence regions implicated in the camp - response are also indicated with cpg2 residing within the cre [ 811 , 29 ] .
the heatmap in figure 4 illustrates the methylation levels of the 9 cpgs under the treatment conditions employed .
methylation levels were significantly different among the cpg sites ranging from 10% to 70% at 0 h and from 13% to 80% at 72 h of culture ( p < 0.001 , figure 4 ) indicating site - specific differential methylation .
the methylation level of cpgs 1 , 2 , 3 , and 7 , but not of cpgs 4 , 5 , 6 , 8 , and 9 increased significantly during the 72 h culture period demonstrating that methylation was dynamic at these sites .
figure 5 shows the methylation of each cpg in the cloned copies of the crh proximal promoter .
the scattered distribution of methylated and unmethylated cpg sites suggests that the partial methylation observed was allele independent both at 0 h and at 72 h of culture .
cells treated with 8-br - camp and 5-aza - dc had similar scattered distribution of methylated cpgs in individual alleles ( not shown ) .
in cells treated with 8-br - camp for 72 h , the cpg sites remained differentially methylated ( from 23% to 73% , p = 0.004 ) , and the methylation levels of individual cpgs were not significantly different from the corresponding sites in the vehicle - treated control ( figure 4 ) .
treatment with the dna methyltransferase inhibitor 5-aza - dc decreased methylation at all cpg sites compared to vehicle , except for cpg 4 , where methylation was relatively low . furthermore , 5-aza - dc abolished the differences between the methylation levels of the individual cpgs .
the methyltransferase inhibitor eliminated the methylation differences among individual cpgs in the presence of 8-br - camp as well ( figure 4 ) .
cotreatment with 8-br - camp prevented , however , the demethylating action of 5-aza - dc at cpgs 1 , and 8 , but not at cpgs 2 , 3 , 5 , 7 , and 9 ( camp versus camp + aza in figure 4 ) .
finally , no individual cpg site exhibited a statistically significant methylation difference in 8-br - camp + 5-aza - dc - treated cells compared to treatment with the methyltransferase inhibitor alone ( aza versus camp + aza in figure 4 ) despite the small , but significant , overall increase in methylation ( aza 72 h versus camp + aza 72 h , in figure 2 ) .
the aim of this study was to explore the involvement of promoter methylation in crh gene regulation in human trophoblast cells .
placental crh expression is predictive of gestational length and is influenced by pregnancy disorders [ 4 , 30 ] .
dna methylation is a developmentally regulated epigenetic modification influenced by environmental inputs [ 31 , 32 ] , which can potentially control crh gene expression during pregnancy and in response to pathogenic factors . in our experiments we used the choriocarcinoma - derived bewo cell line , which is a well - characterized trophoblast model exhibiting increased crh gene expression during spontaneous and camp - induced syncytial differentiation similar to normal trophoblasts .
our dna sequencing data show that the crh proximal promoter sequence is identical in bewo cells and in normal trophoblasts including all reported transcription factor response elements and the methylation sensitive cpg sites ( figure 3 ) .
we have shown by clonal bisulfite sequencing that the cpgs within the crh proximal promoter are partially methylated with significant differences among the individual sites .
the size of the analysed sequence ( 261 bp ) and the methylation level correspond to an intermediate methylation region , which is a genomic feature implicated in tissue specific epigenetic gene regulation .
this is similar to normal trophoblasts , which also exhibit allele independent partial and differential methylation in the crh promoter region .
dna methylation increases in bewo cells during culture and the dna methyltransferase inhibitor 5-aza - dc changes cell phenotype and gene expression levels [ 21 , 22 , 35 ] .
our results show that the crh promoter follows this general trend , which is different from primary trophoblasts , where crh promoter methylation does not change in culture and remains unaltered by 5-aza - dc and 8-br - camp under the conditions where bewo cells show methylation changes . for this reason , bewo cells
are uniquely suited to explore the relationship between crh promoter methylation level and gene activity .
methylation of cpg island promoters is associated generally with the repression of gene activity [ 12 , 14 , 21 ] .
the crh promoter is located in a cpg island shore region relative to a crh intragenic cpg island ( chr8:67,089,250 - 67,089,962 in the grch37/hg19 assembly , ucsc genome browser ) .
our results showed , however , that neither the spontaneous nor the 8-br - camp - evoked increase of crh gene activity was associated with the demethylation of the crh promoter ( figures 1 and 2 ) .
treatment with 5-aza - dc decreased crh promoter methylation and abolished the cpg site - specific methylation differences as expected ( figures 2 and 4 ) , but it also blocked the spontaneous increase of crh gene expression in culture ( figure 1 ) .
8-br - camp - stimulated crh expression strongly in the presence of 5-aza - dc ( figure 1 ) , but the cyclic nucleotide actually increased methylation in 5-aza - dc - treated cells ( aza 72 h versus camp + aza 72 h in figure 2 ) .
thus , crh expression was directly , and not reciprocally , related to promoter methylation under these conditions
. this relationship may be unexpected in view of the well - documented global association between gene repression and promoter methylation , but genome wide trends do not necessarily predict the behavior of individual genes .
in fact , the methylation level of the cpg - poor class of promoters was found to be uncorrelated with gene activity .
the crh promoter falls into the cpg - poor class according to established criteria , with partial methylation in both the hypothalamus and the trophoblast [ 34 , 37 , 38 ] ( figure 2 ) .
methylation reduces crh gene expression in the hypothalamus as expected ; in trophoblastic bewo cells , however , promoter methylation appears to have the opposite effect as detailed before .
there is evidence to suggest that this cell - specific regulation may result from the methylation - dependent change of the functional properties of the camp - response element ( cre ) in the crh promoter ( figure 3 ) .
the cre is critical in regulating the activity of transfected crh promoter - reporter constructs in trophoblast cells [ 911 ] .
it contains a cpg dinucleotide that , when methylated , reduces the affinity of cre to its cognate transcription factor , creb , and renders it unresponsive to camp - stimulation [ 15 , 39 ] . at the same time , the methylated cre has increased affinity to bind the transcription factor c / ebp - alpha , which often activates tissue specific genes during differentiation .
the cpg within the cre was 30% methylated under basal ( 0 h incubation ) conditions ( cpg2 in figure 4 ) .
culturing for 72 h increased cpg2 methylation to 60% concomitantly with enhanced gene expression , while treatment with 5-aza - dc reduced cpg2 methylation to 23% and diminished crh gene activity ( figures 1 and 4 ) .
considering that c / ebp - alpha is expressed in bewo cells , it is reasonable to conjecture that methylation - evoked changes in the transcription factor binding specificity of the cre may have contributed to the enhanced crh expression observed in association with increased promoter methylation .
the cpg2 in the cre was partially methylated under all experimental conditions , which suggests that the unmethylated portion could have mediated stimulation by 8-br - camp using the canonical , creb - dependent pathway .
moreover , the crh proximal promoter contains a second , noncanonical camp - response element ( figure 3 ) , which contributes to the regulation of the gene specifically in the trophoblast [ 9 , 11 ] .
methylation of the cre may thus function to influence the relative contribution of the two camp - response elements , their associated transcription factors , and the coupled signaling pathways to the overall activity of the crh gene under basal and camp - stimulated conditions .
cotreatment with 5-aza - dc strongly augmented the stimulation of crh mrna expression by 8-br - camp , although the dna methyltransferase inhibitor alone had no stimulatory effect under the same culture conditions ( figure 1 ) .
cotreatment with 5-aza - dc , however , decreased cpg methylation in the cre ( cpg2 , figure 4 ) to 43.3% from 70% measured after 8-br - camp treatment ( p = 0.034 , fisher 's exact test , one - sided ) .
the increased proportion of unmethylated cres in the cell population could explain , at least partially , the augmented response to 8-br - camp - stimulation .
moreover , 5-aza - dc decreases dna methylation globally increasing or repressing the activity of numerous genes [ 4143 ] .
this suggests the possibility that 5-aza - dc may potentially influence crh expression indirectly through intervening gene products generating synergy between 8-br - camp and 5-aza - dc .
although gene activation occurs in 5-aza - dc - treated bewo cells [ 22 , 23 , 35 ] , it remains to be established whether transcription factors or other gene products controlled by dna methylation regulate crh expression in trophoblasts .
we have utilized the dynamic changes of dna methylation in the bewo cell line to explore the relationship between this epigenetic chromatin modification and crh gene expression in a trophoblastic cell type .
clonal bisulfite sequencing revealed the cpg site - specific and allele independent partial methylation of the crh proximal promoter and classified it as an intermediately methylated region of the genome .
the data suggest that promoter methylation determines the contribution of the cre , its various associated transcription factors , and a trophoblast specific alternative camp - response element to crh gene regulation .
furthermore , our results are consistent with the possibility that dna methylation controls crh expression indirectly , but any intervening factor that may regulate crh expression by a dna methylation - dependent mechanism remains to be determined .
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corticotropin releasing hormone ( crh ) production by the human placenta increases exponentially as pregnancy advances , and the rate of increase predicts gestational length .
crh gene expression is regulated by camp in trophoblasts through a cyclic amp - response element ( cre ) , which changes its transcription factor binding properties upon methylation .
here we determined whether methylation of the crh proximal promoter controls basal and camp - stimulated crh expression in bewo cells , a well - characterized trophoblastic cell line .
we treated the cells with 8-br - camp and the dna methyltransferase inhibitor 5-aza-2 deoxycytidine ( 5-aza - dc ) and determined the effects on crh mrna level and promoter methylation .
clonal bisulfite sequencing showed partial and allele independent methylation of cpgs in the crh promoter .
crh mrna expression and the methylation of a subset of cpgs ( including cpg2 in the cre ) increased spontaneously during culture .
8-br - camp stimulated crh expression without affecting the increase in methylation .
5-aza - dc decreased methylation and augmented 8-br - camp - stimulated crh expression , but it blocked the spontaneous increase of crh mrna level .
we conclude that the crh promoter is a dynamically and intermediately methylated genomic region in bewo cells .
promoter methylation did not inhibit crh gene expression under the conditions employed ; rather it determined the contribution of alternative camp - independent pathways and camp - independent mechanisms to crh expression control .
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1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusions
|
the three compounds lincomycin ( a natural antibiotic produced by streptomyces lincolnensis and discovered in 1961 ) , clindamycin , and pirlimycin ( two semi - synthetic derivatives of lincomycin ) comprise a group of clinically important antibiotics known as lincosamides . the structure of lincomycin ( fig .
1a ) can be divided into two parts , a pyrrolidine derivative and a six - atom sugar ring ( methylthiolincosamide ) , which are linked via an amide bond in the central part of the molecule .
1b ) is obtained by 7(s)-chloro - substitution of the 7(r)-hydroxyl group of lincomycin , and pirlimycin ( fig .
1c ) is obtained by trimming the propyl group of clindamycin to get an ethyl group .
these compounds are soluble in water and chemically stable both in the dry state and in solution.fig .
1chemical structures of lincosamides : lincomycin ( natural antibiotic , a ) and its semi - synthetic derivatives clindamycin ( b ) and pirlimycin ( c ) chemical structures of lincosamides : lincomycin ( natural antibiotic , a ) and its semi - synthetic derivatives clindamycin ( b ) and pirlimycin ( c ) lincosamides block bacterial protein synthesis , which takes place on the ribosomes .
the bacterial 70s ribosome can be separated into two subunits : large ( 50s ) and small ( 30s ) , named after their sedimentation coefficients .
the small subunit of prokaryotic ribosomes consists of 21 proteins and one 16s rna chain , while the large subunit contains over 30 proteins and two rna chains ( 23s rna and 5s rna ) .
lincosamides bind to the 23s rna ( interacting with the a- and p - trna binding sites ) of the 50s ribosomal subunit and inhibit the peptidyltransferase reaction , i.e. , peptide bond formation .
the main spectrum of action of lincosamides includes bacteria associated with skin infections , and lincosamides are first - choice antibiotics used in veterinary dermatology .
however , although they do have adverse effects , such as nausea , rash , or abdominal pain , lincomycin and clindamycin are also effectively used in human medicine .
lincosamides are effective against gram - positive bacteria , such as staphylococcus , streptococcus , most anaerobic bacteria ( e.g. , bacteroides fragilis ) , and some protozoa . in the latter case
gram - negative bacteria are , in general , resistant to lincosamides , with one important exception : capnocytophaga canimorsus .
lincosamides exhibit excellent pharmacokinetic properties ; they are well absorbed orally and can penetrate well into infected skin .
also , the minimum inhibitory concentration ( mic90 , the minimum concentration needed to inhibit growth overnight in 90% of organisms ) of clindamycin towards streptococcus pyogenes is over 120 times lower than that of tetracycline .
unfortunately , extensive use of these antibiotics has led to increased resistance in many strains of bacteria , which have been developing various mechanisms to counter these drugs .
one known mechanism is structural modification of the drug s target ( ribosome ) through the methylation of 23s rna ( e.g. , base no .
2058 ) and/or mutations ( e.g. , of bases g2057 , a2058 , a2059 , c2452 , and c2611 ) . other mechanisms of resistance are active efflux of the drug across the cell surface , or its enzymatic deactivation
. bacterial resistance together with side effects are the most important reasons for improving known lincosamides and designing modified compounds .
the conformational properties of free clindamycin and lincomycin have been studied using x - ray techniques [ 9 , 10 ] as well as h and c nmr spectroscopy and molecular dynamics . however , verdier et al .
ribosome interactions by two - dimensional transferred nuclear overhauser effect spectroscopy ( trnoesy ) , and showed that the conformation of the lincosamide plays a crucial role in its binding to the ribosome .
they found that the free conformers of clindamycin and linb ( lincosamide nucleotidyltransferase ) enzyme - bound clindamycin are similar .
rajeswaran et al . solved the x - ray structure of lincomycin hydrochloride and found that intra- and intermolecular hydrogen bonds stabilize the structure of the drug . on the other hand ,
there are four crystallographically resolved structures of clindamycin bound to a target : three in complex with the ribosome [ 1315 ] , and one with the linb molecule , the bacterial enzyme responsible for the inactivation of lincosamides by nucleotidylation .
their crystal structures as well as the structure of the native ribosome are available through the protein data bank ( pdb ) .
these crystallographic data form the basis for our theoretical studies of chemical and physical properties of lincosamides .
two out of the three conformers of clindamycin , when bound to the ribosome , show significant differences between antibiotic conformations : in two structures , the pyrrolidynyl propyl group is rotated by 180 relative to the other conformer .
theoretical calculations provide a complementary way to study molecular systems containing an intramolecular hydrogen bond ( ihb ) .
although the accuracy of ab initio calculations is still below the state - of - art accuracy of experimental spectroscopic data , these calculations provide information about the shape of the potential energy surface ( pes ) without the need for any initial assumptions . characterizing the conformational changes and their possible effects on the encounter with and binding to the ribosome are important aspects of understanding the mechanism of action of lincosamides . to the best of our knowledge ,
no systematic and accurate study of the conformational behavior of clindamycin in the gas phase and in solution has been reported .
the aim of the work described in the present paper was to clarify the role of intramolecular hydrogen bonds in clindamycin using quantum chemical calculations .
these ab initio calculations consist of the following steps : the choosing the model structures , geometry optimization , natural bond orbital analysis ( nbo ) , atoms in molecules analysis ( aim ) , and spectroscopic nmr parameter calculations , which are currently among the most popular methods used for conformational analysis .
the methods applied in the calculations are described in the first section of the paper .
we then characterize the conformations and molecular properties , focusing on the intramolecular hydrogen bonds .
four structures of clindamycin are available in the protein data bank ( as of may 2011 ) .
three of these structures are in complex with the bacterial ribosome [ 1315 ] , while one is in complex with linb .
the two ribosome - complexed structures show similar clindamycin conformers , and in one the pyrrolidynyl propyl group is rotated by 180. in this work , we used two significantly different conformers of clindamycin , which were taken from ribosome clindamycin complexes of deinococcus radiodurans ( pdb code 1jzx ) and haloarcula marismortui ( pdb code 1yjn ) .
therefore , based on the known x - ray structures of clindamycin ( fig .
2ball and stick representation showing the superposition of two clindamycin conformers when bound to the ribosome .
black conformer a , gray conformer b ( only heavy atoms are shown for clarity ) ball and stick representation showing the superposition of two clindamycin conformers when bound to the ribosome .
black conformer a , gray conformer b ( only heavy atoms are shown for clarity ) the second step involved optimizing the geometries of all of the antibiotic models constructed , using density functional theory ( dft ) at the b3lyp level [ 18 , 19 ] with the 6 - 31 g basis set and a redundant coordinate algorithm .
, we optimized the investigated molecules without any constraints . in the second case , the two dihedral angles d(c15c16n2c19 ) and d(c16n2c19c20 ) , which are significantly different in both experimentally known conformers ( a and b ; see fig .
2 ) , were kept constant in the optimization procedure to differentiate between the two conformers .
the values of the dihedral angles d(c15c16n2c19 ) and d(c16n2c19c20 ) for conformer a were set to 47 and 180 , while they were set to 119 and 178 , respectively , for conformer b , in accordance with known experimental results .
the corresponding frequency calculations were carried out at the same level in order to confirm the nature of the stationary points .
no imaginary frequencies were observed , which means that the structures of the antibiotics are true minima . these structures , which we called exp - opt , were used during aim , nbo , and nmr calculations ( see below ) . in a third step , in order to explore the conformational landscape of the molecules , we performed a potential energy surface scan along the torsional coordinates mentioned above in a relaxed manner ( i.e. , all other geometrical parameters were optimized at each point ) for both conformers .
to describe the environment of the antibiotic , either the continuum solvation model or an atomic representation of the solvent can be used .
therefore , as a fourth step , in order to study the solvent effect , optimization and frequency calculations were also performed at the b3lyp/6 - 31 g level of theory in combination with the polarizable continuum solvent model based on the integral equation formalism ( ief - pcm ) . in this model ,
the solvent is described by a dielectric constant , which was set to 80 in our work .
the second model that was used to describe the environment of the antibiotic , and mimics the surroundings of the antibiotic in the ribosomal rna , was the model with point ions placed around the antibiotic [ 22 , 23 ] .
the positions of these point charges were obtained from the x - ray structures of the ribosome
the coordinates of residues within 10 of each clindamycin atom were considered , and partial charges were assigned based on the g43b1 gromos96 force field . in this way , we studied the effect of the charged ribosome environment on the conformations of the lincosamides .
next , in order to gain a deeper insight into the nature of the conformational changes , nbo and aim electron density analyses were applied for the two analyzed conformers of clindamycin .
the bond critical points ( bcps ) were characterized in terms of electron density and their laplacian values .
nmr spectroscopy is one of the techniques used to investigate molecular structures and interactions . in the last part of our work , we calculated the nmr chemical shifts and spin spin coupling constants for the gas - phase optimized geometries at the b3lyp/6 - 31 g level .
shielding constants were calculated using the b3lyp / aug - pcs-1 method and the giao routine [ 2630 ] .
the chemical shifts of the i - th nuclei were calculated as \documentclass[12pt]{minimal }
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\begin{document}$$ \delta ( i ) = \sigma_i(x ) - \sigma_i(c ) , $ $ \end{document } where i(c ) and i(x ) are the isotropic parts of the shielding tensors of the i - th nuclei in clindamycin and the i - th nuclei in the reference x molecule , respectively .
spin coupling constants were calculated at the b3lyp / aug - pcj-0 [ 31 , 32 ] level .
the nonstandard basis sets were taken from the emsl basis set library [ 33 , 34 ] .
the nbo calculations were performed with the nbo 5.0 program , while aim calculations were performed using aim2000 .
data were analyzed using gabedit , and the visualizations were carried out with vmd and xdrawchem .
the energies , the zero - point vibrational energies ( zpe ) , and the gibbs free energy ( g ) values based on the harmonic field relative to the most stable one at 298.15 k calculated in the gas phase , taking solvent effects into account , are depicted in table 1 .
the selected geometric parameters obtained from gas - phase calculations for both conformers of clindamycin , lincomycin , and pirlimycin in their bound modes are given in table 2 .
3.table 1total energies ( e0 , in au ) , zero - point energies ( zpe , in kcal / mol ) , and relative gibbs free energies at 298.15 k ( g , in kcal / mol ) .
top : values for the fully optimized a and b conformers of clindamycin , lincomycin , and pirlimycin in vacuum .
bottom : values for the a and b conformers of clindamycin , lincomycin , and pirlimycin ( optimized using a redundant coordinate algorithm in vacuum ) in the pcm model of solvent and in the point ions .
two dihedrals were kept constant , d(c15c16n2c19 ) = 47.0 and d(c16n2c19c20 ) = 180.0 for conformer a , and d(c15c16n2c19 ) = 119.0 and d(c16n2c19c20 ) = 178.0 for conformer b. all calculations were performed at the b3lyp/6 - 31 g levelclindamycinlincomycinpirlimycinabababfully optimized structures e02049.8317372049.8465561665.4584091665.4636602010.5152252010.54063381 zpe323.26323.66332.14332.40305.59305.85 g8.200.02.410.015.690.0structures with frozen dihedrals c15c16n2c19 and c16n2c19c20 e02049.8317042049.8445351665.4567201665.4559602010.5143912010.455988 zpe323.29323.53332.19331.99305.35305.37 g8.050.00.230.05.320.0
e02049.7968862049.8043781665.4182651665.4194562010.4416232010.460224 zpe323.87324.10332.77332.56305.92305.94 g6.580.00.430.05.400.0 e02048.439096652048.448042001664.054851571664.056563142009.116440872009.12370514 zpe329.99329.71339.94339.56312.95312.74 g5.550.01.070.03.860.0table 2selected geometric parameters ( in and degrees ) for the exp - opt structures of both conformers ( a and b ) of clindamycin , lincomycin , and pirlimycin calculated at the b3lyp/6 - 31 g level in vacuumclindamycinlincomycinpirlimycinabababr(s1c11)1.8551.8571.8501.8521.8541.857r(c12o4)1.4221.4151.4211.4131.4221.414r(c12c13)1.5261.5361.4211.5371.5271.536r(c13o5)1.4261.4121.4251.4121.4261.412r(c15o7)1.4431.4321.4511.4381.4411.430r(o7c11)1.4121.4161.4181.8201.4131.415r(c15c16)1.5431.5451.5491.5441.5421.538r(c16c17)1.5431.5361.5581.5551.5421.538r(c17o8)1.4271.419r(c17cl28)1.8331.8521.8321.852r(c17c18)1.5231.5211.5251.5261.5241.520r(c16n2)1.4621.4571.4661.4621.4621.456r(n2c19)1.3711.3621.3651.3701.3721.369r(c19o9)1.2231.2321.2291.2301.2221.231r(c19c20)1.5421.5331.5421.5371.5411.536r(c20n3)1.4621.4521.4821.4571.4601.458r(n3c21)1.4501.4511.4501.4541.4501.454r(n3c22)1.4591.4591.4651.4591.4581.459r(c22c23)1.5371.5371.5311.5331.5381.532r(c23c24)1.5571.5571.5421.5521.5591.551r(c24c20)1.5481.5581.5421.5611.5481.561a(c20c19n2)113.8114.5a(c19n2c16)128.6124.2a(n2c16c15)113.8112.2a(n2c16c17)113.0110.8a(c16c17c18)113.2113.9a(c16c15c14)111.9123.0a(o9c19n2)124.4123.0d(c15c16n2c19)47.0119.047.0119.047.0119.0d(c16n2c19c20)180.0178.0180.0178.0180.0178.0fig . 3ball and stick models of the studied molecules and their atom numbering schemes .
top : clindamycin , middle : lincomycin , bottom : pirlimycin total energies ( e0 , in au ) , zero - point energies ( zpe , in kcal / mol ) , and relative gibbs free energies at 298.15 k ( g , in kcal / mol ) .
top : values for the fully optimized a and b conformers of clindamycin , lincomycin , and pirlimycin in vacuum .
bottom : values for the a and b conformers of clindamycin , lincomycin , and pirlimycin ( optimized using a redundant coordinate algorithm in vacuum ) in the pcm model of solvent and in the point ions .
two dihedrals were kept constant , d(c15c16n2c19 ) = 47.0 and d(c16n2c19c20 ) = 180.0 for conformer a , and d(c15c16n2c19 ) = 119.0 and d(c16n2c19c20 ) = 178.0 for conformer b. all calculations were performed at the b3lyp/6 - 31 g level selected geometric parameters ( in and degrees ) for the exp - opt structures of both conformers ( a and b ) of clindamycin , lincomycin , and pirlimycin calculated at the b3lyp/6 - 31 g level in vacuum ball and stick models of the studied molecules and their atom numbering schemes .
top : clindamycin , middle : lincomycin , bottom : pirlimycin a number of factors influence the structure and stability of the conformers of clindamycin .
our results show that the most important is the energy profit from the formation of the ihb . the presence of hydrogen - bond donors ( o h , n
h , c h ) and hydrogen - bond acceptors ( c = o , cl ) allows for a range of hydrogen - bond combinations and a number of stable forms [ 40 , 41 ] .
because of the internal hydrogen bonds , one conformer is stabilized to a greater extent than the others .
first and foremost , at the b3lyp/6 - 31 g level , the most stable clindamycin b conformer is more energetically favored than the next most stable by as much as 8.0 kcal / mol .
the energetic picture significantly changes for lincomycin and pirlimycin . in the case of lincomycin ,
no single conformer is favored at 298.15 k , while the a conformer of pirlimycin is more stable than b according to the three models used : vacuum , pcm , and point ions .
the clindamycin b conformer in vacuum displays both the longest o6h37 bond ( 0.980 ) and the shortest ihb distance , r(c19o9
h39c15 ) ihb distance in the less stable a conformer is 2.370 , and r(c15h39 ) = 0.970 is the most important component of the conformer s stability .
the differences in the geometries of the two clindamycin conformers are related almost exclusively to the ihbs in the central part of the molecule . for the most stable conformer , b , an eight - atom ring is formed , whereas a six - atom ring is found in the a conformer .
n single bond were obtained by allowing the c15c16n2c19 dihedral angle to vary from 0 to 180 for clindamycin in vacuum and an aqueous pcm phase .
the values of the starting dihedrals were different in the a and b conformers , as shown in fig . 2 .
full geometry optimizations at a fixed dihedral angle with an increment of 10 were carried out .
the graphs of potential energy as a function of the dihedral angle for gas - phase calculations are shown in fig .
both conformers that are stable in the gas phase were analyzed using the ief - pcm / b3lyp/6 - 31 g method .
table 1 confirms that solvation has a relatively small effect on the energy difference between conformers .
the b conformer of clindamycin is favored over the a conformer by 6.6 and 5.6 kcal / mol according to the pcm and point - ion models , respectively.fig .
4changes in the energy ( in au ) of the c15c16n2c19 dihedral angle in both clindamycin conformers , a ( top ) and b ( bottom ) in vacuum ; calculations were performed with the b3lyp/6 - 31 g method changes in the energy ( in au ) of the c15c16n2c19 dihedral angle in both clindamycin conformers , a ( top ) and b ( bottom ) in vacuum ; calculations were performed with the b3lyp/6 - 31 g method in general , the differences in the conformations of the two models of clindamycin can be understood qualitatively in terms of changes in bond lengths , angles , and the electron density distribution over the whole structure .
natural population analysis is recognized as a reliable tool to rationalize different trends observed in molecules containing ihb . in this section
analysis of the mlliken charges for the heavy atoms ( data not shown ) suggests relationships between the charges and geometrical parameters of the two conformers .
the ring with ihb in the b conformer of clindamycin consists of one nitrogen ( n2 ) atom with a charge of 0.53 au , two oxygen atoms ( o9 and o6 ) with charges of 0.54 au and 0.59 au , and carbon atoms with charges ranging from 0.60 au to 0.02 au . in the a conformer of clindamycin , the negative charges of both oxygens are slightly decreased , while both carbon atoms become more positive ( 0.61 au , 0.07 au ) .
such a decrease in negative charge with the changes in torsional angles that occur when moving from the b to the a conformer is related to the fractional transfer of the charge to electronegative oxygen atoms in the b conformer of clindamycin .
the second - order perturbation energy ( e ) due to the interaction energy between the donor and acceptor orbitals in the central part of the molecule together with the charge transfer ( ct ) between two moieties of the molecule are presented in table 3 . the selected orbital interactions ( with a stabilizing effect of over 8 kcal / mol )
are presented in fig . 5 for both clindamycin conformers.table 3selected second - order perturbation energy ( e , in kcal / mol ) between donor and acceptor orbitals and charge transfer ( qi.j , in au ) in the a ( top ) and b ( bottom ) conformers of clindamycin .
calculations were performed for exp - opt structures at the b3lyp/6 - 31 g level in vacuumnbodonor ( i)nboacceptor ( j)e ( kcal / mol)qi.j ( au)alpn2c19o957.270.118lpn3c22h457.890.068lpn3c20h507.730.066lpn3c21h488.310.069lpo4c12c118.520.068lpo6c14c134.320.048lpo6c14h377.110.066lpo9ryc1916.510.142lpo9c20c1920.860.103lpo9c19n226.110.124lpo9c15h390.930.024blpn2c19o960.170.119lpo5c13c129.210.069lpo6c15c149.070.070lpo9ryc1914.250.132lpo9c20c1921.040.106lpo9c19n220.380.112lpo9o6h378.600.074fig .
5representations of hyperconjugative intramolecular interactions , based on the nbo analysis of both clindamycin exp - opt conformers : red dashed line for a ( left ) , blue dashed line for b ( right ) ; b3lyp/6 - 31 g in vacuum selected second - order perturbation energy ( e , in kcal / mol ) between donor and acceptor orbitals and charge transfer ( qi.j , in au ) in the a ( top ) and b ( bottom ) conformers of clindamycin .
calculations were performed for exp - opt structures at the b3lyp/6 - 31 g level in vacuum representations of hyperconjugative intramolecular interactions , based on the nbo analysis of both clindamycin exp - opt conformers : red dashed line for a ( left ) , blue dashed line for b ( right ) ; b3lyp/6 - 31 g in vacuum considering that the charge transfer accompanies the formation of ihb in the nbo model , the donor
acceptor interaction energies e can be taken as a measure of the strength of the intramolecular interaction . in the case of the central h45n2c19o9 group , the ct from the lone pair orbital on n2
is mainly directed to the antibonding c19o9 orbital ( 0.118 in the a conformer and 0.119 au in the b conformer ) .
other important charge - transfer stabilizations are observed between the lone pair orbital of o9 and the antibonding c20c19 orbital ( 0.103 for the a conformer and 0.106 au for the b conformer ) , as well as between the lone pair of the o9 orbital and the antibonding c19n2 orbital ( 0.124 for the a conformer and 0.112 au for the b conformer ) .
additionally , for the a conformer , we found quite a strong interaction between the lone pair orbital on o4 and the antibonding c12c11 orbital ( 0.068 au ) .
the lone pair orbital on o6 interacts with the antibonding c15c14 one ( 0.070 au ) , and the lone pair orbital on o5 with the antibonding orbital c13c12 ( 0.069 au ) . on the other hand ,
the ct that occurs from the lone pair orbital on o9 through the ihb to the antibonding h39c15 orbital ( 0.024 au ) for the a conformer is lower than that for the h37o6 orbital of the b conformer ( 0.074 au ) .
to summarize , nbo analysis indicates that the occupancy of the antibonding c15h39 orbital in the a conformer or the o6h37 orbital of the eight - member moiety in the b conformer should be an overall indicator of conformational stability .
therefore , the charge transfer between the pyrrolidine - derivative ring and the six - atom sugar ( methylthiolincosamide ) , which are linked via an amide bond , is the dominant factor in the greater stability of the b conformer .
the absence or presence of many types of hydrogen bonds can influence the energy properties of molecular conformers .
in many cases , the atoms in molecules ( aim ) method is a practical tool for understanding the properties of hydrogen bonds .
it identifies a unique line of communication between two nuclei , and provides a point describing the nature of this interaction .
topological analysis of the electron density distribution provides evidence for bonding interactions through the discovery of a ( 3 , 1 ) critical point ( bcp ) , which is a key topological descriptor of internuclear interactions , while the laplacian of the electron density values at the critical point bcp is another sensitive measure of the properties of a classical bond .
it should be noted that there is some controversy regarding the use of aim as a diagnostic tool for bonding interactions .
however , typical intermolecular as well as intramolecular h - bonds can be categorized properly , as has been proven in the literature [ 44 , 45 ] .
the popelier criteria [ 44 , 46 ] for hydrogen - bond formation include the requirement that bcp is in the range 0.0020.040 au , and the need for the value of the laplacian at the hydrogen - bond critical point bcp to be between 0.02 and 0.15 au .
a negative laplacian reveals excess potential energy at the bcp , meaning that the electronic charge is concentrated into a bond .
a positive bcp reflects an excess of kinetic energy in a bond , indicating a local depletion of the electron density along a bond path .
in other words , generally , the laplacian of is positive when is locally reduced , and negative if it is locally concentrated .
according to criteria elaborated by the aim theory , we found two types of intramolecular h - bonds in clindamycin : typical hydrogen bonds of type ch oc in the a conformer and oh o = c in the b conformer , and unconventional h - bonds of type oh x ( x = o , cl ) in both conformers ( fig . 6 ) . the numerical values for the electron density ( bcp ) and
figure 7 shows plots of bcp ( top ) and bcp ( bottom ) versus the length of the hydrogen bond ro ... h .
the general shape of the bcp curve is that of an exponential decay , as expected ( see fig .
7 ) that , in accordance with chemical intuition , o ... h is increased in an ihb.fig .
exp - opt conformers of clindamycin ( top : a , bottom : b ) based on critical points obtained from the aim analysis . the brown , red , blue , yellow , purple , and silver beads represent c , o , n , s , cl , and h atoms , respectively .
the light violet and light green beads represent the ( 3 , + 1 ) and ( 3 , 1 ) critical points , respectively .
the h - bonds [ paths connecting the ( 3 , 1 ) critical points ] are marked with dashed linestable 4the lengths of h - bonds and the electron density and laplacian values for selected critical points of the a ( top ) and b ( bottom ) clindamycin exp - opt conformers optimized at the b3lyp/6 - 31 g level in vacuumcritical point no.atom numbers and namesdhbcpcparingscp1n3c20c22c23c240.03810.0689cp2o7c11c12c13c14c150.01850.0301cp3o5h37o6c13c140.01910.0250cp4o7c15c16c17h410.00260.0027h - bondscp5c15o7 h41c172.3220.01550.0164cp6c19o9
7the electron density cp ( top ) and the laplacian cp ( bottom ) as functions of intramolecular hydrogen - bond length in both conformers of clindamycin molecular graphs of both
exp - opt conformers of clindamycin ( top : a , bottom : b ) based on critical points obtained from the aim analysis .
the brown , red , blue , yellow , purple , and silver beads represent c , o , n , s , cl , and h atoms , respectively .
the light violet and light green beads represent the ( 3 , + 1 ) and ( 3 , 1 ) critical points , respectively .
the h - bonds [ paths connecting the ( 3 , 1 ) critical points ] are marked with dashed lines the lengths of h - bonds and the electron density and laplacian values for selected critical points of the a ( top ) and b ( bottom ) clindamycin exp - opt conformers optimized at the b3lyp/6 - 31 g level in vacuum the electron density cp ( top ) and the laplacian cp ( bottom ) as functions of intramolecular hydrogen - bond length in both conformers of clindamycin the electron density at the critical points is equal to 0.034 au for the b conformer ( bcp8 ) and 0.014 au for the a conformer ( bcp6 ) , which is in line with the most stable b structure . bcp , the second measure of the bond properties according to aim , is barely below zero , and remains ca .
0.02 in the b conformer and 0.013 au in the a conformer , which could indicate weak hydrogen - bonding regions
. however , for the ihbs found in this work , the laplacian at the bond critical point tends to be negative ( although small ) , and smaller than that for an intermolecular hydrogen bond , suggesting that the threshold for this descriptor should be revised .
this analysis indicates that the main influence on the stability of the b conformer is the ihb between the two moieties of the molecule .
the experimental c nmr chemical shifts of clindamycin fall within the interval 1690 pm .
the calculated values for the c chemical shifts are in fairly good agreement with the experimental data . as chemical shifts are sensitive to subtle changes in the electronic structure , which depends in a rather complex manner on the molecular structure
, we will now discuss the dependence of the calculated nmr chemical shifts on the conformation and the ihb .
as usual , the central part of the molecule is the most interesting part to consider for this purpose .
the chemical shifts of the carbon atoms are predicted to be located in their usual ranges : (c = o ) near to 185 ppm , (c h ) close to 70 ppm .
(c = o ) exhibits a sensitivity to ihb : the highest value ( 188.6 ppm ) occurs for the b conformer of clindamycin , which is stabilized by the c19o9
intramolecular hydrogen bond more than the a conformer ( 185.1 ppm ) is stabilized by the c19o9
these chemical shifts have also been shown to depend strongly on the local properties of the electron density .
the small absolute value of the chemical shift of n2 in the b conformer is in line with the small absolute value of the charge density on this nucleus ( 0.547 in the a conformer and 0.530 in the b conformer ) .
finally , it is clear that (o ) can be classified into two groups . in the first group , the oxygen acts as the roton acceptor for the oh group , and
its chemical shift is lower for the b conformer than for the a conformer of clindamycin .
the chemical shift of the hydroxyl oxygen o6 is the highest for the a conformer ( considering its absolute value ) , and the same is true of the oxygen o7 in the ring.table 5b3lyp / aug - pcs-1 calculated chemical shifts for both clindamycin exp - opt conformers optimized at the b3lyp/6 - 31 g level in vacuum .
we used h ( tms ) = 31.50 , c ( tms ) = 182.20 , n ( ch3no2 ) = 167.89 , o ( ch3no2 ) = 389.95 ( + 605 ) , s ( cs2 ) = 1131.94 , and cl ( nacl ) = 151.02 as referencesatomchemical shifts ( ppm)experimental value abs1350.15342.59n2293.38285.97n3347.65349.95o463.4751.98o572.5665.78o685.9772.67o734.2132.74o9295.05260.14c1019.720.915.5c1198.74104.4490.0c1277.6679.9270.5c1376.9577.6373.2c1473.1176.8770.9c1572.9578.1471.8c1665.7262.155.8c1771.1377.0360.7c1825.1726.4424.5c19185.06188.59172.4c2078.978.6271.1c2143.2443.4843.4c2271.0470.7464.3c2343.0546.3739.3c2441.4744.9638.7c2543.5944.5337.0c2627.9629.7523.3c2717.618.8916.0cl28341.29336.87 b3lyp / aug - pcs-1 calculated chemical shifts for both clindamycin exp - opt conformers optimized at the b3lyp/6 - 31 g level in vacuum .
we used h ( tms ) = 31.50 , c ( tms ) = 182.20 , n ( ch3no2 ) = 167.89 , o ( ch3no2 ) = 389.95 ( + 605 ) , s ( cs2 ) = 1131.94 , and cl ( nacl ) = 151.02 as references
the calculated oxygen chemical shifts correlate with the charges , but they are of limited diagnostic value due to the large line widths in the oxygen nmr spectra .
some coupling constants vary with changes in conformation ; for example jc15c16 changes from 5.1 to 5.7 hz when moving from the a to the b conformer
. however , more interesting is the jh37o9 sscc transmitted through the c = o h o ihb , which is equal to 5.3 hz in the b conformer .
spin constants j ( in hz ) for both exp - opt conformers of clindamycin , i.e. , a ( top ) and b ( bottom ) , optimized at the b3lyp/6 - 31 g level in vacuum the selected b3lyp / aug - pcj-0 calculated spin
spin constants j ( in hz ) for both exp - opt conformers of clindamycin , i.e. , a ( top ) and b ( bottom ) , optimized at the b3lyp/6 - 31 g level in vacuum
the energies , the zero - point vibrational energies ( zpe ) , and the gibbs free energy ( g ) values based on the harmonic field relative to the most stable one at 298.15 k calculated in the gas phase , taking solvent effects into account , are depicted in table 1 .
the selected geometric parameters obtained from gas - phase calculations for both conformers of clindamycin , lincomycin , and pirlimycin in their bound modes are given in table 2 .
3.table 1total energies ( e0 , in au ) , zero - point energies ( zpe , in kcal / mol ) , and relative gibbs free energies at 298.15 k ( g , in kcal / mol ) .
top : values for the fully optimized a and b conformers of clindamycin , lincomycin , and pirlimycin in vacuum .
bottom : values for the a and b conformers of clindamycin , lincomycin , and pirlimycin ( optimized using a redundant coordinate algorithm in vacuum ) in the pcm model of solvent and in the point ions .
two dihedrals were kept constant , d(c15c16n2c19 ) = 47.0 and d(c16n2c19c20 ) = 180.0 for conformer a , and d(c15c16n2c19 ) = 119.0 and d(c16n2c19c20 ) = 178.0 for conformer b. all calculations were performed at the b3lyp/6 - 31 g levelclindamycinlincomycinpirlimycinabababfully optimized structures e02049.8317372049.8465561665.4584091665.4636602010.5152252010.54063381 zpe323.26323.66332.14332.40305.59305.85 g8.200.02.410.015.690.0structures with frozen dihedrals c15c16n2c19 and c16n2c19c20 e02049.8317042049.8445351665.4567201665.4559602010.5143912010.455988 zpe323.29323.53332.19331.99305.35305.37 g8.050.00.230.05.320.0
e02049.7968862049.8043781665.4182651665.4194562010.4416232010.460224 zpe323.87324.10332.77332.56305.92305.94 g6.580.00.430.05.400.0 e02048.439096652048.448042001664.054851571664.056563142009.116440872009.12370514 zpe329.99329.71339.94339.56312.95312.74 g5.550.01.070.03.860.0table 2selected geometric parameters ( in and degrees ) for the exp - opt structures of both conformers ( a and b ) of clindamycin , lincomycin , and pirlimycin calculated at the b3lyp/6 - 31 g level in vacuumclindamycinlincomycinpirlimycinabababr(s1c11)1.8551.8571.8501.8521.8541.857r(c12o4)1.4221.4151.4211.4131.4221.414r(c12c13)1.5261.5361.4211.5371.5271.536r(c13o5)1.4261.4121.4251.4121.4261.412r(c15o7)1.4431.4321.4511.4381.4411.430r(o7c11)1.4121.4161.4181.8201.4131.415r(c15c16)1.5431.5451.5491.5441.5421.538r(c16c17)1.5431.5361.5581.5551.5421.538r(c17o8)1.4271.419r(c17cl28)1.8331.8521.8321.852r(c17c18)1.5231.5211.5251.5261.5241.520r(c16n2)1.4621.4571.4661.4621.4621.456r(n2c19)1.3711.3621.3651.3701.3721.369r(c19o9)1.2231.2321.2291.2301.2221.231r(c19c20)1.5421.5331.5421.5371.5411.536r(c20n3)1.4621.4521.4821.4571.4601.458r(n3c21)1.4501.4511.4501.4541.4501.454r(n3c22)1.4591.4591.4651.4591.4581.459r(c22c23)1.5371.5371.5311.5331.5381.532r(c23c24)1.5571.5571.5421.5521.5591.551r(c24c20)1.5481.5581.5421.5611.5481.561a(c20c19n2)113.8114.5a(c19n2c16)128.6124.2a(n2c16c15)113.8112.2a(n2c16c17)113.0110.8a(c16c17c18)113.2113.9a(c16c15c14)111.9123.0a(o9c19n2)124.4123.0d(c15c16n2c19)47.0119.047.0119.047.0119.0d(c16n2c19c20)180.0178.0180.0178.0180.0178.0fig . 3ball and stick models of the studied molecules and their atom numbering schemes .
top : clindamycin , middle : lincomycin , bottom : pirlimycin total energies ( e0 , in au ) , zero - point energies ( zpe , in kcal / mol ) , and relative gibbs free energies at 298.15 k ( g , in kcal / mol ) .
top : values for the fully optimized a and b conformers of clindamycin , lincomycin , and pirlimycin in vacuum .
bottom : values for the a and b conformers of clindamycin , lincomycin , and pirlimycin ( optimized using a redundant coordinate algorithm in vacuum ) in the pcm model of solvent and in the point ions .
two dihedrals were kept constant , d(c15c16n2c19 ) = 47.0 and d(c16n2c19c20 ) = 180.0 for conformer a , and d(c15c16n2c19 ) = 119.0 and d(c16n2c19c20 ) = 178.0 for conformer b. all calculations were performed at the b3lyp/6 - 31 g level selected geometric parameters ( in and degrees ) for the exp - opt structures of both conformers ( a and b ) of clindamycin , lincomycin , and pirlimycin calculated at the b3lyp/6 - 31 g level in vacuum ball and stick models of the studied molecules and their atom numbering schemes .
top : clindamycin , middle : lincomycin , bottom : pirlimycin a number of factors influence the structure and stability of the conformers of clindamycin .
our results show that the most important is the energy profit from the formation of the ihb . the presence of hydrogen - bond donors ( o h , n
h , c h ) and hydrogen - bond acceptors ( c = o , cl ) allows for a range of hydrogen - bond combinations and a number of stable forms [ 40 , 41 ] .
because of the internal hydrogen bonds , one conformer is stabilized to a greater extent than the others .
first and foremost , at the b3lyp/6 - 31 g level , the most stable clindamycin b conformer is more energetically favored than the next most stable by as much as 8.0 kcal / mol .
the energetic picture significantly changes for lincomycin and pirlimycin . in the case of lincomycin ,
no single conformer is favored at 298.15 k , while the a conformer of pirlimycin is more stable than b according to the three models used : vacuum , pcm , and point ions .
the clindamycin b conformer in vacuum displays both the longest o6h37 bond ( 0.980 ) and the shortest ihb distance , r(c19o9
h39c15 ) ihb distance in the less stable a conformer is 2.370 , and r(c15h39 ) = 0.970 is the most important component of the conformer s stability .
the differences in the geometries of the two clindamycin conformers are related almost exclusively to the ihbs in the central part of the molecule . for the most stable conformer , b , an eight - atom ring is formed , whereas a six - atom ring is found in the a conformer .
n single bond were obtained by allowing the c15c16n2c19 dihedral angle to vary from 0 to 180 for clindamycin in vacuum and an aqueous pcm phase .
the values of the starting dihedrals were different in the a and b conformers , as shown in fig . 2 .
full geometry optimizations at a fixed dihedral angle with an increment of 10 were carried out .
the graphs of potential energy as a function of the dihedral angle for gas - phase calculations are shown in fig .
both conformers that are stable in the gas phase were analyzed using the ief - pcm / b3lyp/6 - 31 g method .
table 1 confirms that solvation has a relatively small effect on the energy difference between conformers .
the b conformer of clindamycin is favored over the a conformer by 6.6 and 5.6 kcal / mol according to the pcm and point - ion models , respectively.fig .
4changes in the energy ( in au ) of the c15c16n2c19 dihedral angle in both clindamycin conformers , a ( top ) and b ( bottom ) in vacuum ; calculations were performed with the b3lyp/6 - 31 g method changes in the energy ( in au ) of the c15c16n2c19 dihedral angle in both clindamycin conformers , a ( top ) and b ( bottom ) in vacuum ; calculations were performed with the b3lyp/6 - 31 g method
in general , the differences in the conformations of the two models of clindamycin can be understood qualitatively in terms of changes in bond lengths , angles , and the electron density distribution over the whole structure .
natural population analysis is recognized as a reliable tool to rationalize different trends observed in molecules containing ihb . in this section
analysis of the mlliken charges for the heavy atoms ( data not shown ) suggests relationships between the charges and geometrical parameters of the two conformers .
the ring with ihb in the b conformer of clindamycin consists of one nitrogen ( n2 ) atom with a charge of 0.53 au , two oxygen atoms ( o9 and o6 ) with charges of 0.54 au and 0.59 au , and carbon atoms with charges ranging from 0.60 au to 0.02 au . in the a conformer of clindamycin , the negative charges of both oxygens are slightly decreased , while both carbon atoms become more positive ( 0.61 au , 0.07 au ) .
such a decrease in negative charge with the changes in torsional angles that occur when moving from the b to the a conformer is related to the fractional transfer of the charge to electronegative oxygen atoms in the b conformer of clindamycin .
the second - order perturbation energy ( e ) due to the interaction energy between the donor and acceptor orbitals in the central part of the molecule together with the charge transfer ( ct ) between two moieties of the molecule are presented in table 3 .
the selected orbital interactions ( with a stabilizing effect of over 8 kcal / mol ) are presented in fig .
5 for both clindamycin conformers.table 3selected second - order perturbation energy ( e , in kcal / mol ) between donor and acceptor orbitals and charge transfer ( qi.j , in au ) in the a ( top ) and b ( bottom ) conformers of clindamycin .
calculations were performed for exp - opt structures at the b3lyp/6 - 31 g level in vacuumnbodonor ( i)nboacceptor ( j)e ( kcal / mol)qi.j ( au)alpn2c19o957.270.118lpn3c22h457.890.068lpn3c20h507.730.066lpn3c21h488.310.069lpo4c12c118.520.068lpo6c14c134.320.048lpo6c14h377.110.066lpo9ryc1916.510.142lpo9c20c1920.860.103lpo9c19n226.110.124lpo9c15h390.930.024blpn2c19o960.170.119lpo5c13c129.210.069lpo6c15c149.070.070lpo9ryc1914.250.132lpo9c20c1921.040.106lpo9c19n220.380.112lpo9o6h378.600.074fig .
5representations of hyperconjugative intramolecular interactions , based on the nbo analysis of both clindamycin exp - opt conformers : red dashed line for a ( left ) , blue dashed line for b ( right ) ; b3lyp/6 - 31 g in vacuum selected second - order perturbation energy ( e , in kcal / mol ) between donor and acceptor orbitals and charge transfer ( qi.j , in au ) in the a ( top ) and b ( bottom ) conformers of clindamycin .
calculations were performed for exp - opt structures at the b3lyp/6 - 31 g level in vacuum representations of hyperconjugative intramolecular interactions , based on the nbo analysis of both clindamycin exp - opt conformers : red dashed line for a ( left ) , blue dashed line for b ( right ) ; b3lyp/6 - 31 g in vacuum considering that the charge transfer accompanies the formation of ihb in the nbo model , the donor
acceptor interaction energies e can be taken as a measure of the strength of the intramolecular interaction . in the case of the central h45n2c19o9 group , the ct from the lone pair orbital on n2
is mainly directed to the antibonding c19o9 orbital ( 0.118 in the a conformer and 0.119 au in the b conformer ) .
other important charge - transfer stabilizations are observed between the lone pair orbital of o9 and the antibonding c20c19 orbital ( 0.103 for the a conformer and 0.106 au for the b conformer ) , as well as between the lone pair of the o9 orbital and the antibonding c19n2 orbital ( 0.124 for the a conformer and 0.112 au for the b conformer ) .
additionally , for the a conformer , we found quite a strong interaction between the lone pair orbital on o4 and the antibonding c12c11 orbital ( 0.068 au ) .
the lone pair orbital on o6 interacts with the antibonding c15c14 one ( 0.070 au ) , and the lone pair orbital on o5 with the antibonding orbital c13c12 ( 0.069 au ) . on the other hand , the ct that occurs from the lone pair orbital on o9 through the ihb to the antibonding h39c15 orbital ( 0.024 au ) for the a conformer is lower than that for the h37o6 orbital of the b conformer ( 0.074 au ) . to summarize
, nbo analysis indicates that the occupancy of the antibonding c15h39 orbital in the a conformer or the o6h37 orbital of the eight - member moiety in the b conformer should be an overall indicator of conformational stability .
therefore , the charge transfer between the pyrrolidine - derivative ring and the six - atom sugar ( methylthiolincosamide ) , which are linked via an amide bond , is the dominant factor in the greater stability of the b conformer .
the absence or presence of many types of hydrogen bonds can influence the energy properties of molecular conformers . in many cases , the atoms in molecules ( aim ) method is a practical tool for understanding the properties of hydrogen bonds .
it identifies a unique line of communication between two nuclei , and provides a point describing the nature of this interaction .
topological analysis of the electron density distribution provides evidence for bonding interactions through the discovery of a ( 3 , 1 ) critical point ( bcp ) , which is a key topological descriptor of internuclear interactions , while the laplacian of the electron density values at the critical point bcp is another sensitive measure of the properties of a classical bond .
it should be noted that there is some controversy regarding the use of aim as a diagnostic tool for bonding interactions .
however , typical intermolecular as well as intramolecular h - bonds can be categorized properly , as has been proven in the literature [ 44 , 45 ] .
the popelier criteria [ 44 , 46 ] for hydrogen - bond formation include the requirement that bcp is in the range 0.0020.040 au , and the need for the value of the laplacian at the hydrogen - bond critical point bcp to be between 0.02 and 0.15 au .
a negative laplacian reveals excess potential energy at the bcp , meaning that the electronic charge is concentrated into a bond .
a positive bcp reflects an excess of kinetic energy in a bond , indicating a local depletion of the electron density along a bond path .
in other words , generally , the laplacian of is positive when is locally reduced , and negative if it is locally concentrated .
according to criteria elaborated by the aim theory , we found two types of intramolecular h - bonds in clindamycin : typical hydrogen bonds of type ch oc in the a conformer and oh o = c in the b conformer , and unconventional h - bonds of type oh x ( x = o , cl ) in both conformers ( fig .
the numerical values for the electron density ( bcp ) and laplacian ( bcp ) are presented in table 4 .
figure 7 shows plots of bcp ( top ) and bcp ( bottom ) versus the length of the hydrogen bond ro ... h .
the general shape of the bcp curve is that of an exponential decay , as expected ( see fig .
7 ) that , in accordance with chemical intuition , o ... h is increased in an ihb.fig .
exp - opt conformers of clindamycin ( top : a , bottom : b ) based on critical points obtained from the aim analysis .
the brown , red , blue , yellow , purple , and silver beads represent c , o , n , s , cl , and h atoms , respectively .
the light violet and light green beads represent the ( 3 , + 1 ) and ( 3 , 1 ) critical points , respectively .
the h - bonds [ paths connecting the ( 3 , 1 ) critical points ] are marked with dashed linestable 4the lengths of h - bonds and the electron density and laplacian values for selected critical points of the a ( top ) and b ( bottom ) clindamycin exp - opt conformers optimized at the b3lyp/6 - 31 g level in vacuumcritical point no.atom numbers and namesdhbcpcparingscp1n3c20c22c23c240.03810.0689cp2o7c11c12c13c14c150.01850.0301cp3o5h37o6c13c140.01910.0250cp4o7c15c16c17h410.00260.0027h - bondscp5c15o7 h41c172.3220.01550.0164cp6c19o9 h39c152.3700.01460.0131cp7c17cl28
7the electron density cp ( top ) and the laplacian cp ( bottom ) as functions of intramolecular hydrogen - bond length in both conformers of clindamycin molecular graphs of both
exp - opt conformers of clindamycin ( top : a , bottom : b ) based on critical points obtained from the aim analysis .
the brown , red , blue , yellow , purple , and silver beads represent c , o , n , s , cl , and h atoms , respectively . the light violet and light green beads represent the ( 3 , + 1 ) and ( 3 , 1 ) critical points , respectively .
the h - bonds [ paths connecting the ( 3 , 1 ) critical points ] are marked with dashed lines the lengths of h - bonds and the electron density and laplacian values for selected critical points of the a ( top ) and b ( bottom ) clindamycin exp - opt conformers optimized at the b3lyp/6 - 31 g level in vacuum the electron density cp ( top ) and the laplacian cp ( bottom ) as functions of intramolecular hydrogen - bond length in both conformers of clindamycin the electron density at the critical points is equal to 0.034 au for the b conformer ( bcp8 ) and 0.014 au for the a conformer ( bcp6 ) , which is in line with the most stable b structure .
bcp , the second measure of the bond properties according to aim , is barely below zero , and remains ca .
0.02 in the b conformer and 0.013 au in the a conformer , which could indicate weak hydrogen - bonding regions .
however , for the ihbs found in this work , the laplacian at the bond critical point tends to be negative ( although small ) , and smaller than that for an intermolecular hydrogen bond , suggesting that the threshold for this descriptor should be revised .
this analysis indicates that the main influence on the stability of the b conformer is the ihb between the two moieties of the molecule .
the experimental c nmr chemical shifts of clindamycin fall within the interval 1690 pm .
the calculated values for the c chemical shifts are in fairly good agreement with the experimental data . as
chemical shifts are sensitive to subtle changes in the electronic structure , which depends in a rather complex manner on the molecular structure , we will now discuss the dependence of the calculated nmr chemical shifts on the conformation and the ihb . as usual , the central part of the molecule is the most interesting part to consider for this purpose .
the chemical shifts of the carbon atoms are predicted to be located in their usual ranges : (c = o ) near to 185 ppm , (c h ) close to 70 ppm .
(c = o ) exhibits a sensitivity to ihb : the highest value ( 188.6 ppm ) occurs for the b conformer of clindamycin , which is stabilized by the c19o9
intramolecular hydrogen bond more than the a conformer ( 185.1 ppm ) is stabilized by the c19o9
these chemical shifts have also been shown to depend strongly on the local properties of the electron density .
the small absolute value of the chemical shift of n2 in the b conformer is in line with the small absolute value of the charge density on this nucleus ( 0.547 in the a conformer and 0.530 in the b conformer ) .
finally , it is clear that (o ) can be classified into two groups . in the first group ,
the oxygen acts as the roton acceptor for the oh group , and its chemical shift is lower for the b conformer than for the a conformer of clindamycin . the chemical shift of the hydroxyl oxygen o6 is the highest for the a conformer ( considering its absolute value ) , and the same is true of the oxygen o7 in the ring.table 5b3lyp / aug - pcs-1 calculated chemical shifts for both clindamycin exp - opt conformers optimized at the b3lyp/6 - 31 g level in vacuum .
we used h ( tms ) = 31.50 , c ( tms ) = 182.20 , n ( ch3no2 ) = 167.89 , o ( ch3no2 ) = 389.95 ( + 605 ) , s ( cs2 ) = 1131.94 , and cl ( nacl ) = 151.02 as referencesatomchemical shifts ( ppm)experimental value abs1350.15342.59n2293.38285.97n3347.65349.95o463.4751.98o572.5665.78o685.9772.67o734.2132.74o9295.05260.14c1019.720.915.5c1198.74104.4490.0c1277.6679.9270.5c1376.9577.6373.2c1473.1176.8770.9c1572.9578.1471.8c1665.7262.155.8c1771.1377.0360.7c1825.1726.4424.5c19185.06188.59172.4c2078.978.6271.1c2143.2443.4843.4c2271.0470.7464.3c2343.0546.3739.3c2441.4744.9638.7c2543.5944.5337.0c2627.9629.7523.3c2717.618.8916.0cl28341.29336.87 b3lyp / aug - pcs-1 calculated chemical shifts for both clindamycin exp - opt conformers optimized at the b3lyp/6 - 31 g level in vacuum .
we used h ( tms ) = 31.50 , c ( tms ) = 182.20 , n ( ch3no2 ) = 167.89 , o ( ch3no2 ) = 389.95 ( + 605 ) , s ( cs2 ) = 1131.94 , and cl ( nacl ) = 151.02 as references
the calculated oxygen chemical shifts correlate with the charges , but they are of limited diagnostic value due to the large line widths in the oxygen nmr spectra .
some coupling constants vary with changes in conformation ; for example jc15c16 changes from 5.1 to 5.7 hz when moving from the a to the b conformer .
however , more interesting is the jh37o9 sscc transmitted through the c = o h o ihb , which is equal to 5.3 hz in the b conformer . this value is large enough to be measured experimentally.fig .
spin constants j ( in hz ) for both exp - opt conformers of clindamycin , i.e. , a ( top ) and b ( bottom ) , optimized at the b3lyp/6 - 31 g level in vacuum the selected b3lyp / aug - pcj-0 calculated spin
spin constants j ( in hz ) for both exp - opt conformers of clindamycin , i.e. , a ( top ) and b ( bottom ) , optimized at the b3lyp/6 - 31 g level in vacuum
we have quantum chemically characterized the two conformers of each of the known lincosamides clindamycin , lincomycin , and pirlimycin at the b3lyp/6 - 31 g level .
internal rotations in clindamycin were investigated in vacuum and within the framework of the ief - pcm model . using nbo analysis , and with the aid of the aim theory ,
we focused on the sensitivities of electronic structure parameters such as nbo atomic charges , bond critical points , nmr chemical shifts , and spin spin coupling constants to the conformation of clindamycin .
the two most stable conformers of clindamycin exhibit c = oh o intramolecular hydrogen bonds . according to nbo and aim analyses ,
the presence of this internal hydrogen bond between the pyrrolidine - derivative ring and the six - atom sugar ( methylthiolincosamide ) is the main influence on conformer stability in vacuum and in water .
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lincosamides are a class of antibiotics used both in clinical and veterinary practice for a wide range of pathogens .
this group of drugs inhibits the activity of the bacterial ribosome by binding to the 23s rna of the large ribosomal subunit and blocking protein synthesis .
currently , three x - ray structures of the ribosome in complex with clindamycin are available in the protein data bank , which reveal that there are two distinct conformations of the pyrrolidinyl propyl group of the bound clindamycin . in this work , we used quantum mechanical methods to investigate the probable conformations of clindamycin in order to explain the two binding modes in the ribosomal 23s rna .
we studied three lincosamide antibiotics : clindamycin , lincomycin , and pirlimycin at the b3lyp level with the 6 - 31 g * * basis set .
the focus of our work was to connect the conformational landscape and electron densities of the two clindamycin conformers found experimentally with their physicochemical properties .
for both functional conformers , we applied natural bond orbital ( nbo ) analysis and the atoms in molecules ( aim ) theory , and calculated the nmr parameters .
based on the results obtained , we were able to show that the structure with the intramolecular hydrogen bond c = o h o is the most stable conformer of clindamycin .
the charge transfer between the pyrrolidine - derivative ring and the six - atom sugar ( methylthiolincosamide ) , which are linked via an amide bond , was found to be the dominant factor influencing the high stability of this conformer.figuremolecular graph of more stable conformer of clindamycin .
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Introduction
Computational details
Results and discussion
Conformational analysis
Natural bond analysis
Atoms in Molecules analysis
NMR chemical shifts
Conclusions
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the rising prevalence of alzheimer s disease and related dementias ( henceforth referred to as dementia ) is emerging as a leading global health system challenge .
effective early diagnosis and management models are required to mitigate its impact on patients , caregivers , and health - care systems . enhancing primary care capacity
however , dementia care predominantly resides with geriatric specialists , who are in short supply in canada and elsewhere , delaying access to care . to enhance the care of persons with dementia ,
many of these have been established as primary care - based memory clinics ( pcmcs ) .
initial evaluations suggest that pcmcs can provide timelier assessment , lead to a high degree of satisfaction among referring physicians , patients , and caregivers , and streamline access to specialists . in order to retain the fidelity of such programs and consistency with initial training and practice guidelines , and
practice drift and ensure ongoing high quality of care , quality assurance frameworks are needed .
quality indicators ( qis ) , based on best practices , define achievable benchmarks and a quality assurance framework facilitates practice improvement through targeted , educational quality improvement mechanisms . to our knowledge
, there is no quality assurance framework specific to dementia care in primary care - based settings .
this paper describes the results of a consensus approach to identify qis and quality improvement mechanisms in an ontario - wide network of interprofessional pcmcs , and identify potential barriers and facilitators to the implementation of a quality assurance framework .
a delphi technique was deployed to obtain agreement from pcmc clinicians and dementia specialists on preferred qis and quality improvement mechanisms .
the delphi technique is an iterative consensus process , wherein surveys are used to solicit opinions from groups , and responses summarized and redistributed in a subsequent round for consideration .
we identified 38 candidate qis and quality improvement mechanisms for dementia care by reviewing existing clinical guidelines and quality indicator and improvement compendiums developed with standardized methods ( table 1 ) .
respondents were asked to rate the qis and quality improvement mechanisms using a continuous integer 9-point scale , with 1 representing the least important and 9 the most important .
links to the web - based survey were electronically distributed to all pcmc clinicians ( n = 283 ) and ontario specialists through the ontario medical association sections of geriatric medicine ( n = 123 ) and neurologists ( n = 134 ) , and the canadian association of geriatric psychiatrists ( n = 305 ) .
after each round , qis and improvement measures in the lower two tertiles of agreement ( i.e. , with mean ratings less than 7 ) were excluded , and those remaining were reviewed by the authors guided by respondent comments .
qis and quality improvement mechanisms deemed redundant or containing duplicate themes were combined or amended with attention to preserving their intent and conciseness .
data from the preceding round , including number of respondents , rating means , and standard deviations , were included in the subsequent round .
student s t - test was used to identify significant differences between pcmc clinicians and specialists .
spss version 23.0 ( ibm corp . ) was used , with two - sided p values of < .05 as the threshold for statistical significance .
two authors ( gh , vb ) independently analyzed all written comments using descriptive content analysis and incorporated the feedback into the presentation of qis and quality improvement mechanisms in the second delphi round .
a delphi technique was deployed to obtain agreement from pcmc clinicians and dementia specialists on preferred qis and quality improvement mechanisms .
the delphi technique is an iterative consensus process , wherein surveys are used to solicit opinions from groups , and responses summarized and redistributed in a subsequent round for consideration .
we identified 38 candidate qis and quality improvement mechanisms for dementia care by reviewing existing clinical guidelines and quality indicator and improvement compendiums developed with standardized methods ( table 1 ) .
respondents were asked to rate the qis and quality improvement mechanisms using a continuous integer 9-point scale , with 1 representing the least important and 9 the most important .
links to the web - based survey were electronically distributed to all pcmc clinicians ( n = 283 ) and ontario specialists through the ontario medical association sections of geriatric medicine ( n = 123 ) and neurologists ( n = 134 ) , and the canadian association of geriatric psychiatrists ( n = 305 ) .
after each round , qis and improvement measures in the lower two tertiles of agreement ( i.e. , with mean ratings less than 7 ) were excluded , and those remaining were reviewed by the authors guided by respondent comments .
qis and quality improvement mechanisms deemed redundant or containing duplicate themes were combined or amended with attention to preserving their intent and conciseness .
data from the preceding round , including number of respondents , rating means , and standard deviations , were included in the subsequent round .
student s t - test was used to identify significant differences between pcmc clinicians and specialists .
spss version 23.0 ( ibm corp . ) was used , with two - sided p values of < .05 as the threshold for statistical significance .
two authors ( gh , vb ) independently analyzed all written comments using descriptive content analysis and incorporated the feedback into the presentation of qis and quality improvement mechanisms in the second delphi round .
two survey rounds were conducted between april and june 2014 . in the first round , 842 surveys were distributed with a response rate of 21.3% .
the majority of respondents were physicians , and nurses and other health professionals equally represented the remainder of respondents ( table 2 ) .
respondents had an average of 14.6 10.1 years of clinical practice with older adults . among pcmc respondents ,
31% were from urban centres , 42% rural settings , and 25% from mixed urban / rural populations .
in contrast , 73% of specialists worked in urban settings , and 24% served mixed populations .
only 11 neurologists responded to the first survey , and their specialty was not included in round 2 . in round 2 ,
respondents had an average of 17.42 10.08 years of clinical practice with older adults and practice settings were similar to round 1 .
a third delphi round was not conducted due to the substantial drop in response rate between rounds 1 and 2 .
table 3 presents the results of the consensus process and table 4 presents the final list of quality indicators .
quality improvement mechanisms characterized by specialist integration , including case discussions , shared care , observerships , and mentorships , ranked highly ( table 5 ) .
other preferred quality improvement mechanisms included standardized electronic charting forms , self - directed learning activities , and interactive programs .
survey respondents recommended that between 1030% of patients seen in a pcmc also be reviewed by a specialist .
descriptive content analysis identified three themes related to potential barriers and facilitators to the establishment of a quality assurance framework in pcmcs : 1 ) its perceived importance , 2 ) collaboration and role clarity , and 3 ) the implementation process . almost all respondents mentioned the need to maintain high - quality care through ongoing , targeted training on pertinent clinical knowledge and consistency of approach among pcmc teams .
most recognized a formal quality assurance framework and individual qis as essential . great indicators and very necessary [ pcmc clinician , round 1 ] .
our challenge is to maintain evidence based salience , which ultimately facilitates improved quality of life for persons with dementia and their family [ pcmc clinician , delphi round 2 ] .
all are relevant quality indicators for a pcmc [ specialist , round 2 ] . however , a few respondents were unsure why a quality assurance framework was needed at all .
others seemed unfamiliar with the purpose of quality assurance and construed the idea of tracking qis as supplemental work .
is the intent of the questions to assess what we are doing now or [ what ] we think should be the standard ? [ pcmc clinician , round 1 ] .
this is the canadian consensus guideline that every doc should know [ specialist , round 1 ; emphasis added by authors ] .
a second theme pertained to the operationalization and implementation of the quality assurance framework within the context of pcmcs , and identified a lack of clearly delineated responsibilities among referring clinicians , pcmcs , and specialists .
respondents perceived this as problematic because absence of clarity impedes the capture of clinical documentation relevant to qi measurement .
information is sent back to referral physician with recommendations for caregiver and patient mostly related to change in medication or treatment , lifestyle modification and support resources [ pcmc clinician , round 2 ] .
this lack of clarity was considered most problematic with regard to patient follow - up .
patients with dementia with great plans should not need constant surveillance and follow up but ... this is not my experience ... they appear to often benefit from a watchdog team to ensure their decisions are being carried out . [ specialist , round 1 ] .
similar concerns were raised regarding the management of comorbidities . [ my ] assumption is that management of comorbidities is the primary care physician s domain .
recommendations are made for lifestyle changes , however [ congestive heart failure ] and diabetes management are only commented on , suggestions are made to the referring physician [ pcmc clinician , round 2 ] .
it was not clear which health care provider should conduct a physical examination , leading to gaps in assessment . referring family physicians are expected to have completed a complete physical including appropriate neuro exam prior to referral [ pcmc clinician , round 1 ] .
my biggest concern is that the pcmc team assumes that a proper physical and neurologic exam has been done by the referring source [ specialist , round 1 ] .
respondents provided several comments on potential barriers to the implementation process of a quality assurance framework in pcmcs .
one of the main obstacles was the perceived burden of documentation required to implement qis .
[ it will take a lot of work to do the ] searching and documenting as a group of patients needs a process to set up how to search , then doing the search [ pcmc clinician , round 2 ] . integrating qis into existing electronic medical records was touted as a solution , though potentially a resource - intensive one .
making up stamps to collect this information is doable but takes time for someone to make the [ standardized template ] and then to test it
a second issue identified in relation to the implementation of the quality assurance framework is the need to establish benchmarks to properly interpret qi scores .
i think one of the most important quality indicators is a comparison of the pcmc performance versus the specialist , done on both patients referred to the specialist by the pcmc , and unselected patients seen in the clinic that would not have been referred to the specialist [ specialist , round 1 ] .
access to other health care professionals , within pcmcs and in the community , was identified as important for quality care , though access was not perceived as uniform . having a pharmacist on our team is a great asset .... the local [ pharmacists ] volunteer their time to assist us at our once monthly day long clinics [ pcmc clinician , round 2 ] .
the [ social work ] and [ occupational therapy ] members of the team do an excellent job the [ alzheimer s society ] representative has been excellent as well when given an opportunity [ pcmc clinician , round 2 ] .
an important finding is the relative lack of importance ascribed to end - of - life planning . while respondents agreed on its importance , many expressed that such planning discussions were not appropriate for patients with less advanced disease .
palliation and end of life discussion is usually not appropriate at time of our memory clinic initial or follow - up assessment since our patients are not that advanced
. family physician team will do this in following up patient [ pcmc clinician , round 1 ] .
lastly , many respondents expressed a preference for quality improvement mechanisms and learning opportunities characterized by active engagement .
they also identified specialists as agents to promote care quality , particularly in the context of a shared care approach .
this [ case review ] could be done at the same time , as the specialist mentoring the clinic is in a clinic day with the team [ pcmc clinician , round 1 ] .
barriers to the implementation of this framework included role confusion among stakeholders and limited resources .
descriptive content analysis identified three themes related to potential barriers and facilitators to the establishment of a quality assurance framework in pcmcs : 1 ) its perceived importance , 2 ) collaboration and role clarity , and 3 ) the implementation process .
almost all respondents mentioned the need to maintain high - quality care through ongoing , targeted training on pertinent clinical knowledge and consistency of approach among pcmc teams .
most recognized a formal quality assurance framework and individual qis as essential . great indicators and very necessary [ pcmc clinician , round 1 ] .
our challenge is to maintain evidence based salience , which ultimately facilitates improved quality of life for persons with dementia and their family [ pcmc clinician , delphi round 2 ] .
all are relevant quality indicators for a pcmc [ specialist , round 2 ] . however , a few respondents were unsure why a quality assurance framework was needed at all .
others seemed unfamiliar with the purpose of quality assurance and construed the idea of tracking qis as supplemental work .
is the intent of the questions to assess what we are doing now or [ what ] we think should be the standard ? [ pcmc clinician , round 1 ] .
this is the canadian consensus guideline that every doc should know [ specialist , round 1 ; emphasis added by authors ] .
a second theme pertained to the operationalization and implementation of the quality assurance framework within the context of pcmcs , and identified a lack of clearly delineated responsibilities among referring clinicians , pcmcs , and specialists .
respondents perceived this as problematic because absence of clarity impedes the capture of clinical documentation relevant to qi measurement .
information is sent back to referral physician with recommendations for caregiver and patient mostly related to change in medication or treatment , lifestyle modification and support resources [ pcmc clinician , round 2 ] .
this lack of clarity was considered most problematic with regard to patient follow - up .
patients with dementia with great plans should not need constant surveillance and follow up but ... this is not my experience ... they appear to often benefit from a watchdog team to ensure their decisions are being carried out . [ specialist , round 1 ] .
similar concerns were raised regarding the management of comorbidities . [ my ] assumption is that management of comorbidities is the primary care physician s domain .
recommendations are made for lifestyle changes , however [ congestive heart failure ] and diabetes management are only commented on , suggestions are made to the referring physician [ pcmc clinician , round 2 ] .
it was not clear which health care provider should conduct a physical examination , leading to gaps in assessment . referring family physicians are expected to have completed a complete physical including appropriate neuro exam prior to referral [ pcmc clinician , round 1 ] .
my biggest concern is that the pcmc team assumes that a proper physical and neurologic exam has been done by the referring source [ specialist , round 1 ] .
respondents provided several comments on potential barriers to the implementation process of a quality assurance framework in pcmcs .
one of the main obstacles was the perceived burden of documentation required to implement qis .
[ it will take a lot of work to do the ] searching and documenting as a group of patients needs a process to set up how to search , then doing the search [ pcmc clinician , round 2 ] .
integrating qis into existing electronic medical records was touted as a solution , though potentially a resource - intensive one .
making up stamps to collect this information is doable but takes time for someone to make the [ standardized template ] and then to test it [ pcmc clinician , round 2 ] . a second issue identified in relation to the implementation of the quality assurance framework
i think one of the most important quality indicators is a comparison of the pcmc performance versus the specialist , done on both patients referred to the specialist by the pcmc , and unselected patients seen in the clinic that would not have been referred to the specialist
access to other health care professionals , within pcmcs and in the community , was identified as important for quality care , though access was not perceived as uniform . having a pharmacist on our team is a great asset .... the local [ pharmacists ] volunteer their time to assist us at our once monthly day long clinics [ pcmc clinician , round 2 ] .
the [ social work ] and [ occupational therapy ] members of the team do an excellent job the [ alzheimer s society ] representative has been excellent as well when given an opportunity [ pcmc clinician , round 2 ] .
an important finding is the relative lack of importance ascribed to end - of - life planning .
while respondents agreed on its importance , many expressed that such planning discussions were not appropriate for patients with less advanced disease .
palliation and end of life discussion is usually not appropriate at time of our memory clinic initial or follow - up assessment since our patients are not that advanced .
family physician team will do this in following up patient [ pcmc clinician , round 1 ] .
lastly , many respondents expressed a preference for quality improvement mechanisms and learning opportunities characterized by active engagement .
they also identified specialists as agents to promote care quality , particularly in the context of a shared care approach .
this [ case review ] could be done at the same time , as the specialist mentoring the clinic is in a clinic day with the team [ pcmc clinician , round 1 ] .
barriers to the implementation of this framework included role confusion among stakeholders and limited resources .
almost all respondents mentioned the need to maintain high - quality care through ongoing , targeted training on pertinent clinical knowledge and consistency of approach among pcmc teams .
most recognized a formal quality assurance framework and individual qis as essential . great indicators and very necessary [ pcmc clinician , round 1 ] .
our challenge is to maintain evidence based salience , which ultimately facilitates improved quality of life for persons with dementia and their family [ pcmc clinician , delphi round 2 ] .
all are relevant quality indicators for a pcmc [ specialist , round 2 ] . however , a few respondents were unsure why a quality assurance framework was needed at all .
others seemed unfamiliar with the purpose of quality assurance and construed the idea of tracking qis as supplemental work .
is the intent of the questions to assess what we are doing now or [ what ] we think should be the standard ? [ pcmc clinician , round 1 ] .
this is the canadian consensus guideline that every doc should know [ specialist , round 1 ; emphasis added by authors ] .
a second theme pertained to the operationalization and implementation of the quality assurance framework within the context of pcmcs , and identified a lack of clearly delineated responsibilities among referring clinicians , pcmcs , and specialists .
respondents perceived this as problematic because absence of clarity impedes the capture of clinical documentation relevant to qi measurement .
information is sent back to referral physician with recommendations for caregiver and patient mostly related to change in medication or treatment , lifestyle modification and support resources [ pcmc clinician , round 2 ] .
this lack of clarity was considered most problematic with regard to patient follow - up .
patients with dementia with great plans should not need constant surveillance and follow up but ... this is not my experience ... they appear to often benefit from a watchdog team to ensure their decisions are being carried out . [ specialist , round 1 ] .
similar concerns were raised regarding the management of comorbidities . [ my ] assumption is that management of comorbidities is the primary care physician s domain .
recommendations are made for lifestyle changes , however [ congestive heart failure ] and diabetes management are only commented on , suggestions are made to the referring physician [ pcmc clinician , round 2 ] .
it was not clear which health care provider should conduct a physical examination , leading to gaps in assessment .
referring family physicians are expected to have completed a complete physical including appropriate neuro exam prior to referral [ pcmc clinician , round 1 ] .
my biggest concern is that the pcmc team assumes that a proper physical and neurologic exam has been done by the referring source [ specialist , round 1 ] .
respondents provided several comments on potential barriers to the implementation process of a quality assurance framework in pcmcs .
one of the main obstacles was the perceived burden of documentation required to implement qis .
[ it will take a lot of work to do the ] searching and documenting as a group of patients needs a process to set up how to search , then doing the search [ pcmc clinician , round 2 ] .
integrating qis into existing electronic medical records was touted as a solution , though potentially a resource - intensive one .
making up stamps to collect this information is doable but takes time for someone to make the [ standardized template ] and then to test it
a second issue identified in relation to the implementation of the quality assurance framework is the need to establish benchmarks to properly interpret qi scores .
i think one of the most important quality indicators is a comparison of the pcmc performance versus the specialist , done on both patients referred to the specialist by the pcmc , and unselected patients seen in the clinic that would not have been referred to the specialist [ specialist , round 1 ] .
access to other health care professionals , within pcmcs and in the community , was identified as important for quality care , though access was not perceived as uniform .
having a pharmacist on our team is a great asset .... the local [ pharmacists ] volunteer their time to assist us at our once monthly day long clinics [ pcmc clinician , round 2 ] .
the [ social work ] and [ occupational therapy ] members of the team do an excellent job the [ alzheimer s society ] representative has been excellent as well when given an opportunity [ pcmc clinician , round 2 ] .
an important finding is the relative lack of importance ascribed to end - of - life planning . while respondents agreed on its importance
, many expressed that such planning discussions were not appropriate for patients with less advanced disease . palliation and end of life discussion is usually not appropriate at time of our memory clinic initial or follow - up assessment since our patients are not that advanced .
family physician team will do this in following up patient [ pcmc clinician , round 1 ] .
lastly , many respondents expressed a preference for quality improvement mechanisms and learning opportunities characterized by active engagement .
they also identified specialists as agents to promote care quality , particularly in the context of a shared care approach .
this [ case review ] could be done at the same time , as the specialist mentoring the clinic is in a clinic day with the team [ pcmc clinician , round 1 ] .
barriers to the implementation of this framework included role confusion among stakeholders and limited resources .
this study used a consultative process among primary and specialty providers to identify a core group of qis and preferred quality improvement mechanisms for dementia care .
quality assurance can be an effective method to ensure fidelity to best practices and maintain a high standard of care quality .
selected qis address care processes , assessment and reassessment , medication review and management , investigations , non - pharmacological management , pharmacological management , and managing concomitant conditions .
selected quality improvement mechanisms emphasize a desire for multimodal education and closer collaboration with specialists . assessing care quality and publically reporting the findings are increasingly commonplace .
however , quality assurance still mainly focuses on specific care sectors or episodes , rather than on overall care for conditions that require coordination and collaboration across multiple sectors . in that context
critical considerations include educating clinicians about the role of quality assurance , improving role clarity among providers involved in dementia care , expanding access to allied health professionals , and creating standardized electronic medical record templates to simplify qi documentation .
quality assurance must not create additional burden on clinicians often working with limited resources and time , a burden that could also impact the quality of the clinician - patient interaction .
respondents proposed that 1030% of patients seen in pcmcs be reviewed with a specialist , preferably in a shared care approach .
closer integration of urban specialists with rural pcmcs represents a tremendous opportunity to extend quality dementia care , particularly to rural patients .
, it will be necessary to identify barriers ( e.g. , allocation of funding ) and facilitators ( e.g. , resources for coordination , electronic medical records , telemedicine ) .
another important finding was the relatively low rankings of qis related to end - of - life planning with dementia patients .
in addition to potential discomfort among clinicians to address such issues , this finding may also stem from the understandable desire to maintain hope and engagement early in the course of the illness , though undue delays may leave patients and caregivers less able to meaningfully participate in such discussions . the most important identified barrier to the implementation of the quality assurance framework
is the need for greater clarity on responsibilities of referring family physicians , pcmc clinicians , and specialists , particularly with respect to follow - up , physical examination , and care of complex comorbidities .
quality assurance often targets relatively simple conditions , such as hypertension , or restricts its scope to specific aspects , processes or locations of care .
in contrast , dementia , like all major chronic conditions , follows a course of progressive decline punctuated by increasingly frequent health complications ( related to dementia itself , as well as to exacerbations of concurrent comorbidities ) , multiple care transitions , progressive caregiver stress and health service utilization , and ultimately death .
optimal dementia care thus requires a systems approach of integration and coordination . addressing greater role clarity among all dementia stakeholders , a task that with proper resources could be coordinated from within pcmcs , requires immediate attention , in order to ensure a stable clinical infrastructure that is able to safely and effectively address the needs of these patients , wherever they may be and whenever they arise . until such integration is achieved
first , only two delphi rounds were conducted because the response rate fell markedly after the first .
however , the response rate remained above what is considered appropriate for delphi surveys , and ratings of individual qis , except those related to physical examination , remained stable between rounds .
a second limitation is that the survey was solely distributed within the previously described network of pcmcs , though this model is widely implemented across ontario .
as the qis were selected by both primary and specialist providers , they are likely applicable to other dementia care settings . third , participation of neurologists in the first round was low and their response was not solicited in the second round .
the relatively lower participation of neurologists in this work , compared to geriatricians and geriatric psychiatrists , is notable and requires further investigation .
fourth , allied health providers were under - represented and , given their importance in dementia care , additional work is required to further understand and develop their role in an integrated system of dementia care .
fifth , patient and caregivers were not surveyed regarding what they consider to be important qis .
finally , candidate qis and quality improvement mechanisms were not identified through a systematic literature review , but the use of published guidelines and compendiums likely identified the most important elements of quality dementia care , which would thus have remained highly ranked .
first , only two delphi rounds were conducted because the response rate fell markedly after the first . however , the response rate remained above what is considered appropriate for delphi surveys , and ratings of individual qis , except those related to physical examination , remained stable between rounds .
a second limitation is that the survey was solely distributed within the previously described network of pcmcs , though this model is widely implemented across ontario .
as the qis were selected by both primary and specialist providers , they are likely applicable to other dementia care settings . third , participation of neurologists in the first round was low and their response was not solicited in the second round .
the relatively lower participation of neurologists in this work , compared to geriatricians and geriatric psychiatrists , is notable and requires further investigation .
fourth , allied health providers were under - represented and , given their importance in dementia care , additional work is required to further understand and develop their role in an integrated system of dementia care .
fifth , patient and caregivers were not surveyed regarding what they consider to be important qis .
finally , candidate qis and quality improvement mechanisms were not identified through a systematic literature review , but the use of published guidelines and compendiums likely identified the most important elements of quality dementia care , which would thus have remained highly ranked .
while this study has identified qis and quality improvement mechanisms to assess care quality for dementia , findings underscore the importance of system integration for the provision of quality dementia care , with specifically defined and mutually understood roles among stakeholders and , where necessary , the reallocation of existing resources to support this approach to care .
an approach whereby clear roles are negotiated among dementia stakeholders can provide sufficient flexibility to meet regional needs ( especially in rural areas where access to specialists is more limited ) , foster more effective collaboration and accountability , and thus facilitate the delivery and measurement of care quality for dementia . within that context , proper field - testing , validation , and evaluation of selected qis and quality improvement mechanisms
this work has significant implications on the organization of care for aging patients with complex conditions .
primary and specialist providers share the responsibility of providing and supporting integrated dementia quality care . as such
, the care of persons with cognitive impairment would be enhanced by the development of a practical and realistically feasible quality assurance framework , under whose umbrella both pcmc and specialist services are integrated , and which ensures high fidelity to intended design and best practices , and thus maintains a high level of care quality .
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backgroundprimary care - based memory clinics ( pcmcs ) have been established in several jurisdictions to improve the care for persons with alzheimer s disease and related dementias .
we sought to identify key quality indicators ( qis ) , quality improvement mechanisms , and potential barriers and facilitators to the establishment of a quality assurance framework for pcmcs.methodswe employed a delphi approach to obtain consensus from pcmc clinicians and specialist physicians on qis and quality improvement mechanisms .
thirty - eight candidate qis and 19 potential quality improvement mechanisms were presented to participants in two rounds of electronic delphi surveys .
written comments were collected and descriptively analyzed.resultsthe response rate for the first and second rounds were 21.3% ( n = 179 ) and 12.8% ( n = 88 ) , respectively .
the majority of respondents were physicians .
fourteen qis remained after the consensus process .
ten quality improvement mechanisms were selected with those characterized by specialist integration , such as case discussions and mentorships , being ranked highly .
written comments revealed three major themes related to potential barriers and facilitators to quality assurance : 1 ) perceived importance , 2 ) collaboration and role clarity , and 3 ) implementation process.conclusionwe successfully utilized a consultative process among primary and specialty providers to identify core qis and quality improvement mechanisms for pcmcs . identified quality improvement mechanisms highlight desire for multi - modal education . system integration and closer integration between pcmcs and specialists were emphasized as essential for the provision of high - quality dementia care in community settings .
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INTRODUCTION
METHODS
Protocol and Process
Data Collection
Data Analysis
RESULTS
Respondent Comments
1. Need for and Relevance of Quality Assurance for Dementia Care
2. Collaboration and Role Clarity
3. Process of QA Framework Implementation in PCMCs
DISCUSSION
Limitations of the Study
CONCLUSIONS
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copd is a common , usually progressive disease characterized by chronic inflammation of the airways and persistent airflow limitation .
its prevalence is increasing worldwide , and it is expected to be the third largest global cause of mortality by the year 2030.1 in japan , a large epidemiological study ( the nippon copd epidemiological study ) reported that the prevalence of airflow limitation was 10.6% , and at least 8.6% of subjects were sought to have copd.2 several studies have shown that the prevalence of potentially undiagnosed airflow limitation in both western35 and asian6 countries is ~ 3%15% .
many patients with copd continue to be underdiagnosed and untreated.2,7 since copd is a preventable and treatable disease , the importance of early detection has been emphasized.8 the use of simple copd screening questionnaires to detect persistent airflow limitation may help in the early diagnosis of copd .
these tools reliably detect airflow limitation in the general population and may facilitate the early , accurate diagnosis of copd in general practice settings.9,10 some copd diagnostic questionnaires have already been reported.1114 the copd population screener ( copd - ps ) , which was developed by a clinician working group in the united states , is a five - item , self - administered questionnaire that was validated for screening individuals in the general population who are at high risk of copd .
it is composed of three copd - related items ( breathlessness , productive cough , and activity limitation ) and one question , each regarding smoking history and age.15 in a previous population - based study , we verified the validity of the japanese version of the copd - ps questionnaire for the identification of individuals at increased risk of airflow limitation.16 this instrument is easy to score and would be suitable for large - scale screening of possible airflow obstruction ( ao ) .
another copd screening tool , the international primary care airway group ( ipag ) questionnaire,11 is in use worldwide .
it consists of eight items and takes more time to administer than the copd - ps questionnaire .
the purpose of this study was to compare these two questionnaires in the general japanese population , as no previous research has done so , as well as to assess the ability of both instruments to discriminate between subjects with and without persistent ao .
this study was based on data from the hisayama study , which is an ongoing population - based epidemiologic study designed to investigate the morbidity , mortality , and risk factors of cardiovascular and smoking - related diseases in the town of hisayama , japan .
the town is located in a suburban area adjacent to fukuoka city , a large urban center on kyushu island in the southern part of japan .
the population of the town is ~8,000 and has been stable for over 50 years .
according to national census data , the distributions of age and occupations in hisayama have been almost identical to those across japan since the 1960s.17 this cross - sectional study compared the screening efficacy of the copd - ps and ipag questionnaires in copd patients . in 2012 ,
registered subjects 40 years of age and older were solicited to participate in a town - wide health checkup that included spirometry . of the 2,643 subjects who were enrolled between june 2012 and october 2012 ,
307 were excluded for the following reasons : 105 had physician - diagnosed asthma , 22 had a previous lung resection , 159 had poor studied data , and 21 had records with missing data .
the final analysis included data for 2,336 subjects with fully completed copd - ps and ipag questionnaires and valid spirometry measurements .
the subjects who provided informed consent to participate in the health checkup independently completed the japanese versions of the copd - ps and ipag questionnaires , and then , in addition to their usual clinical tests , underwent spirometry using a chestgraph hi-105 spirometer ( chest mi , tokyo , japan ) .
each subject performed at least three forced vital capacity ( fvc ) maneuvers according to the recommended method .
the results were assessed by two pulmonary physicians , who visually inspected the flow volume curve and excluded subjects without at least two satisfactory tests .
the highest forced expiratory volume in 1 second ( fev1 ) and fvc values were used for analysis .
the subjects who had pre - bronchodilator ( bd ) fev1/fvc < 0.70 were eligible for post - bd testing , in which spirometry was performed 15 minutes after inhalation of salbutamol ( glaxosmithkline , tokyo , japan ) via a metered - dose inhaler with a spacer , according to the procedure recommended.18 persistent ao was defined as having a post - bd fev1/fvc < 0.70 . the subjects with persistent ao were categorized according to the global initiative for chronic obstructive lung disease criteria ( mild , fev1 80% predicted ; moderate , 50% fev1 < 80% predicted ; severe , 30% fev1 < 50% predicted ; very severe , fev1
< 30% predicted).19 the study protocol was approved by the institutional review board for clinical research of kyushu university ( numbers 2137 , 2482 , and 24123 ) and of kagoshima university ( numbers 156 and 279 ) , and all subjects provided their written informed consent prior to participation in the study .
the copd - ps is a brief , reliable , self - scored questionnaire to identify individuals likely to have copd .
it consists of five items , three assessing copd - related symptoms on a 5-point scale , one on cigarette use ( 3-point scale ) , and one on the subject s age ( four categories ) .
these five items are scored 0 , 1 , or 2 with a summed total score ranging from 0 to 10 . in the japanese version of the copd - ps questionnaire , a cutoff point of 4
has been found to be useful for copd screening.16 the ipag questionnaire is also a self - scored questionnaire and has been validated in smokers as a screening tool for copd diagnosis.11 the ipag questionnaire is composed of eight items : one on the subject s age ( four categories ) , one on body mass index ( 3-point scale ) , one on cigarette use ( 4-point scale ) , and five on symptoms / history ( 2- or 3-point scales ) .
each question is scored individually , with a summed total score ranging from 0 to 38 .
a suggested cutoff score of 17 is used for smokers in general health checkup settings and general practices in japan . however , there is no cutoff score for persistent ao in the general japanese population , including never - smokers , and we therefore investigated this issue in this study .
baseline data for the demographic characteristics of the study population and the informant questionnaires were evaluated in descriptive analyses . the kruskal
spearman correlations were used to examine the strength of associations between informant and performance measures . for the subjects who used a bd ,
another way to evaluate the utility of screening tests is with the likelihood ratio;20 likelihood ratios range from 0 to infinity ; larger numbers provide more convincing evidence of a disease , smaller numbers indicate that the disease is less likely , and ratios close to one lack diagnostic value .
likelihood ratios ( positive and negative ) were calculated for both the copd - ps and the ipag questionnaires .
receiver operating characteristic ( roc ) curves and areas under the roc curves ( aucs ) were generated to reflect graphically and quantitatively the ability of the copd - ps and the ipag questionnaires to discriminate between subjects with and without persistent ao using the delong method.21 another method proposed by pencina et al22 was also utilized for this purpose ; this approach assesses the ability of a model to reclassify case and control subjects , respectively , on the basis of the individual - estimated probability of an event .
the ability of the model to reclassify is summarized by the net reclassification improvement ( nri ) and integrated discrimination improvement ( idi ) .
we defined four strata dividing the risk of persistent ao into four quartiles , namely , q1 , q2 , q3 , and q4 .
the nri considers only the changes in the estimated prediction probabilities that imply a change from one category to another .
in contrast , the idi does not require a prior definition of strata risk and considers the change in the estimation prediction probabilities as a continuous variable .
we computed the nri and idi and examined the accuracy of the two screening questionnaires in diagnosing ao .
all statistical analyses were performed using r version 3.1.0.23 the results were considered statistically significant when p<0.05 .
this study was based on data from the hisayama study , which is an ongoing population - based epidemiologic study designed to investigate the morbidity , mortality , and risk factors of cardiovascular and smoking - related diseases in the town of hisayama , japan .
the town is located in a suburban area adjacent to fukuoka city , a large urban center on kyushu island in the southern part of japan .
the population of the town is ~8,000 and has been stable for over 50 years .
according to national census data , the distributions of age and occupations in hisayama have been almost identical to those across japan since the 1960s.17
this cross - sectional study compared the screening efficacy of the copd - ps and ipag questionnaires in copd patients . in 2012 , registered subjects 40 years of age and older were solicited to participate in a town - wide health checkup that included spirometry .
of the 2,643 subjects who were enrolled between june 2012 and october 2012 , 307 were excluded for the following reasons : 105 had physician - diagnosed asthma , 22 had a previous lung resection , 159 had poor studied data , and 21 had records with missing data .
the final analysis included data for 2,336 subjects with fully completed copd - ps and ipag questionnaires and valid spirometry measurements .
the subjects who provided informed consent to participate in the health checkup independently completed the japanese versions of the copd - ps and ipag questionnaires , and then , in addition to their usual clinical tests , underwent spirometry using a chestgraph hi-105 spirometer ( chest mi , tokyo , japan ) .
each subject performed at least three forced vital capacity ( fvc ) maneuvers according to the recommended method .
the results were assessed by two pulmonary physicians , who visually inspected the flow volume curve and excluded subjects without at least two satisfactory tests .
the highest forced expiratory volume in 1 second ( fev1 ) and fvc values were used for analysis .
the subjects who had pre - bronchodilator ( bd ) fev1/fvc < 0.70 were eligible for post - bd testing , in which spirometry was performed 15 minutes after inhalation of salbutamol ( glaxosmithkline , tokyo , japan ) via a metered - dose inhaler with a spacer , according to the procedure recommended.18 persistent ao was defined as having a post - bd fev1/fvc < 0.70 .
the subjects with persistent ao were categorized according to the global initiative for chronic obstructive lung disease criteria ( mild , fev1 80% predicted ; moderate , 50% fev1 < 80% predicted ; severe , 30% fev1 < 50% predicted ; very severe , fev1 < 30% predicted).19 the study protocol was approved by the institutional review board for clinical research of kyushu university ( numbers 2137 , 2482 , and 24123 ) and of kagoshima university ( numbers 156 and 279 ) , and all subjects provided their written informed consent prior to participation in the study .
the copd - ps is a brief , reliable , self - scored questionnaire to identify individuals likely to have copd .
it consists of five items , three assessing copd - related symptoms on a 5-point scale , one on cigarette use ( 3-point scale ) , and one on the subject s age ( four categories ) .
these five items are scored 0 , 1 , or 2 with a summed total score ranging from 0 to 10 . in the japanese version of the copd - ps questionnaire ,
the ipag questionnaire is also a self - scored questionnaire and has been validated in smokers as a screening tool for copd diagnosis.11 the ipag questionnaire is composed of eight items : one on the subject s age ( four categories ) , one on body mass index ( 3-point scale ) , one on cigarette use ( 4-point scale ) , and five on symptoms / history ( 2- or 3-point scales ) .
each question is scored individually , with a summed total score ranging from 0 to 38 .
a suggested cutoff score of 17 is used for smokers in general health checkup settings and general practices in japan .
however , there is no cutoff score for persistent ao in the general japanese population , including never - smokers , and we therefore investigated this issue in this study .
baseline data for the demographic characteristics of the study population and the informant questionnaires were evaluated in descriptive analyses . the kruskal
spearman correlations were used to examine the strength of associations between informant and performance measures . for the subjects who used a bd ,
another way to evaluate the utility of screening tests is with the likelihood ratio;20 likelihood ratios range from 0 to infinity ; larger numbers provide more convincing evidence of a disease , smaller numbers indicate that the disease is less likely , and ratios close to one lack diagnostic value .
likelihood ratios ( positive and negative ) were calculated for both the copd - ps and the ipag questionnaires .
receiver operating characteristic ( roc ) curves and areas under the roc curves ( aucs ) were generated to reflect graphically and quantitatively the ability of the copd - ps and the ipag questionnaires to discriminate between subjects with and without persistent ao using the delong method.21 another method proposed by pencina et al22 was also utilized for this purpose ; this approach assesses the ability of a model to reclassify case and control subjects , respectively , on the basis of the individual - estimated probability of an event .
the ability of the model to reclassify is summarized by the net reclassification improvement ( nri ) and integrated discrimination improvement ( idi ) .
we defined four strata dividing the risk of persistent ao into four quartiles , namely , q1 , q2 , q3 , and q4 .
the nri considers only the changes in the estimated prediction probabilities that imply a change from one category to another .
in contrast , the idi does not require a prior definition of strata risk and considers the change in the estimation prediction probabilities as a continuous variable .
we computed the nri and idi and examined the accuracy of the two screening questionnaires in diagnosing ao .
all statistical analyses were performed using r version 3.1.0.23 the results were considered statistically significant when p<0.05 .
the baseline characteristics of the 2,336 subjects stratified by airflow limitation category following post - bd spirometry are presented in table 1 .
the majority of subjects ( 88.9% ) showed an initial fev1/fvc 0.70 . following post - bd spirometry ,
almost all the ao subjects ( 94.0% ) were classified as having mild or moderate copd ; only 6.0% of the ao subjects had severe or very severe copd .
the ao subjects were older , were more likely to be men , had lower body mass index , had a higher number of pack - years smoked , and were more likely to be former or current smokers ( table 1 ) . the mean and
for the ao and non - ao subjects , respectively , the mean scores were 3.9 and 2.4 , while the median scores were 4 and 2 .
the ipag and the copd - ps questionnaires were correlated with fev1/fvc ( r=0.356 and r=0.301 , respectively , p<0.001 ) , and the two questionnaires correlated with each other ( r=0.622 , p<0.001 ; table 2 ) . the previously identified cutoff point of 4 was used for the copd - ps questionnaire .
the crude odds ratio ( or ) of the copd - ps questionnaire for ao was 5.52 , and the sensitivity of copd - ps questionnaire was 66.7% and the specificity was 73.4% .
in this population - based study that included never - smokers , a cutoff point of 20 on the ipag questionnaire would be adequate for screening for ao ( table 3 ) .
the crude or of the ipag questionnaire for ao , using a cutoff point of 20 , was 6.56 .
if a cutoff point of 17 was used on the ipag questionnaire , the sensitivity was higher ( 86.0% ) but both the specificity and auc were much lower , 46.2% and 0.66% , respectively .
if a cutoff point of 20 was used instead of 17 , the sensitivity was slightly lower ( 71.3% ) but both the specificity and auc were higher , 75.2% and 0.72% , respectively .
roc curves were generated to measure the properties of the copd - ps and the ipag questionnaires in discriminating subjects without ao from those with ao .
the auc values obtained from the roc curve by discriminating ao from no ao were 0.747 ( 95% confidence interval [ ci ] , 0.7070.788 ) for the copd - ps questionnaire and 0.775 ( 95% ci , 0.7350.816 ) for the ipag questionnaire ( figure 1 ) .
there was no significant difference in the auc values with the two questionnaires ( p=0.09 ) . between the copd - ps and ipag questionnaires ,
no statistically significant differences were founded in terms of sensitivity , specificity , or positive and negative predictive values .
however , compared with the copd - ps questionnaire , the ipag questionnaire had superior likelihood ratios for both a positive ( 2.51 vs 2.59 ) and negative test ( 0.45 vs 0.40 ) , with nonoverlapping cis ( table 4 ) .
reclassifications of subjects with and without ao are summarized in table 5 . for 672 ( 30.7% ) subjects without ao ,
classification improved using the model with the copd - ps questionnaire , and for 549 ( 25.1% ) subjects , it became worse , with the net gain in reclassification proportion of 0.056 . for subjects with ao
, classification was improved in 21 subjects ( 14.0% ) and less accurate in 28 subjects ( 18.7% ) , with the net gain in reclassification proportion of 0.047 .
thus , the categorical nri was 0.0096 ( 95% ci , 0.08680.106 ; p=0.845 ) .
the continuous nri and idi for predicting the presence of ao using the model with the copd - ps questionnaire against the ipag questionnaire were 0.107 ( 95% ci , 0.2730.058 ; p=0.203 ) and 0.014 ( 95% ci , 0.0330.006 ; p=0.182 ) , respectively , and these were not statistically significant ( table 5 ) .
the copd - ps and the ipag questionnaires had only a marginal difference in their ability to discriminate between the subjects with and without ao .
the baseline characteristics of the 2,336 subjects stratified by airflow limitation category following post - bd spirometry are presented in table 1 .
the majority of subjects ( 88.9% ) showed an initial fev1/fvc 0.70 . following post - bd spirometry ,
almost all the ao subjects ( 94.0% ) were classified as having mild or moderate copd ; only 6.0% of the ao subjects had severe or very severe copd .
the ao subjects were older , were more likely to be men , had lower body mass index , had a higher number of pack - years smoked , and were more likely to be former or current smokers ( table 1 ) . the mean and
for the ao and non - ao subjects , respectively , the mean scores were 3.9 and 2.4 , while the median scores were 4 and 2 .
the ipag and the copd - ps questionnaires were correlated with fev1/fvc ( r=0.356 and r=0.301 , respectively , p<0.001 ) , and the two questionnaires correlated with each other ( r=0.622 , p<0.001 ; table 2 ) .
the previously identified cutoff point of 4 was used for the copd - ps questionnaire .
the crude odds ratio ( or ) of the copd - ps questionnaire for ao was 5.52 , and the sensitivity of copd - ps questionnaire was 66.7% and the specificity was 73.4% . in this population - based study that included never - smokers , a cutoff point of 20 on
the crude or of the ipag questionnaire for ao , using a cutoff point of 20 , was 6.56 .
if a cutoff point of 17 was used on the ipag questionnaire , the sensitivity was higher ( 86.0% ) but both the specificity and auc were much lower , 46.2% and 0.66% , respectively .
if a cutoff point of 20 was used instead of 17 , the sensitivity was slightly lower ( 71.3% ) but both the specificity and auc were higher , 75.2% and 0.72% , respectively .
roc curves were generated to measure the properties of the copd - ps and the ipag questionnaires in discriminating subjects without ao from those with ao .
the auc values obtained from the roc curve by discriminating ao from no ao were 0.747 ( 95% confidence interval [ ci ] , 0.7070.788 ) for the copd - ps questionnaire and 0.775 ( 95% ci , 0.7350.816 ) for the ipag questionnaire ( figure 1 ) .
there was no significant difference in the auc values with the two questionnaires ( p=0.09 ) . between the copd - ps and ipag questionnaires ,
no statistically significant differences were founded in terms of sensitivity , specificity , or positive and negative predictive values .
however , compared with the copd - ps questionnaire , the ipag questionnaire had superior likelihood ratios for both a positive ( 2.51 vs 2.59 ) and negative test ( 0.45 vs 0.40 ) , with nonoverlapping cis ( table 4 ) . reclassifications of subjects with and without ao are summarized in table 5 . for 672 ( 30.7% ) subjects without ao ,
classification improved using the model with the copd - ps questionnaire , and for 549 ( 25.1% ) subjects , it became worse , with the net gain in reclassification proportion of 0.056 . for subjects with ao , classification was improved in 21 subjects ( 14.0% ) and less accurate in 28 subjects ( 18.7% ) , with the net gain in reclassification proportion of 0.047 .
thus , the categorical nri was 0.0096 ( 95% ci , 0.08680.106 ; p=0.845 ) .
the continuous nri and idi for predicting the presence of ao using the model with the copd - ps questionnaire against the ipag questionnaire were 0.107 ( 95% ci , 0.2730.058 ; p=0.203 ) and 0.014 ( 95% ci , 0.0330.006 ; p=0.182 ) , respectively , and these were not statistically significant ( table 5 ) .
the copd - ps and the ipag questionnaires had only a marginal difference in their ability to discriminate between the subjects with and without ao .
in the present population - based study , the japanese version of the copd - ps questionnaire was compared with the ipag questionnaire in a general japanese population at the age of 40 years or older .
the ipag questionnaire had superior likelihood ratios of both positive and negative tests compared with those of the copd - ps questionnaire .
however , in comparison with two questionnaires , no significant differences were founded in the auc values obtained from the roc curves discriminating between subjects with and without ao and in the sensitivity , specificity , or positive and negative predictive values .
the two questionnaires were correlated with each other , fev1/fvc , and for both of them , the ors were significantly greater than 1.0 .
these findings suggest that the copd - ps and the ipag questionnaires are useful screening tools for detecting persistent ao . in addition , the subjects with a 4 point on the copd - ps questionnaire and those with a 20 point on the ipag questionnaire are at increased risk for ao .
overall , the categorical and continuous nri and idi for predicting the presence of ao using the model with the copd - ps questionnaire were 0.0096 ( 95% ci , 0.08680.106 ; p=0.845 ) , 0.107 ( 95% ci , 0.2730.058 ; p=0.203 ) , and 0.014 ( 95% ci , 0.0330.006 ; p=0.182 ) , respectively .
reclassifications showed the ipag questionnaire to be superior to the copd - ps questionnaire ; however , there was no significant difference between the two questionnaires .
a cutoff point of 17 on the ipag questionnaire is used in general health checkup settings and general practices in japan.24 in this study , we found that a cutoff point of 20 was superior .
the reason for this discrepancy is not clear , although it may be due at least in part to the backgrounds of the study subjects .
the present study was population based and included never - smokers . in contrast , in a previous study that enrolled subjects in general health checkup settings , a cutoff point of 17 resulted in a higher sensitivity of 86.0% and a smaller auc of 0.66 ( lower than 0.70 ) than a cutoff point of 20.24 the present study was implemented in the town of hisayama , in which the age and occupational distributions of the population have been almost identical to those of japan as a whole , and thus , we recommend a cutoff point of 20 for the general japanese population .
there were only marginal differences between the two questionnaires in terms of ability to discriminate between subjects with and without ao .
moreover , the copd - ps questionnaire consists of fewer items than the ipag questionnaire and requires less time to complete .
it takes ~5 minutes to fill out copd - ps questionnaire , however , the completion of the ipag questionnaire takes ~510 minutes . in this regard ,
the japanese version of the copd - ps questionnaire should be an adequate measure for large - scale screening for possible ao .
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backgroundthe incidence of chronic obstructive pulmonary disease ( copd ) is increasing worldwide . in japan and other countries ,
epidemiological studies have found that many patients with copd are underdiagnosed and untreated , and thus , early detection and treatment of copd has been emphasized .
screening questionnaires may have utility in the initial detection of copd.objectivethis study aimed to validate and compare the copd population screener ( copd - ps ) and the international primary care airway group ( ipag ) questionnaires in a general japanese population.patients and methodseligible subjects 40 years of age and older living in the town of hisayama were solicited to participate in a health checkup in 2012 .
all subjects 4079 years of age without physician - diagnosed asthma or lung resection were recruited , and 2,336 subjects who fully completed both questionnaires and who had valid spirometry measurements were analyzed .
persistent airflow obstruction ( ao ) was defined by a postbronchodilator forced expiratory volume in 1 second / forced vital capacity < 0.70 .
receiver operating characteristic curves , net reclassification improvement , and integrated discrimination improvement were used to examine the ability of the copd - ps and ipag questionnaires to discriminate between subjects with and without ao.resultsthe overall area under the receiver operating characteristic curve for the copd - ps questionnaire was 0.747 ( 95% confidence interval [ ci ] , 0.7070.788 ) and for the ipag was 0.775 ( 95% ci , 0.7350.816 ) , with no significant difference ( p=0.09 ) .
the net reclassification improvement and integrated discrimination improvement were 0.107 ( 95% ci , 0.2730.058 ; p=0.203 ) and 0.014 ( 95% ci , 0.0330.006 ; p=0.182 ) , respectively.conclusionthe five - item copd - ps questionnaire was comparable to the eight - item ipag for discriminating between subjects with and without ao . the copd - ps is a simple and useful screening questionnaire for persistent ao .
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Introduction
Methods
Study population
Study design
COPD-PS
IPAG questionnaire
Statistical analysis
Results
Participant characteristics
Discriminating subjects with and without AO
Discussion
Conclusion
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personalized medicine is defined by the use of genomic signatures of patients to assign effective therapies in order to achieve the best medical outcomes for individual patients , thus improving public health . despite the variety of clinical , morphological , and molecular parameters
used to classify human malignancies , patients receiving the same diagnosis can have markedly different clinical courses and treatment responses . since there is no simple way to determine who will have an adverse reaction ,
the current system of one - size - fits - all- diagnoses is simply not good enough .
an increasing number of studies have demonstrated sex differences in drug reactions to the same drug treatment .
migeon implied that males and females responded differently to drug treatments and that sex plays a key role in cancer .
in addition , females are historically less studied subjects due to the complication of estrous cycle , and therefore such studies would further benefit women 's health and promote public health .
recent advancements in biotechnology have accelerated the search for molecular biomarkers useful in the diagnosis and treatment of disease .
molecular biomarkers of disease risk and status are critical to an accurate treatment by identifying patients most likely to benefit from particular drugs or experience adverse reactions . because medicine is always practiced on individuals rather than populations , the goal is to change the assignment of therapies from a population - based approach to an individualized approach .
gene - expression data can be used to identify patients with a good disease prognosis , thereby preventing some patients from unnecessary therapies and toxicity .
for example , gene - expression profiling was used to predict clinical outcomes in pediatric patients with acute myeloid leukemia and to find genes whose aberrant expression leads to a poor prognosis .
thus , accurate classification of prognosis of patients leads to efficient cancer treatment and prolonged survival of patients .
classification algorithms are needed for biomedical decision making in clinical assignment of patients to treatment therapies based on individual risk factors and disease characteristics . since many of those genes are not relevant , feature selection is a commonly addressed problem in classification [ 3 , 4 ] .
the goal of gene selection is to identify a set of genes which plays an important role in the classification .
a common approach is to select a fixed number of the highest ranked genes based on t - test - like statistics , some discrimination measures [ 6 , 7 ] , or classification algorithms including support vector machines ( svm ) [ 8 , 9 ] and random forest ( rf ) [ 10 , 11 ] .
development of a biomarker classifier involves two distinct components : ( 1 ) a procedure for building a classifier and ( 2 ) validation / evaluation procedure for estimating the error rate of biomarker .
the most important consideration is the validation / evaluation of a biomarker classifier to assess whether it can accurately and unbiasedly predict new samples based on a set of selected features .
two methods are commonly used to develop and assess performance of a classifier : the split - sample procedure and cross - validation procedure . in the split - sample procedure , the sample dataset
is randomly split into two subsets : a training set for model building and a test set for performance assessment .
that is , the training dataset is used for building the biomarker classifiers and the test set is used for evaluating the classifier .
however , samples are often insufficient to split into two sample sets of approximately equal size for model building and model testing , and a cross - validation ( cv ) procedure is commonly used for performance assessment [ 12 , 13 ]
. a cv can be regarded as a generalization of the split - sample method .
it involves repeatedly splitting the data into a training set containing most of the samples and applying the prediction rule to the test set of the remaining samples to estimate the prediction accuracy rate .
the prediction accuracy is the average accuracy of the numerous training - test partitions . for model building ,
given that differences in the biology of lung cancer and other diseases exist between men and women [ 14 , 15 ] , investigations to identify genomic biomarkers for clinical assignment of therapies on an individual patient basis are crucial . in this paper , the ratio of between - group to within - group sums of squares ( bw ratio , ) gene selection algorithm is used to obtain a feasible set of influential genes via variable importance ranking procedure in the training phases of 20 trials of 10-fold cv within leave - one - out cross - validation ( loocv ) procedure as illustrated below .
the genes with largest bw ratios are ranked high as significant genes . in the development of biomarker classifier
, the predictive accuracy of the classifier must be evaluated on a separate set of data . to derive an unbiased accuracy estimate , a nested cross - validation procedure , 20 trials of 10-fold cv within loocv ,
is used in this paper . in other words , in each loocv , 90% of the
wherefore genes are selected only using learning data sets of size ( n 1 ) each , and in the evaluation / validation step a never - touched and left - out single observation in loocv is used to assess the predictive performance of selected genes . in this way , each test case is never used for gene selection . to avoid a bias due to partitions of data ,
if the performance is assessed using the very same data that are used for developing the classifier , this obviously leads to a biased down estimate of classification error . in other words , if one applies a method to the original data with 20 trials of 10-fold cv only as a way of building the classifier and selects a set of genes and then on that very same data calculates the classification error , it clearly leads to a biased upward estimate of classification accuracy .
three publicly available data sets of interest in this paper are pediatric acute myeloid leukemia ( aml ) , b - cell chronic lymphocytic leukemia ( b - cll ) , and primary cutaneous melanoma .
the data sets are downloaded from the brb - arraytools data archive for human cancer gene expression located at the website http://linus.nci.nih.gov/~brb/dataarchive_new.html .
sex differences in disease rates or in rates of adverse reactions to treatment are common , which we intend to exploit to obtain sex - specific biomarkers from gene - expression data .
we hypothesize that genomic biomarkers developed from the sex - specific application of classification algorithms will further improve class prediction accuracy .
the summary of our algorithm to find sex - specific predictive / prognostic genomic biomarkers for efficacy or toxicity in individualized treatment of patients for serious diseases is as follows . in each loocv trial , firstly , the data is partitioned into a test data set with one observation and the remaining data as the learning data set .
the learning data set is further separated into male and female patients ' learning data sets .
thus , this process will be applied n times , where n represents the total number of patients .
secondly , within each trial of loocv , twenty trials of 10-fold cv are conducted for each set of male and female patients in the learning data set . at each 10-fold cv step ,
two sets of top - ranked genes , one for male patients ( smi ) and one for female patients ( sfi ) , are obtained via the bw ratio to differentiate types of patients such as diseased versus nondiseased .
this can be done by applying variable importance ranking approach in order to extract most influential genes and by combining a measure of importance in each gene .
a score taking the average of the measure in cross - validation ( cv ) is prioritized in the list of genes according to variable importance .
thirdly , the mutually exclusive sets of male - specific genes and female - specific genes are obtained .
fourthly , within each loocv trial , after the sets of potential sex - specific genes are determined in the second and the third steps , they are fitted to a model with diagonal linear discriminant analysis ( dlda ; ) using each learning data set for male and female patients .
finally , the performance is measured with the test data with one observation in each loocv trial .
this method is implemented in r. the r code is available upon request . within each trial of loocv , each set of top - ranked genes for male and female patients is obtained in the second and the third steps as follows .
first , in order to build genomic biomarkers , 20 trials of 10-fold cv are applied to each learning data set for males and females , where 90% were randomly selected without replacement as a set for each trial of cv .
next , for each trial of 10-fold cv , the bw ratio was applied to this 90 percent learning set and the top 25 ranked genes were selected in each process with the target endpoint of a dataset .
the bw ratio for a gene j is defined as
( 1)bwj=iki(yi = k)(xkjxj)2iki(yi = k)(xijxkj)2 ,
where x-j=ixij / n indicates the average value of a gene j across all the training samples , x - kj=i(yi = k)xij / nk indicates the average value of a gene j for a class k , and i indicates an observation . here
this criterion has been shown to be reliable for variable selection from high - dimensional data sets [ 6 , 12 ] .
next , to avoid selection bias from a pattern of selection of learning samples , we repeat the entire process 20 times by shuffling samples at every 10-fold cv . in order to obtain the variable importance ranking , 200 sets of top 25 ranked genes were combined so that the maximum possible rank score of a gene would be 200 .
a set of genes that has been selected at least once ( rank score > 0 ) was obtained separately for males and females .
these most influential genes used in the classification process are identified in order to extract a feasible set of sex - specific genes . for both male and female learning data sets ,
the final product of the 20 trials of 10-fold cv described in each loocv is the sets of genes selected for male and female patients in the learning data set smj = { g1 , , gxmj } and sfj = { g1 , , gxfj } , j = 1 , 2 , , n , respectively .
the sets smj and sfj contain prognostic / predictive genes for male and female patients , respectively
. there would be n sets of selected genes . within each trial of loocv , genes that are commonly identified between male - specific and female - specific genes
finally , the test sample with one observation is tested using these sex - specific genes in each loocv . at the end of the entire loocv
, each selected gene has a combined variable importance score that is less than or equal to n. to verify sex - specific biomarkers , we consider the following four different cases : we classify the outcome of ( 1 ) male patients with a set of male - specific genes smj,(2 ) female patients with a set of male - specific genes smj,(3 ) male patients with a set of female - specific genes sfj , and ( 4 ) female patients with a set of female - specific genes sfj .
we anticipate that data with a set of male - specific genes have higher predictable power to predict male patients than the data with female - specific genes .
similarly , data with female - specific genes have higher predictable power to predict female patients than the data with male - specific genes . upon completion of loocv
, the performance of sex - specific biomarkers is obtained . in order to validly evaluate the performance of a gene set selected by the proposed method ,
cv utilizes resampling without replacement of the entire data set to repeatedly develop classifiers on a training set and to evaluate these classifiers on a separate test set and then averages the results over the resamples .
in this section , we apply the proposed algorithm to the following genomic data sets to find the most meaningful sex - specific predictive / prognostic genomic biomarkers for improving individualized treatment of patients and for evaluating the biomarkers from the proposed algorithm . current chemotherapy enables a high percentage of pediatric patients with aml to enter complete remission ( cr ) , but a large number of them experience relapse ( r ) with resistant disease . because of the wide heterogeneity of aml , predicting a patient 's risk for treatment failure or relapse at the time of diagnosis is critical for the optimal treatment . this gene - expression data set consists of 54 aml pediatric patients ( < 15 years old ) with an oligonucleotide microarray containing 12,566 probe sets and it is also available at ftp://ftp.ncbi.nih.gov/pub/geo/data/soft/gds/gds1059.soft.gz .
patients with cr for more than 3 years are classified as having a good prognosis , while patients experienced relapse within 1 year of the first cr are considered as having a poor prognosis . in this data set , there are 28 patients with cr and 25 patients with r. since a five - month male patient experienced induction failure ( no cr achievement ) within 3 months of the start of treatment is considered neither cr nor r , we exclude this subject . therefore , there are 32 male patients and 21 female patients in this data set . with the prognostic endpoint ( r / cr ) ,
the average accuracy of 66% ( sd 3.0% ) for pediatric patient classification was obtained when no gene selection was introduced . when a set of 200 genes was selected in a learning phase of each cv ,
the average accuracy of 68.0% ( sd 3.0% ) was achieved . when a set of 20 genes was selected in a learning phase of each cv ,
the average accuracy of 71.0% ( sd 5.0% ) was obtained . since it appeared to be no substantial difference between accuracy and the number of genes selected , a feasible set of 25 genes in the learning phase of each cv
was collectively ranked by the bw ratio to find sex - specific biomarkers . at the end of loocv trials , a set of male - specific genes that were selected at least 75% of the time in the entire loocv were 1882_g_at ( mecom : mds1 and evi1 complex locus ) , 37902_at ( cryz : crystallin , zeta ( quinone reductase ) ) , 38789_at ( tkt : transketolase ( wernicke - korsakoff syndrome ) ) , 39105_at ( vasp : vasodilator - stimulated phosphoprotein ) , 40844_at ( ctr9 : ctr9 , paf1/rna polymerase ii complex component , homolog ( s. cerevisiae ) ) , 36981_at ( srp9 : signal recognition particle 9 kda ) , 36338_at ( luzp1 : leucine zipper protein 1 ) , 31870_at ( cd37 : cd37 antigen ) , 1624_at ( rap1gds1 : rap1 , gtp - gdp dissociation stimulator 1 ) , and 39142_at ( nudt21 : cleavage and polyadenylation specific factor 5 , 25 kda ) .
these ten top - ranked male - specific genes were selected as potential male - specific genomic biomarkers to classify male patients into r / cr . among them
38789_at ( tkt : transketolase ) and 36338_at ( est ) were also included in the thirty - five genes associated with prognosis of pediatric aml identified by yagi et al . .
in order to select a feasible set of sex - specific biomarkers a cut - off criterion of 75 percent is used . since
every dataset may have a different sample size , the number of genes selected is given by the percentage , which is a rank score of the selected genes greater than 75% of the sample size in our case .
for example , if a sample size is 100 , then genes that have rank scores greater than 75 have been selected .
similarly , nine top - ranked genes for classifying female patients into r / cr were 40601_at ( tm2d1 : tm2 domain containing 1 ) , 36330_at ( ccbl1 : cysteine conjugate beta- lyase ; cytoplasmic ( glutamine transaminase k , kynurenine aminotransferase ) ) , 40586_at ( eef1e1 : eukaryotic translation elongation factor 1 epsilon 1 ) , 36648_at ( crsp9 : cofactor required for sp1 transcriptional activation , subunit 9 , 33 kda ) , 32351_at ( gpr20 : g protein - coupled receptor 20 ) , 1718_at ( arpc2 : actin - related protein 2/3 complex , subunit 2 , 34 kda ) , 38622_at ( mtg1 : mitochondrial gtpase 1 homolog ( s. cerevisiae ) ) , 36496_at ( impa2 : inositol(myo)-1(or 4)-monophosphatase 2 ) , and 38337_at ( znf193 : zinc finger protein 193 ) .
these nine top - ranked genes which were selected at least 75% of the time in the entire loocv were considered as potential female - specific genomic biomarkers to classify female patients into r / cr .
data with male - specific genes showed higher prediction accuracy ( 71.9% ) to classify male patients than the accuracy ( 43.8% ) to classify male patients from data with female - specific genes .
similarly , data with female - specific genes showed higher prediction accuracy ( 76.2% ) to classify female patients than the accuracy ( 61.9% ) to classify female patients from data with male - specific genes .
as shown in table 1 , sensitivity and specificity were also higher in using sex - specific genes for each sex .
genomic aberrations and mutational status of the immunoglobulin variable heavy chain ( vh ) gene have been shown to be among the most important predictors for outcome in patients with b - cll .
b - cll is the most common leukemia in the western world , and due to its clinical heterogeneity ( wide range of life expectancy ) and correlations to genomic aberrations , it is important to predict a patient 's vh mutation status at the time of diagnosis for optimal treatment .
in addition , the study presented by haslinger et al . suggested that the genomic signature for vh mutational status might be sex related .
the gene - expression data consists of 100 b - cll patients with an oligonucleotide microarray containing around 12,000 probe sets , and it is available at http://linus.nci.nih.gov/~brb/dataarchive_new.html .
patients are classified as either vh - mutated or unmutated ( m / nm ) .
there are 62 males ( 33 m and 29 nm ) and 38 females ( 18 m and 20 nm ) , with a total of 51 mutated and 49 unmutated patients . in step 1 , for each data set with male only and female only patients using the target endpoint ( i.e. , vh - mutated ( m ) versus unmutated ( nm ) ) , we separately selected and ranked 25 potential prognostic genes for males and for females in every cv trial and separately combined ranks of these genes for males and females during the learning phase of 20 trials of 10-fold cv within each loocv trial . in every loocv trial , we prioritized and combined the final top - ranked 200 genes from the male patients result ( sm ) and 200 genes from the female patients result ( sf ) as explained in section 3.1 . at the end of the entire loocv trials , a set of potential sex - specific genes
are obtained for each sex by prioritizing and combining n sets of top - ranked 200 genes .
after deletion of overlapped genes in both males and females , eleven potential male - specific genes were obtained to classify male patients into m / nm . they were 41209_at ( lpl : lipoprotein lipase ) , 41755_at ( cobll1 : cobl - like 1 ) , 39878_at ( pcdh9 : protocadherin 9 ) , 38211_at ( zbtb20 : zinc finger and btb domain containing 20 ) , 39488_at ( pcdh9 : protocadherin 9 ) , 36886_f_at ( kir2dl3 : killer cell immunoglobulin - like receptor , two domains , long cytoplasmic tail , 3 ) , 32140_at ( sorl1 : sortilin - related receptor , l(dlr class ) a repeats - containing ) , 33535_at ( p2rx1 : purinergic receptor p2x , ligand - gated ion channel , 1 ) , 39967_at ( ldoc1 : leucine zipper , downregulated in cancer 1 ) , 32842_at ( bcl7a : b - cell cll / lymphoma 7a ) , and 36899_at ( satb1 : special at - rich sequence binding protein 1 ( binds to nuclear matrix / scaffold - associating dna 's ) ) . for female patients ,
only five genes were selected at least 75% of the time in the entire loocv trial as potential female - specific genomic biomarkers to classify female patients into m / nm . they were 33745_at ( phkg2 : phosphorylase kinase , gamma 2 ( testis ) ) , 38152_at ( loh11cr2a : loss of heterozygosity , 11 , chromosomal region 2 , gene
a ) , 34142_at ( pde8a : phosphodiesterase 8a ) , 39593_at ( fgl2 : fibrinogen - like 2 ) , and 217_at ( klk2 : kallikrein 2 , prostatic ) . to verify the sex - specific genomic biomarkers , we considered the performance of four different cases in the loocv trials as described in section 2 .
data with male - specific genomic biomarkers showed higher accuracy ( 67.7% ) to classify male patients into m / nm than the accuracy to classify male patients into m / nm from data with female - specific genomic biomarkers ( 40.3% ) .
however , female data with female - specific genes showed prediction accuracy similar to random guess close to 50% .
therefore , there is insufficient evidence to support that the selected female - specific genes were female - specific genes ( see table 2 ) with 38 female patients in this data set .
although cutaneous melanoma represents a small subset , it is the most life - threatening neoplasm of the skin , and its incidence and mortality have been increasing worldwide .
the key underlying molecular events have not been clearly elucidated , which may explain why no target has been developed and why almost no clinical benefits from new therapies have been clearly demonstrated in patients with melanoma since the late 1970s .
additionally , gene - expression profiling data for human primary cutaneous melanomas are scarce because of the lack of retrospective collections of frozen tumors .
this gene - expression data set was collected from 83 patients corresponding to the training data set and 17 patients corresponding to the validation data set .
the probes are from tumor tissue and from reference tissue that are differentially labeled by the incorporation of cyanine 3 ( cy3 ) and cyanine 5 ( cy5 ) , respectively .
set , the endpoint was patient prognosis and survival along with tumor stages , defined as follows . in stage
i , cure rates are excellent with surgical removal , since they are the least likely to spread . in stage ii , melanomas can be cured , but the success rate lags behind that of stage i because a small number of cancer cells may have spread to distant sites . in stage iii , since the tumor has started to metastasize ( the spreading of a disease from one organ or part to another nonadjacent organ or part ) , the survival rate for these stages is lower than the earlier ones .
stage iv is associated with metastasis beyond the regional lymph nodes to distant sites in the body , such as the lung , liver , or brain , or to distant areas of the skin .
based on the tumor size , descriptions , and number of lymph nodes the stages are categorized in two classes .
a class of high survival and small tumor size ( hs / st ) is defined and composed of stages 1 and 2 .
the second is defined as low survival and non - small tumor size ( ls / nst ) , which is composed of stages 3 and 4 .
tables 3 and 4 show the distribution of the primary cutaneous melanoma data based on sex , the defined clinical endpoint hs / st and ls / nst for the training data and validation data . among 83 patients in the training dataset ,
there are 27 males ( 12 hs / st and 15 ls / nst ) and 56 females ( 30 hs / st and 26 ls / nst ) .
there are 42 cases of hs / st and 41 cases of ls / nst in total .
after preprocessing the data the final gene count is 4641 genes . among 17 patients in the validation data
set , there are 8 males ( 1 hs / st and 7 ls / nst ) and 9 females ( 1 hs / st and 8 ls / nst )
. there are 2 cases of hs / st and 15 cases of ls / nst in total .
since the validation set was separately provided , the sex - specific genes were selected via 20 trials of 10-fold cv in the learning set .
the following ten male - specific genes were selected at least 75% of the time during 20 trials of 10-fold cv : a_23_p128263 ( prb1 : proline - rich protein bstni subfamily 1 ) , a_23_p83838 ( ca8 : carbonic anhydrase viii ) , a_24_p212990 ( mgc70863 : similar to rpl23ap7 protein ) , a_23_p333650 ( rad9b : rad9 homolog b ( s. cerevisiae ) ) , a_32_p125251 ( n / a ) , a_23_p108835 ( ypel5 : yippee - like 5 ( drosophila ) ) , a_32_p150856 ( loc407835 : mitogen - activated protein kinase kinase 2 pseudogene ) , a_23_p11936 ( ubxn11 : ubx domain protein 11 ) , a_24_p169976 ( n / a ) , and a_32_p3998 ( znf600 : zinc finger protein 600 ) . for female patients ,
the following eight female - specific genes were selected from 20 trials of 10-fold cv : a_23_p69497 ( clec3b : c - type lectin domain family 3 , member b ) , a_24_p118884 ( n / a ) , a_23_p152420 ( kiaa0182 ) , a_24_p265177 ( phc3 : polyhomeotic - like 3 ( drosophila ) ) , a_23_p251421 ( cdca7 : cell division cycle associated 7 ) , a_23_p211738 ( ubp1 : upstream binding protein 1 ( lbp-1a ) ) , a_23_p47377 ( hsd17b12 : hydroxysteroid ( 17-beta ) dehydrogenase 12 ) , and a_32_p113646 ( cdna flj45341 fis , clone brhip3009672 ) . using a given validation set ,
the sex - specific genes were verified . as shown in the confusion matrix in table 5 , there was no difference in the performance from female data with female genes compared with the performance of female data with male genes .
however , there was only one misclassification for male patients with male genes as shown in table 6 , while there were four misclassifications for male patients with female genes as shown in table 7 .
therefore , we conclude that there exists evidence of male - specific genes in a classification of primary cutaneous melanoma .
large inter individual differences in benefit from chemotherapy highlight the need to develop predictive genomic biomarkers for selecting the right treatment for the right patient .
inappropriate chemotherapy can result in the selection of more resistant and aggressive tumor cells . to date
, no reliable genomic biomarkers have been developed to provide the physician with prechemotherapy information to accurately predict the efficacy of a specific therapy .
we proposed a procedure to find sex - specific prognostic and predictive genomic biomarkers in order to assign individualized treatments in a personalized paradigm using variable importance ranking via combination of 20 trials of 10-fold cv and loocv .
the proposed procedure was applied to data sets obtained from the brb arraytools data human cancer archive .
however , the issue arose out of there being not enough samples , and the data was unbalanced ( i.e. , more males than females or more positives ( disease patients ) than negatives ( nondisease patients ) ) . while patient 's sex information was not specified for many of the publicly available data sets adding to that the difficulty of searching for data , we found the following three genomic data sets that had sex information : ( 1 ) pediatric patients with aml , ( 2 ) b - cell chronic lymphocytic leukemia ( b - cll ) , and ( 3 ) primary cutaneous melanoma . in one application , pediatric patients with aml were classified by the algorithms as having either a good or poor prognosis , in terms of the likelihood of induction failure or relapse within one year of the first complete remission , based on gene - expression profiles .
if this were brought into clinical application , a patient with a confidently predicted good prognosis might want to elect out of adjuvant chemotherapy and its associated debilitating side effects . with current rule - based decisions ,
the overall average accuracy of this data set with a variable selection from pooled patients ( males and females ) was about 71.0% .
however , using male - specific genes found by the proposed procedure , the accuracy was improved to about 72% as we found in the model validation studies . similarly , using female - specific genes found by the proposed procedure ,
the average accuracy was improved to about 76% ( see table 1 ) . in the b - cell chronic lymphocytic leukemia ( b - cll )
dataset we found male - specific prognostic genomic biomarkers associated with b - cell chronic lymphocytic leukemia and its average classification accuracy was improved to about 68% .
there was no substantial evidence to find female - specific prognostic genomic biomarkers in this data set .
similarly , male - specific predictive genomic biomarkers associated with a classification of primary cutaneous melanoma were found with the classification accuracy of about 88% .
the scope of our paper was to find sex - specific genomic biomarkers , if any , imbedded in the data instead of finding genomic biomarkers from the data .
if commonly identified genes were kept in the proposed procedure , our procedure was not sex - specific genomic biomarker classifier involving two populations ( males and females ) any more but rather it became genomic biomarker classifier involving one combined population .
in fact , even though commonly identified genes were kept , it did not necessarily improve the classification accuracy .
for a counterexample , for the pediatric aml data of yagi et al . , the accuracy for female patients with female - specific genes by keeping the commonly identified genes was 62% ; however , the accuracy without commonly identified genes was 76% .
for the male patients with male - specific genes by keeping the commonly identified genes the accuracy was 59% , but the accuracy without the commonly identified genes was 72% .
for the related note , the accuracy of this data can be found anywhere between 59% and 66% with gene preprocessing and between 58% and 71% with gene selection .
it is not an easy task to find sex - specific genes , let alone verifying and proving that they are indeed sexspecific .
we have presented a procedure for finding sex - specific prognostic and predictive genomic biomarkers in order to assign individualized treatments in a personalized paradigm .
the procedure is shown to have good sensitivity and specificity in the sense that the sex - specific genes obtained can improve prediction accuracy in classification of individual patient 's prognosis .
the proposed procedure to discover predictive and prognostic sex - specific genomic biomarkers for individualized treatment of diseases can play a critical role in developing safer and more effective therapies that replace one - size - fits - all drugs with treatments that focus on specific patient needs .
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numerous studies have demonstrated sex differences in drug reactions to the same drug treatment , steering away from the traditional view of one - size - fits - all medicine .
a premise of this study is that the sex of a patient influences difference in disease characteristics and risk factors . in this study , we intend to exploit and to obtain better sex - specific biomarkers from gene - expression data .
we propose a procedure to isolate a set of important genes as sex - specific genomic biomarkers , which may enable more effective patient treatment .
a set of sex - specific genes is obtained by a variable importance ranking using a combination of cross - validation methods .
the proposed procedure is applied to three gene - expression datasets .
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1. Introduction
2. Method for Identifying Sex-Specific Predictive/Prognostic Genomic Biomarkers
3. Results: Identification and Evaluation of Predictive/Prognostic Genomic Biomarkers
4. Discussion and Conclusions
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inadequate organization has been identified as an important cause of inappropriate courses of treatment for patients in the health care system [ 1 , 2 ] .
a response to this challenge has been to focus on integrated health care and co - ordination between the actors involved in the process [ 37 ] .
an illustrative example of this can be found when looking at frail elderly patients on their discharge from hospital .
studies show that readmission can be diminished by increased collaboration between hospital , district nurses and general practitioners [ 8 , 9 ] .
follow - up home visits by the district nurse and general practitioner to frail elderly patients after discharge have shown promising results [ 10 , 11 ] .
the aim of follow - up home visits to elderly and frail persons is to enable them to remain in their own home , avoid readmission to hospital , admission to nursing homes or other forms of sheltered housing and to improve the functional ability and general wellbeing of the elderly .
a follow - up home visit comprises a home visit by a general practitioner ( gp ) and district nurse within about one week after discharge from hospital .
the visit is expected to last approximately one hour , a relatively long period compared to other types of doctor - patient contact .
this visit can be supplemented with two subsequent home visits or visits to the gp if this is considered necessary .
the latest study from denmark has shown that the readmission rate in the intervention group was 40% while it was 53% in the control group .
at the same time , studies have shown that there are indications that patients prefer primary care follow - up to specialist care .
these results are overall convincing and , as a result , on - going work is being done by health authorities to test and further develop follow - up home visits as an initiative , including a programme in copenhagen carried out from 20082011 .
the copenhagen programme was conducted as a randomized controlled trial which implies strict procedures for the implementation process .
a total of approximately 300 patients from bispebjerg hospital ( a major hospital in copenhagen ) were included in the intervention and control group .
approximately 100 follow - up home visits to patients in the intervention group have been made . in the remaining approximately 200 cases in the intervention group it has not been possible to carry out a follow - up home visit , first of all because gps could not meet the requirements . in this feasibility study gps
were not carefully selected for the programme as they were in an earlier study , and , as a result , more gps chose not to take part in the programme .
the effect of the copenhagen programme will be reported in 2012 by the danish institute of health services research .
this paper will focus on the implementation of follow - up home visits using a qualitative methodological approach .
we know some of the general dynamics in the area but we do not know more precisely what the practical limitations of the home visitor 's programmes are and how these limitations could make a difference to the outcomes assessed [ 14 , 15 ] . why do so few gps take part in the follow - up home visit programme ?
why is the documented effect of such visits not motivation enough for gps to encourage them to do so ?
addressing such questions could help us understand how to design and implement these visits in order to fully exploit the potential benefits of the intervention .
moreover , such results could generate useful hypotheses which could be addressed in future trials attempting to examine the effects of home visiting programmes .
the aim of this study is to explore one particular aspect of follow - up home visits : the motivation and rationale of local care providers to invest time and effort in follow - up home visits .
what is the main motivation of gps , district nurses and hospital staff to take part in a cross - sectoral activity like follow - up home visits ?
the case study is placed in a danish setting . in order to understand the organizational dynamics involved a short description of the setting
is needed : the danish health care system is mainly a public system based on general taxation , and characterized by universal access to health care services .
gps have a central position in the system since they have a kind of gatekeeper function , they are usually the first professional confronted with the patient 's problems and they see it as their responsibility to guide patients through the system .
gps are self - employed , general practices are small and typically privately owned ; only a minority of gps are organized in health centres .
home care organizations ( district nurses ) have occupied an ever more central role since a major administrative reform in 2007 gave the local level the main responsibility for preventive health care .
co - ordination between organizational units in primary and secondary health care is determined by agreements between regional and municipality authorities , which include procedures for such cases as when frail and elderly people are discharged from hospital .
in general , the danish system is considered quite integrated , in particular because the climate of trust in denmark makes co - operation possible [ 7 , 16 ] . as a result ,
follow - up home visits and other cross - sector interventions , such as follow - home arrangements and case managers have been quite regularly implemented on an experimental basis as a possible alternative to hospital - based measures .
the interventions have shown promising results but studies in the area also point to problems in connection with implementing the interventions [ 7 , 8 , 10 , 12 , 17 , 18 ] .
empirical data were gathered to develop hypotheses and theoretical concepts . at the same time , however , a rough theoretical framework was used as an underlying foundation in the study to ensure that a relevant focus and approach were applied .
resource dependency theory tells us that network formation can largely be explained by an organization 's need to reduce costs or gain resources and power .
organizations only work together when administrative and economic structures are designed which makes co - operation a profitable pursuit .
organizations thus typically follow their own agenda rather than involve themselves in cross - sectoral activities . in the health sector some support can be found for this basic claim .
studies have shown that network formation in general is limited and poor co - ordination predominant in the area [ 13 , 21 ] .
resource dependency theory also tells us , however , that organizations need to reduce uncertainty . to reduce uncertainty , organisations tend to co - operate with organisations they trust and depend on to achieve common goals [ 19 , 22 ] .
hence , in order to understand co - operation you also need to look at the question of trust and the quality of inter - organizational networks , as done in inter - organizational network theory .
this perspective has shown us that we need to have a comprehensive view of the factors which make health sector organisations work together .
cross - sectoral programs and inter - organizational activities are not based purely on partners ' own needs and resources [ 23 , 24 ] .
motivation plays a key role in inter - organizational theory [ 22 , 25 ] .
working together is a challenging task ; it is typically regarded as uncertain whether it will bring benefits equivalent to the resources invested .
motivation and commitment to the process are therefore needed among the actors involved in order to keep things moving and overcome the uncertainties inherent in the process . what motivates organizations to work together ?
the literature points to two aspects in particular : the object needs to be seen as highly relevant and benefits should be obvious for all involved [ 22 , 25 ] .
this paper is based on an evaluation report made by one of the authors for health and social care , city of copenhagen .
the interviews were structured around the following questions :
to which extent do the actors in the programme regard the content of and target group for follow - up home visits as relevant?how do the actors in the programme view communication and workflow in relation to a successful implementation of follow - up home visits ? are the benefits obvious ?
to which extent do the actors in the programme regard the content of and target group for follow - up home visits as relevant ? how do the actors in the programme view communication and workflow in relation to a successful implementation of follow - up home visits ?
two focus groups and seven individual interviews were conducted in march may 2010 , giving a total of 23 respondents .
the focus group approach was chosen because the interaction between some of the key respondents ( hospital staff , district nurses ) was considered to be of importance , since a key goal in the study was to understand the shared views on collaboration among these key actors in the programme .
one focus group interview was conducted with seven employees at bispebjerg hospital who were involved in the follow - up home visit programme .
recruitment criteria were designed in order to include a suitable spread of respondents , i.e. to ensure that members of all staff groups directly involved in the project were represented .
we ensured that the project co - ordinator at the hospital responsible for the selection of patients for the programme was represented in the focus group . at the same time we also ensured that a doctor and nurses from the most relevant wards were represented .
another focus group interview was conducted with nine district nurses who had been involved in the project .
nurses from different districts in copenhagen were selected for the focus group in order to ensure that different ways of organising follow - up home visits were represented .
gps were not as motivated to take part in the research project as district nurses and hospital staff , so a less time - consuming method than focus groups was considered appropriate . as it turned out , in - depth interviews proved to be highly relevant for gps because gps had divergent and less firm views about follow - up home visits than other groups in the study and the methodological design allowed these views to be fully expressed .
five individual interviews were held with gps involved in the project . both gps with a positive attitude towards follow - up home visits and gps with a less positive attitude were recruited to ensure that a wide range of attitudes was included in the study .
these recruitment criteria were based on data registered by the city of copenhagen showing which gps had refused to participate in follow - up home visits and which had agreed . at the same time we ensured that the gps represented covered different districts in copenhagen .
two individual interviews were held with employees from referrals in the city of copenhagen home nursing services .
there is a purchaser - provider split in the city of copenhagen , and the referrals purchase while the district nurses provides .
the two referrals came from different districts and had different positions in the organization , ensuring that the most important experiences from this organization were covered .
the focus group interviews took place in a neutral meeting room arranged by the project manager , and the in - depth interviews took place in the gps ' offices .
the focus group interviews lasted between 90 and 120 minutes , and the in - depth interviews between 25 and 60 minutes . both were recorded with a digital voice recorder and transcribed verbatim .
the interview transcripts were analyzed under the two headings which form the basic empirical questions in the study : relevance of collaboration and benefits of collaboration .
quotes from the interviews were grouped under each heading in order to find the quotes which were most illustrative of the views of gps , district nurses and hospital staff . to ensure reliability ,
results were presented and discussed in a steering group with representatives from the main professional groups involved in the programme ( gps , district nurses and hospital staff ) .
a guide suggesting the ideal content of a follow - up home visit has been designed as part of the programme .
gps and district nurses have been instructed to follow this guide in order to ensure that the same standards and procedures are used in the programme but ultimately it is an individual judgment on the part of the gp and district nurse to decide whether all elements in the guide are relevant in each specific case .
the main elements of the guide are : a medication review , a general health assessment ( including a number of relevant tests ) , and an assessment of the need for follow - up arrangements ( home care , rehabilitation etc . ) .
the gps generally see the follow - up home visit as a relevant type of contact .
a gp said : it is very relevant to make home visits because many elderly patients find it very difficult to go to their gp .
, it can give the patient extra resources when you visit the patient on the patient 's home ground .
the district nurses also generally see the follow - up home visits as a relevant type of contact .
the gps find that the medication review is an especially important element in follow - up home visits .
a gp said : we should be able to see what type of medication the patient has . but the patient may have been given some additional medicine by the hospital that we are not aware of , and in this case a home visit is a good way of finding out whether there is a problem with the different types of medicine the patient takes .
at the same time the gps also stress that follow - up home visits are not only about reviewing medication : the purpose of a follow - up home visit is first of all about making a patient review .
it is not only the medicine we should look at , but the patient in this sense follow - up home visits are very relevant .
the district nurses also found that the follow - up home visit was a good way of assessing the health situation of the patient : the guide creates a good structure for the home visit .
the doctor and the district nurse can on the basis of the home visit agree what their respective responsibilities are .
the two subsequent visits / contacts recommended in the programme are only seldom carried out with the participation of the gp . as a result , the relationship between the gp , the district nurse and the patient is typically not further developed after the first home visit . at the hospital
the general view was also that the content of the follow - up home visit was relevant . at the same time , however , several respondents expressed the need for co - ordination between the hospital and the prime sector : the guide is quite voluminous .
we have already made a lot of the tests at the hospital so there is no need to repeat these at home .
you should make sure that the gp has read the discharge summary before the follow - up home visit
the target group of the programme is elderly and frail patients just out of from hospital .
a number of specific criteria have been formulated : the patient has to be 65 years or older , been in specific wards at bispebjerg hospital , have returned to his or her own home , have experienced deteriorating health , have a limited social network , and experienced many readmissions . at the hospital
a nurse at the hospital expressed it this way : it is typically this type of patient that we see again and again : deteriorating health and a small social network .
the gps and district nurses also found that the patient group was very relevant even if they did not find that the use of strict inclusion criteria was the best way to find the most relevant patients .
other patients would have benefited from a follow - up home visit but they were not included in the programme gps receive discharge letters and medicine lists from the hospital when one of their patients is discharged , ideally within three days .
this form of communication works fine but is , according to gps and district nurses in the study , not sufficient when it comes to elderly and frail patients : it would be natural if the doctor at the hospital contacted the gp directly before the patient is discharged .
a follow - up home visit could be agreed and the confirmation of the home visit could be sent to the gp along with discharge letters etc .
a gp said : the hospital has contact with the patient at the time of discharge .
in the present situation there is not much direct communication between the hospital and the gp after the discharge of a patient in the target group .
it is mainly if there are questions about medicine lists that the gp contacts the hospital and that is not without problems : if something is missing from the medicine lists or if the district nurse says that the new medicine list from the hospital is different from the old one you need to contact the hospital .
but it is often difficult to find out who is responsible and it is difficult to sort out the problem ( gp ) .
the problem , according to several respondents , is that nobody is in charge of this transition phase and , as a result , communication problems occur : we need an anchor with overall responsibility for the patient when the patient is discharged from hospital .
a fax from the hospital to the home nursing services is not enough ( doctor , hospital ) . in the city of copenhagen
the home nursing services are responsible for organizing follow - up home visits after the hospital has selected patients which fit the target group and discharged them .
it is quite new for district nurses to have this kind of responsibility but according to district nurses it is a responsibility they enjoy having : follow - up home visits are all about the patient leaving hospital earlier and about home nurses meeting the patient earlier on and having more responsibility .
the study showed that there was some frustration among gps because they in many cases were not given full information about the patient by the hospital and the home nursing services and , as a result , they could not be very outreaching .
today i find we often do not hear what is going on in the daily life of our patients . the district nurses know more what is going on than we do .
the health and social care administration of the city of copenhagen has sent pamphlets to gps informing them about follow - up home visits , and other forms of information have also been used .
these activities were set up to inform gps about the reason for follow - up home visits and to motivate gps to take part in them .
such information is essential since follow - up home visits are new to many gps .
the study indicated that many gps react negatively when contacted by the district nurses and that motivation to take part in follow - up home visits was low :
i have experienced very negative - minded doctors when calling to arrange a follow - up home visit . in these cases
i received a reprimand and was told that it was certainly not his [ the gp 's ] duty to participate in such a visit
the gps who were favourably inclined towards follow - up home visits especially valued that communication between gps and district nurses was strengthened .
a gp said : you get to talk that is the biggest gain , to get the dialogue .
we work in two parallel organizational systems . by not having the dialogue we as gps miss some observations .
the gps want , however , more information about the efficacy of follow - up home visits .
gps in the study find it highly motivating to receive such information if results show an effect on readmission and general health : if research clearly shows that follow - up home visits have a positive effect it would really give us a moral incentive to participate in these visits .
information could be better ( gp ) . at the hospital the staff involved in the programme
a nurse at the hospital said : follow - up home visits are especially important in the case of patients where it has not been possible to offer the most appropriate treatment .
it is really nice to know that there is this form of support when patients get home .
communication about the implementation and results of follow - up home visits could , however , be better according to hospital staff .
doctors and nurses at the hospital miss feedback from the follow - up home visits , as illustrated in the following quote : at meetings we have often asked for the
good story. it would make it much more meaningful for us if we were told that mrs .
that is the most annoying that we are not told what the results of follow - up home visits are
a guide suggesting the ideal content of a follow - up home visit has been designed as part of the programme .
gps and district nurses have been instructed to follow this guide in order to ensure that the same standards and procedures are used in the programme but ultimately it is an individual judgment on the part of the gp and district nurse to decide whether all elements in the guide are relevant in each specific case .
the main elements of the guide are : a medication review , a general health assessment ( including a number of relevant tests ) , and an assessment of the need for follow - up arrangements ( home care , rehabilitation etc . ) .
the gps generally see the follow - up home visit as a relevant type of contact .
a gp said : it is very relevant to make home visits because many elderly patients find it very difficult to go to their gp .
, it can give the patient extra resources when you visit the patient on the patient 's home ground .
the district nurses also generally see the follow - up home visits as a relevant type of contact .
the gps find that the medication review is an especially important element in follow - up home visits .
a gp said : we should be able to see what type of medication the patient has . but the patient may have been given some additional medicine by the hospital that we are not aware of , and in this case a home visit is a good way of finding out whether there is a problem with the different types of medicine the patient takes .
at the same time the gps also stress that follow - up home visits are not only about reviewing medication : the purpose of a follow - up home visit is first of all about making a patient review .
it is not only the medicine we should look at , but the patient in this sense follow - up home visits are very relevant .
the district nurses also found that the follow - up home visit was a good way of assessing the health situation of the patient : the guide creates a good structure for the home visit .
the doctor and the district nurse can on the basis of the home visit agree what their respective responsibilities are .
the two subsequent visits / contacts recommended in the programme are only seldom carried out with the participation of the gp . as a result , the relationship between the gp , the district nurse and the patient is typically not further developed after the first home visit . at the hospital
the general view was also that the content of the follow - up home visit was relevant . at the same time , however , several respondents expressed the need for co - ordination between the hospital and the prime sector : the guide is quite voluminous .
we have already made a lot of the tests at the hospital so there is no need to repeat these at home .
you should make sure that the gp has read the discharge summary before the follow - up home visit
the target group of the programme is elderly and frail patients just out of from hospital .
a number of specific criteria have been formulated : the patient has to be 65 years or older , been in specific wards at bispebjerg hospital , have returned to his or her own home , have experienced deteriorating health , have a limited social network , and experienced many readmissions . at the hospital
a nurse at the hospital expressed it this way : it is typically this type of patient that we see again and again : deteriorating health and a small social network .
the gps and district nurses also found that the patient group was very relevant even if they did not find that the use of strict inclusion criteria was the best way to find the most relevant patients .
other patients would have benefited from a follow - up home visit but they were not included in the programme
gps receive discharge letters and medicine lists from the hospital when one of their patients is discharged , ideally within three days .
this form of communication works fine but is , according to gps and district nurses in the study , not sufficient when it comes to elderly and frail patients : it would be natural if the doctor at the hospital contacted the gp directly before the patient is discharged .
a follow - up home visit could be agreed and the confirmation of the home visit could be sent to the gp along with discharge letters etc .
a gp said : the hospital has contact with the patient at the time of discharge .
there is not much direct communication between the hospital and the gp after the discharge of a patient in the target group .
it is mainly if there are questions about medicine lists that the gp contacts the hospital and that is not without problems : if something is missing from the medicine lists or if the district nurse says that the new medicine list from the hospital is different from the old one you need to contact the hospital .
but it is often difficult to find out who is responsible and it is difficult to sort out the problem ( gp ) .
the problem , according to several respondents , is that nobody is in charge of this transition phase and , as a result , communication problems occur : we need an anchor with overall responsibility for the patient when the patient is discharged from hospital .
a fax from the hospital to the home nursing services is not enough ( doctor , hospital ) . in the city of copenhagen
the home nursing services are responsible for organizing follow - up home visits after the hospital has selected patients which fit the target group and discharged them .
it is quite new for district nurses to have this kind of responsibility but according to district nurses it is a responsibility they enjoy having : follow - up home visits are all about the patient leaving hospital earlier and about home nurses meeting the patient earlier on and having more responsibility .
the study showed that there was some frustration among gps because they in many cases were not given full information about the patient by the hospital and the home nursing services and , as a result , they could not be very outreaching .
today i find we often do not hear what is going on in the daily life of our patients . the district nurses know more what is going on than we do .
the health and social care administration of the city of copenhagen has sent pamphlets to gps informing them about follow - up home visits , and other forms of information have also been used .
these activities were set up to inform gps about the reason for follow - up home visits and to motivate gps to take part in them .
such information is essential since follow - up home visits are new to many gps .
the study indicated that many gps react negatively when contacted by the district nurses and that motivation to take part in follow - up home visits was low :
i have experienced very negative - minded doctors when calling to arrange a follow - up home visit . in these cases
i received a reprimand and was told that it was certainly not his [ the gp 's ] duty to participate in such a visit
the gps who were favourably inclined towards follow - up home visits especially valued that communication between gps and district nurses was strengthened .
a gp said : you get to talk that is the biggest gain , to get the dialogue .
the gps want , however , more information about the efficacy of follow - up home visits . gps in the study find it highly motivating to receive such information if results show an effect on readmission and general health : if research clearly shows that follow - up home visits have a positive effect it would really give us a moral incentive to participate in these visits .
the staff involved in the programme is very positive towards follow - up home visits .
a nurse at the hospital said : follow - up home visits are especially important in the case of patients where it has not been possible to offer the most appropriate treatment .
it is really nice to know that there is this form of support when patients get home .
communication about the implementation and results of follow - up home visits could , however , be better according to hospital staff .
doctors and nurses at the hospital miss feedback from the follow - up home visits , as illustrated in the following quote : at meetings we have often asked for the
good story. it would make it much more meaningful for us if we were told that mrs .
that is the most annoying that we are not told what the results of follow - up home visits are
the analysis illustrates that it is a big challenge to co - ordinate activities across organizational boundaries and motivate actors to participate in cross - sectoral health programs .
resources clearly matter ( fees for gps etc . ) but the paper also points to other key factors which need to be taken into consideration when trying to motivate partners to engage actively in a cross - sectoral programme like a follow - up home visit programme . in the following these key factors will be discussed under three sub - headings : the focus of collaboration , a partnership of equals , and the will to co - operate .
the analysis indicated that it is beneficial to have agreed upon a common structure for follow - up home visits .
the guide specifying the ideal content of these visits ensures that common standards and procedures focusing on the patient 's overall situation are followed by all professionals involved . as a result , gps and
district nurses do not follow their usual agendas but are forced to focus on these commonly agreed standards , a type of procedure unusual for the health sector .
collaboration requires partners to subscribe to common standards , and the fixed structure of follow - up home visits is a powerful way of defining these standards . the medication review is a very illustrative example of how gps and district nurses can work closely together and achieve a shared understanding of the patient 's intake of medicine .
the study indicates that the gp gains a very realistic picture of the patient 's medicine intake during the home visit , a picture the gp could not as easily have gained in a different setting . as a result ,
communication between the gp and the district nurse about a patient 's medicine is typically improved in the treatment following the visit .
this is very motivating for gps and district nurses because communication about changes in medication has been a long - standing problem in danish health care [ 6 , 23 ] .
the results also showed that follow - up home visits aimed at the right target group , and this motivated care providers to take part in the intervention .
studies have shown that frail elderly patients benefit significantly from follow - up home visits and other types of home visits , and that is a notion shared by most gps and district nurses in the program .
thus , the results indicate that the object at the centre of these efforts the focus of collaboration needs to be clearly defined and agreed upon if the actors involved are to be motivated to invest in an extra - ordinary and cross - sectoral activity like follow - up home visits .
inter - organizational studies in the area generally support such a conclusion but also point out that it is a difficult task [ 18 , 26 , 27 ] .
providers in the health sector typically have different incentives , different organizational cultures and working practices and that makes it difficult for them to reach an agreement regarding the focus of their collaborative efforts .
they are responsible for contacting the gp , organizing the visit , and co - ordinating the activities following the visit .
the results of the study indicate that district nurses are highly motivated and view these visits as part of a dominant trend in health care , a trend putting more emphasis on primary sector treatment which has evolved as a response to a growing pressure on health services to provide cheaper care than traditional hospital care [ 6 , 8 ] .
this is not surprising since district nurses generally are very positive towards this kind of nurse - led follow - up care and studies have shown that they produce positive results .
gps generally did not feel they had a central role in follow - up home visits and , as a result , felt no responsibility for them .
moreover , follow - up home visits break with gps ' normal routine because they need to leave their practice and they need to co - ordinate their calendar with a district nurse .
this is time - consuming and , from a narrow cost - benefit analysis , probably not as rewarding as other activities in general practice . as a result , a programme like the follow - up home visit programme needs to be specific and out - reaching to motivate gps to take part .
hospital staff is responsible for selecting patients who need a follow - up home visit after leaving hospital but , despite this essential role , they do not see how they contribute to the overall goals of the programme .
the interviews showed that hospital staff lacks information about the actual visits , and how this type of intervention fits into the treatment given at the hospital .
the main argument for this is that the gp would be more fully informed about the treatment of the patient at the hospital and follow - up home visits could , as a result , focus more explicitly on what is most needed .
this form of communication between the hospital and gps could give both hospital staff and gps a greater sense of responsibility for follow - up home visits and , as a result , increase the motivation to make these visits .
it is important for all providers to have an essential role in follow - up home visits and feel a sense of responsibility in order to feel motivated .
if the specific roles and responsibilities of the partners are not clear people feel frustrated and motivation suffers .
there needs to be a shared responsibility a partnership of equals to engage all partners in a cross - sectoral activity like follow - up home visits .
research has shown that an interorganizational relationship is at its most effective when it is in equilibrium , a state in which the organizational network is balanced in terms of roles and functions [ 22 , 29 ] .
research has also shown , however , that there typically are asymmetrical power relations and it is impossible for all members to participate as equals in collaborative processes .
for gps in particular the partnership of equals in follow - up home visits is clearly not equal enough .
they are trained to have the prime responsibility for their patients in primary care and find the current state of affairs demotivating .
the interviews showed that it is important that all partners can see the benefits of working together and understand how they can contribute to achieve shared goals .
gps in particular need to be convinced that an intervention like follow - up home visits can add something extra to their normal practice .
danish gps are influenced by a culture of individualism since they are typically small and privately owned entities with few rules and procedures as in larger organizational units , like health centres , and it typically requires an extra effort to motivate gps to take part in a larger scheme , like a follow - up home visit programme .
the results from the study showed that hospital staff also has problems in seeing how they get extra value out of working in partnership .
hospital staff is given no feedback after follow - up home visits , and need a clearer notion of how this type of intervention fits into the treatment given at the hospital .
as hospital stays are shortened and follow - up care in the primary health sector is given a larger role in the treatment of elderly and frail patients in particular , the need in the secondary health sector for information about the treatment given in the primary sector is growing .
district nurses do not need this type of information to the same extent because they follow the patients more closely and have a clearer notion than gps and hospital staff of the impact of follow - up home visits . in order to create more will to co - operate regarding follow - up home visits it seems necessary for partners to have a shared understanding of common goals and values , a recommendation also found in other inter - organizational studies in the area [ 20 , 26 , 31 ] .
basically , the partners in the danish follow - up home visit programme share the understanding that health services need to be more efficient and more coherent . in particular
there is a common understanding that the complex needs of frail elderly patients are not adequately dealt with by traditional health care services and require an extra - ordinary and cross - sectoral approach .
follow - up home visits could be the answer to this challenge and the programme could benefit from the basic goodwill among collaborative partners but communication has been insufficient . in particular , there has been a lack of opportunity and incentives for collaborative partners to learn by working together .
the learning potentials in integrated care for older people are big , and health care professionals are generally motivated to take part if the learning potential of the experience is clearly communicated .
the analysis indicated that it is beneficial to have agreed upon a common structure for follow - up home visits .
the guide specifying the ideal content of these visits ensures that common standards and procedures focusing on the patient 's overall situation are followed by all professionals involved . as a result
, gps and district nurses do not follow their usual agendas but are forced to focus on these commonly agreed standards , a type of procedure unusual for the health sector .
collaboration requires partners to subscribe to common standards , and the fixed structure of follow - up home visits is a powerful way of defining these standards . the medication review is a very illustrative example of how gps and district nurses can work closely together and achieve a shared understanding of the patient 's intake of medicine .
the study indicates that the gp gains a very realistic picture of the patient 's medicine intake during the home visit , a picture the gp could not as easily have gained in a different setting . as a result , communication between the gp and the district nurse about a patient 's medicine
this is very motivating for gps and district nurses because communication about changes in medication has been a long - standing problem in danish health care [ 6 , 23 ] .
the results also showed that follow - up home visits aimed at the right target group , and this motivated care providers to take part in the intervention .
studies have shown that frail elderly patients benefit significantly from follow - up home visits and other types of home visits , and that is a notion shared by most gps and district nurses in the program .
thus , the results indicate that the object at the centre of these efforts the focus of collaboration needs to be clearly defined and agreed upon if the actors involved are to be motivated to invest in an extra - ordinary and cross - sectoral activity like follow - up home visits .
inter - organizational studies in the area generally support such a conclusion but also point out that it is a difficult task [ 18 , 26 , 27 ] .
providers in the health sector typically have different incentives , different organizational cultures and working practices and that makes it difficult for them to reach an agreement regarding the focus of their collaborative efforts .
they are responsible for contacting the gp , organizing the visit , and co - ordinating the activities following the visit .
the results of the study indicate that district nurses are highly motivated and view these visits as part of a dominant trend in health care , a trend putting more emphasis on primary sector treatment which has evolved as a response to a growing pressure on health services to provide cheaper care than traditional hospital care [ 6 , 8 ] .
this is not surprising since district nurses generally are very positive towards this kind of nurse - led follow - up care and studies have shown that they produce positive results .
gps generally did not feel they had a central role in follow - up home visits and , as a result , felt no responsibility for them .
moreover , follow - up home visits break with gps ' normal routine because they need to leave their practice and they need to co - ordinate their calendar with a district nurse .
this is time - consuming and , from a narrow cost - benefit analysis , probably not as rewarding as other activities in general practice . as a result , a programme like the follow - up home visit programme needs to be specific and out - reaching to motivate gps to take part .
hospital staff is responsible for selecting patients who need a follow - up home visit after leaving hospital but , despite this essential role , they do not see how they contribute to the overall goals of the programme .
the interviews showed that hospital staff lacks information about the actual visits , and how this type of intervention fits into the treatment given at the hospital .
the main argument for this is that the gp would be more fully informed about the treatment of the patient at the hospital and follow - up home visits could , as a result , focus more explicitly on what is most needed .
this form of communication between the hospital and gps could give both hospital staff and gps a greater sense of responsibility for follow - up home visits and , as a result , increase the motivation to make these visits .
it is important for all providers to have an essential role in follow - up home visits and feel a sense of responsibility in order to feel motivated .
if the specific roles and responsibilities of the partners are not clear people feel frustrated and motivation suffers .
there needs to be a shared responsibility a partnership of equals to engage all partners in a cross - sectoral activity like follow - up home visits .
research has shown that an interorganizational relationship is at its most effective when it is in equilibrium , a state in which the organizational network is balanced in terms of roles and functions [ 22 , 29 ] .
research has also shown , however , that there typically are asymmetrical power relations and it is impossible for all members to participate as equals in collaborative processes .
for gps in particular the partnership of equals in follow - up home visits is clearly not equal enough .
they are trained to have the prime responsibility for their patients in primary care and find the current state of affairs demotivating .
the interviews showed that it is important that all partners can see the benefits of working together and understand how they can contribute to achieve shared goals .
gps in particular need to be convinced that an intervention like follow - up home visits can add something extra to their normal practice .
danish gps are influenced by a culture of individualism since they are typically small and privately owned entities with few rules and procedures as in larger organizational units , like health centres , and it typically requires an extra effort to motivate gps to take part in a larger scheme , like a follow - up home visit programme .
the results from the study showed that hospital staff also has problems in seeing how they get extra value out of working in partnership .
hospital staff is given no feedback after follow - up home visits , and need a clearer notion of how this type of intervention fits into the treatment given at the hospital .
as hospital stays are shortened and follow - up care in the primary health sector is given a larger role in the treatment of elderly and frail patients in particular , the need in the secondary health sector for information about the treatment given in the primary sector is growing .
district nurses do not need this type of information to the same extent because they follow the patients more closely and have a clearer notion than gps and hospital staff of the impact of follow - up home visits . in order to create more will to co - operate regarding follow - up home visits it seems necessary for partners to have a shared understanding of common goals and values , a recommendation also found in other inter - organizational studies in the area [ 20 , 26 , 31 ] .
basically , the partners in the danish follow - up home visit programme share the understanding that health services need to be more efficient and more coherent . in particular there is a common understanding that the complex needs of frail elderly patients are not adequately dealt with by traditional health care services and require an extra - ordinary and cross - sectoral approach .
follow - up home visits could be the answer to this challenge and the programme could benefit from the basic goodwill among collaborative partners but communication has been insufficient . in particular , there has been a lack of opportunity and incentives for collaborative partners to learn by working together .
the learning potentials in integrated care for older people are big , and health care professionals are generally motivated to take part if the learning potential of the experience is clearly communicated .
the findings support a basic claim in inter - organizational theory that other factors than resources matter if we want to understand what drives efforts at collaboration .
this analysis shows that the focus of collaboration needs to be clearly defined and agreed upon , there needs to be a high degree of equality between the professionals involved , and there has to be a will to co - operate based on a shared understanding of values and learning potentials .
one lesson for current policy is that motivational factors need to be addressed in future collaborative programs in order to fully exploit the potential health benefits .
the follow - up home visit programme in copenhagen suffered from a lack of motivation among key actors ( especially gps ) to participate and , as a result , readmissions to hospital can be expected to be higher than anticipated prior to the programme .
susanna bihari axelsson , senior lecturer , associate professor nordic school of public health , box 12133 , nya varvet building 25 , se-402 42 gteborg sweden janne seemann , assistant professor , phd , department of sociology and social work , aalborg university , denmark j.d.h .
van wijngaarden , dr , assistant professor , institute of health policy and management , erasmus university rotterdam , p.o .
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objectivesthe aim of follow - up visits by the general practitioner and district nurse ( within a week after discharge from hospital ) is to reduce hospital readmissions and improve the overall wellbeing of the patient .
there is strong evidence that these programmes are effective , but are difficult to implement because of a number of organizational obstacles , including co - ordination between the organizations involved in the process . in this paper
we look at the factors that affect motivation to participate in a cross - sectoral programme in copenhagen , denmark , implementing follow - up home visits to elderly persons.theory and methodsthe analysis is based on inter - organizational network theory in an attempt to explain the role of motivation in network formation between organizational systems .
the empirical findings are based on focus groups and in - depth interviews with hospital staff , general practitioners , and district nurses.resultscare providers are motivated to collaborate by a number of factors .
the focus of collaboration needs to be clearly defined and agreed upon , there needs to be a high degree of equality between the professionals involved , and there has to be a will to co - operate based on a shared understanding of values and learning potentials.conclusionsthe study concludes that we need to focus on specific care fields and actors to reduce complexity in the area and more fully understand what motivates care providers to participate in cross - sectoral activities , such as a follow - up home visit programme .
one lesson for current policy is that motivational factors need to be addressed in future collaborative programmes in order to fully exploit the potential health benefits .
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Introduction
Theoretical framework
Methodology
Results
Relevance of collaboration
Benefits from collaboration
Discussion
The focus of collaboration
A partnership of equals
The will to co-operate
Conclusions
Reviewers
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alzheimer s disease ( ad ) is a progressive neurodegenerative disorder starting with mild memory loss and manifesting in severe cognitive decline .
neuropathologically , the disease is characterized by an extensive deposition of amyloid ( a ) peptides in extracellular amyloid plaques and by intracellular neurofibrillary tangles ( nfts ) of hyper - phosphorylated tau protein .
sporadic late - onset ad is the most common form of dementia in the elderly and is strongly associated with age . in rare cases
( < 5% ) , ad is inherited and results in an early disease onset .
one genetic risk factor for late - onset ad that has been reported throughout many studies is apolipoprotein e ( apoe ) which physiologically functions as a ligand in receptor - mediated endocytosis of lipoprotein particles [ 2 , 3 ] .
several single nucleotide polymorphisms in apoe lead to alterations in the coding sequence and result in three common isoforms called apoe2 , apoe3 and apoe4 with the e4 allele being an ad risk factor and e2 being protective [ 46 ] .
strong evidence suggests that apoe influences ad via its effect on a metabolism ; however , the details of this process have to be fully elucidated .
positional cloning studies of familial ad ( fad ) cases have identified mutations in three genes , amyloid precursor protein ( app ) , presenilin 1 and 2 ( ps1 , ps2 ) , which are tightly linked to the generation of a peptides .
proteolytic processing of amyloid precursor protein ( app ) by bace ( -site app cleaving enzyme ) is followed by cleavage by -secretase containing presenilins and results in the release of a peptides of different length . a40 and a42 ( 40 or 42 amino acids , respectively )
are the most prominent a peptides and since the c - terminus has important implication for toxicity , the longer a42 peptide exhibits stronger neurotoxic properties .
the fad - linked mutations cause an elevated production of amyloidogenic a , which is the basis for the amyloid hypothesis of ad , stating that a peptides are the key - players in neuronal dysfunction and subsequent neurodegeneration in ad .
recently , it has been suggested that the soluble oligomeric forms of a42 are the potentially harmful species and that these oligomers affect various important cellular mechanisms , resulting in decreased functionality and survival of neuronal cells .
however , a peptides are not exclusively generated under pathological conditions as they are part of the normal cellular metabolism and a significant load of amyloid plaques is also observed in brains of healthy aged individuals [ 10 , 11 ] .
this challenges the amyloid hypothesis of ad and up to date the nature of the disease - relevant alteration in aged neurons remains unclear .
although alois alzheimer already described protein deposits in the brain of demented patients , the question why distinct proteins that are potentially causative for the disease accumulate in discrete brain regions in the course of aging remains unanswered .
today , we know that the progressive deposition of misfolded and aggregation - prone proteins in defined regions of the nervous system is not unique to ad , but a well - described characteristic of several neurodegenerative disorders , including parkinson s disease ( pd ) , huntington s disease ( hd ) and amyotrophic lateral sclerosis ( als ) . recently , it has been proposed that the accumulation of disease - relevant proteins in aggregates might actually even be considered as a protective mechanism to dispose these proteins and remove them from the cellular metabolism [ 13 , 14 ] . generally , within the crowded cellular environment
, proteins are always at risk for denaturation . changing genetic and environmental factors , as during pathology or aging , challenge the integrity of the cellular protein homeostasis ( proteostasis ) . therefore , the stability and metabolism of the proteome needs to be controlled carefully and is carried out by a complex network of several hundred evolutionarily conserved proteins .
this network , including molecular chaperones , the ubiquitin proteasome system ( ups ) and the autophagy system is stringently regulated and indispensable for the maintenance of proteostasis .
all main players of this network are ubiquitously expressed and consequently also present in neuronal tissue .
molecular chaperones , most be prominently represented by the heat shock protein 70 ( hsp70 ) family and their regulators , are responsible for correct folding and refolding of proteins as well as for the transport of misfolded proteins to the protein - degradation systems .
the chaperone network is controlled by the activity of transcription factors , e.g. heat shock transcription factor 1 ( hsf1 ) , which rapidly adjusts the cellular chaperone capacity during stress conditions .
the ups is the major protein - degradation machinery of the cell and controls the metabolism of cytosolic proteins and the degradation of misfolded proteins .
proteins that are destined for proteasomal degradation become specifically tagged with the protein ubiquitin in a well - organized enzymatic cascade .
interestingly , also a ubiquitin - independent way of proteasomal degradation has been described recently that mostly involves small and misfolded proteins . as an additional mechanism for protein degradation
the major autophagic systems described so far are macroautophagy and chaperone - mediated autophagy ( cma ) .
macroautophagy is a complex process that involves the formation of a membrane structure , the autophagosome , which engulfs bulk cellular material ( proteins and organelles ) and subsequently fuses with the lysosome .
cma involves the constitutively expressed heat shock cognate 70 ( hsc70 ) that targets client proteins with a specific consensus motif directly to the lysosome .
in fact , hsp70 is also an important factor to assure selectivity of macroautophagy of degradation - prone proteins in a distinct pathway , mediated via the co - chaperone bcl2-associated athanogene 3 ( bag3 ) . aging and disease challenge the proteostasis network and the accumulation of instable proteins results in wide - spread protein aggregation [ 21 , 22 ] .
our discussion presented here attempts to integrate two major factors that are associated with neurodegeneration , protein homeostasis and aging .
most importantly , we focus on the role of specific pathways which are needed to restore and maintain proteostasis during aging and their interplay with a special focus on impaired proteostasis in ad , which may add to a novel view and understanding of ad pathogenesis .
since recently many excellent reviews on various aspects providing in depth discussions have been presented , we are concentrating here on the integration of these different aspects .
therefore , we frequently refer to review work with the recommendation of further reading rather than trying to fully cover the discussed issues by their original publications .
cells permanently encounter the problem to maintain the integrity and functionality of the proteome . within the crowded cellular environment ,
the correct conformation of proteins must be controlled and misfolded and irreversibly damaged proteins must be efficiently refolded or removed .
central players of the protein homeostasis system are molecular chaperones that sense misfolded proteins and , when refolding fails , direct them to the protein - degradation pathways .
molecular chaperones are specified as proteins that interact with and participate in folding or refolding of non - native proteins .
therefore , chaperones help unfolded proteins to achieve their functional conformation without being present in the final structure [ 15 , 2327 ] .
they exert a multitude of activities , including de novo folding , refolding of denatured proteins , transport to subcellular compartments , oligomeric assembly and disposal by proteolytic degradation [ 28 , 29 ] .
different classes of chaperones work together to form co - operative networks and are often termed heat shock proteins ( hsps ) , because they are sensitively up - regulated under stress conditions in which the amount of misfolded proteins is increased .
chaperones , which are involved in protein folding and refolding , such as hsp70 , hsp90 and chaperonin ( hsp60 ) , promote folding activity through atp / cofactor - regulated binding and release cycles [ 15 , 31 ] .
they recognize short hydrophobic amino acid stretches of misfolded proteins and can co - operate with atp - independent chaperones , e.g. small hsps ( shsps ) , to prevent protein aggregation .
hsp70 proteins are central players in proteostasis control and increasing hsp70 levels prevent protein aggregation in various disease models that are based on the expression of aggregation - prone proteins [ 32 , 33 ] .
the activity of hsp70 is atp dependent and is controlled by chaperones of the hsp40 family as well as nucleotide exchange factors ( nefs ) [ 34 , 35 ] .
after atp hydrolysis , a nef binds to the atpase domain of hsp70 and catalyzes the nucleotide exchange , which results in substrate release .
thus , chaperone binding to the hydrophobic region of misfolded proteins transiently blocks aggregation and the subsequent atp - dependent release allows controlled folding of the client protein .
hsp40/dnajs , hsc70-interacting protein ( hip ) , bcl2-associated athanogene 1 ( bag1 ) and bag3 are prominent co - chaperones that control the atp - dependent activity of hsp70 .
dnaj proteins generally enhance the atpase activity of hsp70 , but their action can lead to different fates of their bound substrates .
dnajb1 is an example for a dnaj protein that supports substrate folding , whereas dnajb2-bound clients are degraded by the proteasome .
the co - chaperone hip stabilizes the adp - bound state of hsp70 and co - operates with hsp70 in protein folding .
other hsp70-binding cofactors are the ubiquitin ligases carboxy terminus of hsc70-interacting protein ( chip ) and parkin , which provide a link between hsp70 , co - chaperones and the ups , resulting in client ubiquitination and degradation by the proteasome [ 34 , 38 , 39 ] .
recently , it has been shown that bag3 interacts with hsp70 and directs the chaperone and its substrates into the autophagic pathway , providing an association of hsp70 , co - chaperones and autophagy .
an extensive description of the hsp70 machinery , the exact structure - function relationship between hsp70 and its various cofactors , has been excellently reviewed elsewhere .
the functional variability of the chaperone system that is exerted by a multitude of single proteins highlights the complexity of this proteostasis network .
however , limited knowledge exists on the exact composition and capacity of the chaperone system within a cell or a certain tissue .
a general view assumes that total levels of cellular chaperones exceed the actual requirements and that a sufficient amount of chaperones is free of clients .
thus , the network has excess capacity to initially deal with sudden additional chaperone requirements , as exhibited during stress conditions .
an opposite view proposes that the capacity of the chaperone network is always closely titrated to the actual demand of chaperone activity and that the system is otherwise rapidly adapted to conditions of increased requirements .
the analysis of the chaperone capacity of neuronal and muscular tissue of caenorhabditis elegans ( c. elegans ) demonstrates that these tissues indeed display different folding activities and that neurons are particularly sensitive to protein denaturation during heat stress . however , the adaptation of the chaperone network during aging is critical as it is well acknowledged that protein aggregation and disruption of proteostasis are characteristic for aged cells .
the periodical application of mild heat stress decreases mortality in drosophila melanogaster and c. elegans , which is mediated by hsp70 activity and the overexpression of hsp70 in c. elegans increases the life span of the nematode [ 4345 ] .
several reports have analyzed chaperone protein or mrna levels in aged cells and found increased or basal amounts , whereas the stress - mediated induction of chaperone expression is impaired .
the transcription of chaperone genes in response to stress conditions is controlled by the transcription factor hsf1 , which shows an impaired dna - binding potential in aged cells .
a reduced activity of hsf1 in c. elegans results in a shortened life span and , conversely , the enhanced expression of the transcription factor increases the life span .
activity is also essential for the extended life span of the extremely long - lived daf-2/insulin / igf-1 receptor mutants of c. elegans [ 47 , 48 ] .
thus , hsf1 and chaperone activity can promote longevity , demonstrating a clear association of chaperones , proteostasis and aging .
some of the factors mentioned so far are involved in linking chaperone functions with cellular protein - degradation pathways , the ups and autophagy , for the removal of misfolded proteins . besides protein aggregation ,
one factor that induces ubiquitination is protein damage caused by free radical oxygen species ( ros ) and oxidative stress .
most likely , irreversible oxidation may activate chaperones and the ups to induce protein repair of misfolded proteins and lead to ubiquitination and protein degradation . during aging ,
in particular , the mitochondrial respiratory chain is strongly linked to the production of ros and as one consequence may cause protein dysfunction , apoptosis , necrosis , aging and disease [ 49 , 50 ] .
protein oxidation leads to a change in protein conformation and function and chaperones may sense such changes and in turn activate the ups as a quality control system .
depending on the degree of oxidation , irreversible oxidation and loss of protein function may lead to degradation and/or accumulation of damaged proteins and to the formation of so - called aggresomes , which display a high autophagic activity [ 51 , 52 ] .
the ups is a complex enzymatic pathway that starts with the ligation of ubiquitin , a 76-amino - acid - long and highly conserved protein , to other cellular proteins and thus labels them for degradation .
initially the c - terminal end of ubiquitin is activated by atp - dependent phosphorylation and formation of a thiol ester via an activating enzyme , e1 .
it is then transferred to a thiol group of an ubiquitin - carrier protein , e2 .
the e3 ligase directs ubiquitin from e2 to an -amino group of the target protein [ 53 , 54 ] .
the c - terminus of an additional ubiquitin protein can be ligated onto one of the seven lysine residues within the attached ubiquitin molecule . for degradation via the proteasome
ubiquitin linkages between either the c - terminus and lysine residues k48 or k63 are the major recognition signals for proteasomal degradation .
ubiquitin chains also occur among other lysine residues , whereas ubiquitin extension via k6 is associated with dna repair , k11 with endoplasmatic reticulum - associated protein degradation and cell cycle regulation , k27 with ubiquitin fusion degradation , k29 with lysosomal degradation and k33 with kinase modification .
monoubiquitination can modify the activity of the protein transport machinery and when attached to transmembrane proteins can serve as a sorting signal to direct their movement between different cellular compartments [ 5659 ] . the polyubiquitinated proteins destined for degradation
are processed by the multienzymatic proteasome complex : first they become deubiquitinated and then degraded by the 26s proteasome , a system that is composed of various proteasome subunits .
the eukaryotic 20s proteasome core complex consists of four heptameric rings comprising two classes of seven non - identical but homologous subunits .
the outer rings contain alpha - type subunits with gating function for substrate entrance and product release , while the beta - subunits exhibit peptide hydrolyzing activity .
the 19s regulatory complex binds to the 20s catalytic core in a flexible manner and consists of six atpases , forming a ring at the entrance of the core and exerting chaperone - like activity .
the ups is the major degradation system in the cell for the degradation of short - lived , misfolded and defective proteins .
an accumulation of polyubiquitinated proteins is reported for numerous neurological disorders , which suggests that ups dysfunction plays a prominent role in the pathogenesis of neurodegenerative diseases and moreover in aging . since proteasomal
activity decreases during aging over all , the degradation demand of the cell increases , which results in an age - associated induction of the autophagy pathway that delivers substrates for degradation ultimately to lysosomes [ 20 , 63 ] .
autophagy is negatively regulated via the evolutionarily conserved enzymatic mammalian target of rapamycin complex 1 ( mtorc1 ) . in turn , if mtor activity is inhibited by rapamycin , the autophagy pathway is switched on .
rapamycin exerts its stimulatory effect on autophagy by preventing mtor phosphorylation at ser-2448 and thereby subsequently blocks mtor signaling .
so far , many different autophagic systems have been described including macroautophagy and cma that form the main pathways .
macroautophagy is a multi - step process by which cytosolic material is sequestered into a double - layered membrane structure , the autophagosome , and is delivered to the lysosome for degradation .
it is a highly orchestrated process and involves autophagy - related ( atg ) proteins .
more than 30 genes have been identified in genetic analyses of yeast mutants which have defects in autophagic function .
after autophagy stimulation through mtor inhibition , the formation of phagophores ( autophagosome precursors / pre - autophagosomal structures ) is initiated in a not yet completely understood mechanism .
a subset of atg proteins is required for autophagosome formation : first , the atg9 system including atg9 , the atg1 kinase complex ( atg1 and atg13 ) , atg2 and atg18 ; second , the phosphatidylinositol 3-oh kinase ( pi(3)k ) complex consisting of vacuolar protein sorting ( vps)34 , vps15 , beclin 1/atg6 and atg14 and third , the ubiquitin - like protein ( ubl ) system composed of two ubl proteins ( lc3/atg8 and atg12 ) , an e1 enzyme ( atg7 ) , two analogues of e2 ubiquitin - conjugating enzymes ( atg10 and atg3 ) , an lc3/atg8-modifying protease ( atg4 ) , the protein target of atg12 attachment ( atg5 ) and atg16 .
beclin 1/atg6 regulates autophagic trafficking and membrane trafficking in a variety of physiological and pathological processes .
microtubule - associated protein 1 light chain 3 ( lc3/atg8 ) is commonly used as a marker for autophagic activity . with its e1-like activity , atg7 converts cytosolic lc3-i to the membrane - associated lc3-ii , which then is conjugated to phosphatidylethanolamine .
lc3-ii binds the inner and outer membrane of the forming autophagosome and provides a physical link between the autophagosome and lc3 interacting proteins , such as sequestosome 1 ( p62/sqstm1 ) or neighbor of brca1 gene 1 ( nbr1 ) .
these so - called autophagy receptors can simultaneously bind to ubiquitinated proteins and lc3 , employing their lc3-interacting motif and ubiquitin - associated domain .
the identification of autophagy receptors , such as p62/sqstm1 and nbr1 has provided a molecular link between ubiquitination and autophagy .
thus , p62/sqstm1 and nbr1 are possibly also important bridging factors between ups and autophagy . through self - oligomerization , which is stimulated by ubiquitin binding , p62/sqstm1 sequesters ubiquitinated substrates in form of inclusion bodies .
these inclusions are then specifically engulfed by the autophagosome membrane by recruiting lc3 [ 69 , 70 ] . while macroautophagy is considered as a rather unspecific robust degradation process , cma is a highly selective lysosomal pathway that removes a distinct subset of proteins containing a pentapeptide lysosome - targeting motif .
these substrates can on the one hand directly be translocated into the lysosome after docking to the lysosomal receptor lysosomal - associated membrane protein 2a or on the other hand unfolded by a chaperone complex containing hsc70 and the co - chaperones bag1 , hip , hsp70hsp90 organizing protein ( hop ) and hsp40/dnajb1 .
these co - chaperones provide a direct link to the ups and the folding and refolding activity of molecular chaperones .
in the course of aging , a variety of proteins tend to aggregate and impair the ups directly .
therefore , these proteins , most of which are ubiquitinated , can not be handled by the proteasome and have to be degraded by different protein clearance pathways .
recently , it has been shown that the cellular protein quality control ( pqc ) of polyubiquitinated proteins by proteasomal and autophagic systems is regulated by the hsp70 co - chaperones bag1 and bag3 , respectively .
proteasome activity decreases in an age - dependent manner in a cell model of replicative senescent human fibroblasts and is associated with an increased autophagic activity in aged cells .
this correlates with decreased levels of bag1 and increased levels of bag3 in aged cells , an age - related bag1/bag3 switch that serves as an induction control of the macroautophagic pathway ( bag3-mediated selective macroautophagy ) and may be considered as a backup of the ups in pqc ( [ 40 , 72 ] ) .
therefore , the expression shift from bag1 to bag3 during aging , but also upon acute stress ( e.g. proteasome inhibition , oxidative stress ) , can be considered as a physiologically important adaptive response [ 20 , 40 ] .
the bag3-mediated selective pathways have been shown to be involved in a variety of disease causing processes . in a model for hd ,
bag3 in concert with hspb8 facilitates the disposal of polyq43-huntingtin by stimulating macroautophagy and this complex is also involved in z - disc maintenance in flies , mice and men [ 73 , 74 ] . additionally , the transport of target proteins to the aggresome , a compartment with high autophagic activity , has been shown to be mediated by bag3 .
this aggresome - targeting pathway involving bag3 and hsp70 is distinct from other before - described mechanisms as it does not depend on substrate ubiquitination . taken together the molecular chaperone machinery , the ups and the autophagy system are involved in pqc and maintaining proteostasis within aging and disease to prevent protein misfolding and aggregation .
a summary of the main routes for protein degradation is shown in fig . 1 .
as aggregated proteins are found in ad patients and aging is a prevailing risk factor for ad it is important to further characterize how these pathways are regulated and how misregulation possibly contributes to the pathology of ad.fig .
1the ubiquitin proteasome system ( ups ) and various autophagy routes as the main pathways for protein degradation ( regular turnover ) and of misfolded and aggregated proteins ( and mitochondria ) the ubiquitin proteasome system ( ups ) and various autophagy routes as the main pathways for protein degradation ( regular turnover ) and of misfolded and aggregated proteins ( and mitochondria )
ad is characterized by the accumulation of intracellular a ( oligomers ) , extracellular high molecular weight deposits of a peptides ( fibrilliar forms ) , and the intracellular aggregation of hyper - phosphorylated tau . indeed ,
modification of the cellular proteostasis and the metabolism of a and tau have been proven to cause neuronal dysfunction and cell death .
the malfunctioning proteostasis control results in the accumulation of misfolded and aggregated proteins , which is a general hallmark of aging and neurodegeneration , as observed in pd , hd , als and ad . aging in particular is characterized by decreasing proteostasis capacity and increasing protein damage which are in combination a major challenge for the cell due to the inability to maintain metastable proteins in folded states . to counteract a cascade of protein destabilization caused by metastable proteins which escaped the pqc
the proteostasis network including molecular chaperones , the ups and autophagic pathways must manage the increasing burden of protein misfolding to maintain proteome stability .
since aging is the major risk factor of ad [ 7880 ] , the age - dependent loss of proteostasis might be an important contributor to the pathology of ad .
an overview of proteins that affect proteostasis in ad is shown in table 1.table 1proteins affecting proteostasis in alzheimer s diseaseproteinfunctionreferencea40/42decreases proteasome activity , modulates autophagy through mtor signaling[99102 , 104 , 143146]tauinhibitory effect of tau aggregates on proteasome activityhsf1induces app gene during stress[81 , 82]grp78 ( er isoform of hsp70)modulates app maturation and reduces a40/42 secretion[84 , 85]hsps ( hsp22 , 27 , 70 , 90)small hsps ( hsp22 , hsp27 ) bind to fibrillar amyloid plaques and inhibit their fibrillarisation ; hsp70 , hsp90 inhibit early stages of amyloid aggregation[88 , 89]chipe3 enzyme for phosphorylated tau[93 , 117]bag1regulates proteasomal degradation of tau together with hsp70ps1increases production of a42 , essential for lysosomal proteolysis and autophagy by enabling the acidification of lysosomes required for protease activation[163 , 165]ubb+1potently inhibits the degradation of polyubiquitinated substrates and therefore induces neuronal cell death[120 , 121]uch - l1dub needed for proteasomal degradation of client proteins , oxidized and down - regulated in ad[124 , 125]ubqln1ubqln1 activity is necessary to regulate the production of app and app fragments[127 , 128]mtorinhibitory and/or activating modulation of mtor through a42 but not a40[143146]beclin1beclin1 is down - regulated in ad brains and consequently increases app levels and its metabolites[161 , 162]a amyloid beta , app amyloid precursor protein , hsf1 heat shock transcription factor 1 , grp78 glucose - related protein 78 , hsp heat shock protein , chip carboxy terminus of hsc70-interacting protein , bag1 bcl2-associated athanogene 1 , ps1 presenilin 1 , uch - l1 ubiquitin carboxy terminal hydrolase isozyme 1 , ubqln1 ubiquilin 1 , mtor mammalian target of rapamycin , dub deubiquitination enzyme , er endoplasmic reticulum proteins affecting proteostasis in alzheimer s disease a amyloid beta , app amyloid precursor protein , hsf1 heat shock transcription factor 1 , grp78 glucose - related protein 78 , hsp heat shock protein , chip carboxy terminus of hsc70-interacting protein , bag1 bcl2-associated athanogene 1 , ps1 presenilin 1 , uch - l1 ubiquitin carboxy terminal hydrolase isozyme 1 , ubqln1 ubiquilin 1 , mtor mammalian target of rapamycin , dub deubiquitination enzyme , er endoplasmic reticulum the two main chaperone scaffolds are hsp70 and hsp90 which are accompanied by a variety of co - chaperones specifying substrate binding and release and are transcriptionally regulated through heat shock elements ( hse ) via the transcription factor hsf1 .
interestingly , promoter studies of the app gene showed hses within its promoter region suggesting an impact of hsps on ad pathology [ 81 , 82 ] .
as app is a membrane - associated protein it maturates in the endoplasmic reticulum ( er ) and the golgi apparatus .
there , the ectodomain of app associates with the luminal localized er chaperone glucose - related protein 78 ( grp78 , the er isoform of hsp70 ) and this interaction impairs its maturation resulting in a reduced generation of a40 and a42 [ 84 , 85 ] .
the cytosolic hsp70 co - chaperone chip interacts with intracellular domain of app in the er and golgi compartments providing a link between molecular chaperones and the ups in app processing .
in addition to the er and golgi - associated hsps , the cytosolic chaperones are as well linked to app and the a metabolism .
the shsps hsp22 and hsp27 have been shown to bind to fibrillar a to inhibit further fibrillarization .
moreover , hsp70 and hsp90 inhibit early stages of a aggregation [ 88 , 89 ] .
hsps have also been associated with extracellular a as they can be released by either active secretion mechanisms or from cells undergoing necrosis [ 90 , 91 ] . in ad brains ,
furthermore , extracellular hsp90 and hsp70 increased the amount of a42 peptides in microglia after 1 day and decreased the amount after 3 days in vitro .
these observations suggest that hsp - induced microglial activation may have a neuroprotective role by facilitating a clearance .
besides extracellular a accumulation , the second major hallmark of ad is the intracellular aggregation of tau .
post - translational modifications of tau , e.g. hyper - phosphorylation , affect the conformation of the protein and promote the aggregation state .
these post - translational modifications impact the interaction of tau with microtubules , and thus , there may be specific forms of tau that are preferred chaperone substrates relative to others .
hsp27 , hsp70 and chip are reported to recognize abnormal tau and reduce its concentration by assisting its degradation and dephosphorylation [ 9294 ] .
hsp27 preferentially binds to hyper - phosphorylated but not to non - phosphorylated tau and is cross - linked with tau in nfts from ad brains [ 94 , 95 ] .
protein levels of soluble tau positively correlate with hsp27 , hsp40 , hsp90 , alphab - crystallin and chip levels in ad brains .
vice versa hsp protein levels are inversely correlated with levels of tau oligomers , which are an intermediate of tau filaments . in different cellular models
, it has been shown that increased hsp70 and hsp90 levels promote tau solubility and microtubule binding .
tau seems to directly bind to hsp70 and this interaction is mediated by the hsp70 co - chaperone bag1 pointing to a tight interplay between molecular chaperones and the ups in counteracting tau aggregation [ 97 , 98 ] .
collectively , these data suggest that up - regulation of molecular chaperones may suppress formation of ntfs by partitioning tau into a productive folding pathway and thereby preventing tau aggregation . the ubiquitin
proteasome system is the main degradation pathway in the cell and it has been shown that a40 and a42 can block proteasome function in vitro .
a40 binds to the inner surface of proteasomes and inhibits 20s chymotrypsin - like activity .
a42 can inhibit proteasome function to an extent that is comparable to a well - known proteasome inhibitor [ 99102 ] .
it still has to be elucidated which form of a is now affecting proteasome function .
there is experimental evidence that a oligomers but not monomers or fibrils impair long - term potentiation in vivo . in a cell
free proteasome activity assay , it has been shown that indeed a40 and a42 oligomers , but not monomers , decreased proteolytic activity of the proteasome in a dose - dependent manner .
there is evidence suggesting that a is degraded by the proteasome because inhibition of the proteasome with lactacystin caused a significant increase in a42 levels in cultured neurons and astrocytes .
consistent with these results , it has been shown that the 20s proteasome degrades a40 and a42 in vitro and that inhibition of the proteasome in cultured cells increases intracellular a40 and a42 level .
so far , it is still a matter of debate how the cytoplasmic and nuclear localized proteasome could physically interact with extracellular or luminal localized a in vivo . in cultured neurons
, it has been shown via immunoelectron microscopy that 20s proteasome subunits are detectable in the outer membranes and inner vesicles of multivesicular bodies . in normal and ad brain
, a42 has also been shown to accumulate predominantly in multivesicular bodies , indicating that these structures are the possible interaction site of a and the proteasome in vivo . further supporting the influence of the ups on a metabolism
thus , a decrease in proteasome activity would increase -secretase activity and thereby a production .
this illustrates a clear association between a peptides and the proteostasis network , highlighting the importance of chaperones and the ups in a metabolism .
the protein tau is degraded by the 26s and 20s proteasome in vitro and the use of proteasome inhibitors in cells and animal models leads to an accumulation of tau [ 108111 ] .
furthermore , mass spectrometry studies on isolated tau from inclusion bodies show k48 and k63 ubiquitin linkages on tau , which are the recognition signals for degradation via the ups [ 112114 ] .
it has also been shown that the 20s proteasome co - precipitated with tau aggregates and that the amount of pulled down tau aggregates was higher in samples with low proteasome activity , suggesting an inhibitory interaction between tau aggregates and proteasome activity .
in addition , in vitro studies show that tau aggregates isolated from human ad brain directly inhibit the proteasome .
in contrast , non - aggregated tau isolated from ad brain or from control brain did not interfere with proteasome activity .
these data show that different aggregation states of tau modify its turnover via the proteasome .
it has been shown that tau co - immunoprecipitates with chip , an e3 ubiquitin ligase , that ubiquitinates tau for subsequent degradation via the proteasome and soluble phosphorylated tau accumulates in brains of chip knockout mice [ 93 , 116 , 117 ] . in ad ,
the mechanism of stabilization and accumulation of hyper - phosphorylated tau may involve inhibition of tau interaction with chip .
in addition to phosphorylation , tau is also post - translationally acetylated and this acetylation impairs proteosomal degradation and enhances accumulation of tau .
moreover , changes in the combination of proteasome subunits have been reported in ad brain , resulting in an altered proteasome activity .
taken together , these data clearly indicate that proteasome activity is necessary for tau turnover and that aggregated tau inhibits proteasomal function .
ubiquitin immunoreactivity accumulates in aggregates in ad brains and it has also been shown that some of these structures enclose ubiquitin - b mutant protein ( ubb+1 ) .
overexpression of this mutant , which contains a c - terminal amino acid extension , leads to an impairment of the proteasome and induces neuronal cell death [ 120 , 121 ] .
oxidative stress is one factor that leads to protein damage and subsequently to protein ubiquitination and degradation via the proteasome .
it is interesting to note that the ubiquitin carboxy - terminal hydrolase l1 ( uch - l1 ) is oxidized in ad patients and is down - regulated in specific brain regions of early ad cases [ 122 , 123 ] .
uch - l1 functions as a deubiquitination enzyme ( dub ) that hydrolyses ubiquitin from polyubiquitinated proteins to liberate and stabilize ubiquitin monomers and it promotes proteasomal degradation .
interestingly , when overexpressed , uch - l1 reverses behavioral deficits in ad model mice consistent with an impairment of the ups in ad [ 124 , 125 ] .
several lines of evidence implicate a tight regulation of the ups and a strong interaction with autophagic pathways to maintain proteostasis .
for example , tau seems to be a substrate of the ups and of the autophagic system in vivo .
recently , it has been shown that truncated tau ( tauc ) is rapidly degraded via the autophagic system whereas non - truncated tau is favorably degraded via the ups .
in addition , tau gets targeted for degradation through hsp70 regardless of its phosphorylation state involving the molecular chaperone machinery , which in turn interacts with the ups - related proteins bag1 and chip , both known to be involved in tau degradation .
taken together , these data indicate that decreased ups function may be involved ( or even partially causative ) for ad pathogenesis .
this view is further strengthened by recent genetic evidence showing a positive association between ad and several single nucleotide polymorphisms in an ubiquitin - like protein called ubiquilin 1 ( ubqln1 ) .
ubqln1 is capable of preventing the aggregation of app both in vitro and in vivo [ 127 , 128 ] .
interestingly , mutations in another member of the ubiquilin family , ubiquilin 2 ( ubql2 ) , are associated with the motor neuron disease als , which supports a general role of the ups in neurodegeneration .
in fact , already in 1967 suzuki and terry reported dystrophic neurite swellings filled with vacuolar structures positive for acid phosphatase .
these structures have been shown to be unique for ad and ultrastructural imaging of ad brains revealed that they consist of lysosomes and a bulk of autophagic vacuoles ( avs ) [ 131 , 132 ] .
these histological changes reflect either an increased synthesis of components of the lysosomal system , a disturbed clearance of avs , or both , as the accumulation of vesicles can be initiated by blocking lysosomal activity in primary neuronal cell lines [ 133135 ] .
a general disturbance of neurite transport mechanism is not causative for the accumulation of avs as the transport of organelles such as mitochondria appears unaltered under conditions of lysosomal inhibition .
neuronal tissue might be extraordinarily susceptible for disturbances within the autophagic - lysosomal system , because this network enables neurons to perform highly specific functions such as vesicle release and synaptic transmission .
furthermore , post - mitotic neurons have to cope with elevated levels of stress and damaged organelles as well as misfolded or aggregated proteins during their lifetime , cellular aging and disease progression , which highlight the importance of an effective degradation system in this particular cell type . besides changes of the autophagic system on a histological level , also molecular regulators of autophagic degradation are altered during ad progression .
genes that represent negative regulators of autophagy are repressed , whereas positive modulators are more likely to be up - regulated in the brain of ad patients .
however , enhanced expression of autophagy activators does not result in a persistent increase of autophagic activity as the autophagic efficacy declines during the course of the disease , resulting in an accumulation of undegraded proteins .
the most prominent physiological modulator of mtor signaling is caloric restriction ( cr ) meaning the energy content of food is reduced without compromising the supply of essential nutrients .
attenuation of ad as well as a prolonged lifespan in healthy humans mediated by cr might not only be due to reduced metabolic toxicity and decreased vulnerability to metabolic diseases like diabetes , but may also rely on cytoprotective effects of mtor - mediated autophagic activity [ 141 , 142 ] .
interestingly , a42 , but not a40 , affects mtor signaling and is as well affected by mtor modulation itself . the discussion , if a silences or enhances mtor signaling is still ongoing : some studies show inhibitory effects of a on mtor signaling , while others show an activation [ 143146 ] . in a murine neuroblastoma cell line ( n2a ) and
hippocampal slice cultures exposed to a42 , in appswe / ps1 , a double - transgenic mouse model for ad , and in lymphocytes of ad patients , the phosphorylation of mtor as well as the phosphorylation of its substrate p70s6-kinase is reduced [ 144 , 145 ] .
furthermore , in the tg2576 app - overexpressing mice , the a-mediated reduction of mtor signaling has been shown to be accompanied by a decline in synaptic activity that can be rescued by mtor up - regulation .
in contrast , cells which produce high levels of a oligomers show an enhanced mtor activity which can be counteracted by decreasing a formation or administration of rapamycin .
similar results have been seen in triple transgenic ad mice ( 3tg - ad mice ) , combining a and tau - pathology of ad , which show a reduction in tau phosphorylation and improved learning and memory after rapamycin treatment .
interestingly , the effects on mtor signaling are only observed in brain areas such as hippocampus and cortex which are also affected in brains of ad patients , but not in others .
the beneficial effect of rapamycin treatment and , therefore , autophagy stimulation on learning and memory is also reported for a further ad mouse model .
these animals show a recovery of the autophagic flux by enhanced autophagic activity in the hippocampus after administration of rapamycin .
interestingly , already in 1992 , haass and colleagues reported a lysosomal pathway that is involved in app processing and potentially responsible for the generation of amyloid - bearing fragments in ad via the generation of an extensive array of app c - terminal fragments ( app - ctfs ) which are found in lysosomes .
later , alterations in the metabolism of app have been shown in models for glycosphingolipid storage disease , where a mtor - independent increase in the autophagic vacuole - associated protein lc3-ii occurs , indicating an impaired lysosomal flux .
this suggests an anti - amyloidogenic role of lysosomal proteolysis in post - secretase app - ctf catabolism , which does not directly involve macroautophagy .
the accumulation of sphingolipids , which characterizes lysosomal lipid storage disorders , has also been shown to decrease the lysosome - dependent degradation of app - ctfs and to stimulate -secretase activity .
thus , sphingolipids might trigger increased generation of a via impairment of the autophagic lysosomal system and contribute to neurodegeneration in sporadic ad . pharmacological enhancement of autophagy or induction of autophagy via starvation greatly decreased the levels of a peptides and app - ctfs in a -secretase independent manner .
consequently , after inhibition of autophagy , a significant accumulation of a peptides and app - ctfs has been observed .
the upcoming data support the involvement of autophagy in the clearance of a and app - ctfs .
the autophagy receptor p62/sqstm1 shows a reduced expression in ad patients , as well as in 3tg - ad - expressing mice .
this might be caused by oxidative stress and subsequent dna damage of the p62/sqstm1 promoter , which leads to reduced p62/sqstm1 transcription [ 151 , 152 ] . because guanine is the nucleobase which is most susceptible to oxidation , the p62/sqstm1 promoter is exceptionally prone to oxidative stress , as it is rich of gc regions [ 153 , 154 ] .
interestingly , tau is degraded via the autophagic - lysosomal system , whereby tau aggregates are cleared via macroautophagy .
as tau possesses two putative cma - targeting motifs , soluble tau is most likely degraded via cma .
hyper - phosphorylated tau accumulates in p62/sqstm1-knockout mice , indicating an interplay of oxidative stress and the clearance of protein aggregates in ad .
another important modulator of autophagic activity is beclin 1 which is a hub - protein - forming multiprotein complexes that possess different functions according to their composition [ 157160 ] .
beclin 1 is down - regulated in the brain of ad patients even in cases of mild cognitive impairment .
in addition , mutant app - overexpressing mice with a heterozygous deletion of beclin 1 show enhanced microglial activation and an accumulation of lysosomes .
moreover , the knockdown of beclin 1 in cultured cells increases the levels of app and its cleavage products which are accompanied by impaired autophagic flux .
however , the overexpression of mutant app has no effect on beclin 1 expression in cells and mice [ 161 , 162 ] .
recently , it has been shown that ps1 is important for a normal autophagic - lysosomal function independent of a .
ps1 has been found to be important for the acidification of the lysosomal compartment and thus to be crucial for lysosomal proteolysis and autophagic function . in neurons lacking ps1 ,
the subunit v01a of the vacuolar atpase is not correctly delivered to the lysosomes and not properly integrated into the lysosomal membrane .
this subunit is needed for correct function of the proton pump , which is responsible for lysosomal acidification .
-secretase lacking ps1 is not able to sufficiently facilitate n - glycosylation of the v01a subunit in the er , which is crucial for efficient transport to the lysosomes and assembly of the functional proton pump . as a result , v01a is entrapped in the er and cells loose lysosomal function .
in addition , an accumulation of avs is seen in ps1 hypomorphic mice and the pharmacological block of lysosomal acidification leads to similar accumulation of lysosomes and avs in primary neuronal cell lines .
the first abnormalities of the lysosomal - endosomal system occur prior to other hallmarks of the disease such as amyloid deposits in the neocortex and persist until massive impairments of the autophagic system emerge in the later stages of the disease .
taken together , all these findings suggest an important role for autophagy in the pathogenesis of ad .
the two main chaperone scaffolds are hsp70 and hsp90 which are accompanied by a variety of co - chaperones specifying substrate binding and release and are transcriptionally regulated through heat shock elements ( hse ) via the transcription factor hsf1 .
interestingly , promoter studies of the app gene showed hses within its promoter region suggesting an impact of hsps on ad pathology [ 81 , 82 ] .
as app is a membrane - associated protein it maturates in the endoplasmic reticulum ( er ) and the golgi apparatus .
there , the ectodomain of app associates with the luminal localized er chaperone glucose - related protein 78 ( grp78 , the er isoform of hsp70 ) and this interaction impairs its maturation resulting in a reduced generation of a40 and a42 [ 84 , 85 ] .
the cytosolic hsp70 co - chaperone chip interacts with intracellular domain of app in the er and golgi compartments providing a link between molecular chaperones and the ups in app processing .
in addition to the er and golgi - associated hsps , the cytosolic chaperones are as well linked to app and the a metabolism .
the shsps hsp22 and hsp27 have been shown to bind to fibrillar a to inhibit further fibrillarization .
moreover , hsp70 and hsp90 inhibit early stages of a aggregation [ 88 , 89 ] .
hsps have also been associated with extracellular a as they can be released by either active secretion mechanisms or from cells undergoing necrosis [ 90 , 91 ] . in ad brains ,
furthermore , extracellular hsp90 and hsp70 increased the amount of a42 peptides in microglia after 1 day and decreased the amount after 3 days in vitro .
these observations suggest that hsp - induced microglial activation may have a neuroprotective role by facilitating a clearance .
besides extracellular a accumulation , the second major hallmark of ad is the intracellular aggregation of tau .
post - translational modifications of tau , e.g. hyper - phosphorylation , affect the conformation of the protein and promote the aggregation state .
these post - translational modifications impact the interaction of tau with microtubules , and thus , there may be specific forms of tau that are preferred chaperone substrates relative to others .
hsp27 , hsp70 and chip are reported to recognize abnormal tau and reduce its concentration by assisting its degradation and dephosphorylation [ 9294 ] .
hsp27 preferentially binds to hyper - phosphorylated but not to non - phosphorylated tau and is cross - linked with tau in nfts from ad brains [ 94 , 95 ] .
protein levels of soluble tau positively correlate with hsp27 , hsp40 , hsp90 , alphab - crystallin and chip levels in ad brains .
vice versa hsp protein levels are inversely correlated with levels of tau oligomers , which are an intermediate of tau filaments . in different cellular models
, it has been shown that increased hsp70 and hsp90 levels promote tau solubility and microtubule binding .
tau seems to directly bind to hsp70 and this interaction is mediated by the hsp70 co - chaperone bag1 pointing to a tight interplay between molecular chaperones and the ups in counteracting tau aggregation [ 97 , 98 ] .
collectively , these data suggest that up - regulation of molecular chaperones may suppress formation of ntfs by partitioning tau into a productive folding pathway and thereby preventing tau aggregation .
the ubiquitin proteasome system is the main degradation pathway in the cell and it has been shown that a40 and a42 can block proteasome function in vitro .
a40 binds to the inner surface of proteasomes and inhibits 20s chymotrypsin - like activity .
a42 can inhibit proteasome function to an extent that is comparable to a well - known proteasome inhibitor [ 99102 ] .
it still has to be elucidated which form of a is now affecting proteasome function .
there is experimental evidence that a oligomers but not monomers or fibrils impair long - term potentiation in vivo . in a cell
free proteasome activity assay , it has been shown that indeed a40 and a42 oligomers , but not monomers , decreased proteolytic activity of the proteasome in a dose - dependent manner .
there is evidence suggesting that a is degraded by the proteasome because inhibition of the proteasome with lactacystin caused a significant increase in a42 levels in cultured neurons and astrocytes .
consistent with these results , it has been shown that the 20s proteasome degrades a40 and a42 in vitro and that inhibition of the proteasome in cultured cells increases intracellular a40 and a42 level .
so far , it is still a matter of debate how the cytoplasmic and nuclear localized proteasome could physically interact with extracellular or luminal localized a in vivo . in cultured neurons
, it has been shown via immunoelectron microscopy that 20s proteasome subunits are detectable in the outer membranes and inner vesicles of multivesicular bodies . in normal and ad brain ,
a42 has also been shown to accumulate predominantly in multivesicular bodies , indicating that these structures are the possible interaction site of a and the proteasome in vivo . further supporting the influence of the ups on a metabolism
thus , a decrease in proteasome activity would increase -secretase activity and thereby a production .
this illustrates a clear association between a peptides and the proteostasis network , highlighting the importance of chaperones and the ups in a metabolism .
the protein tau is degraded by the 26s and 20s proteasome in vitro and the use of proteasome inhibitors in cells and animal models leads to an accumulation of tau [ 108111 ] .
furthermore , mass spectrometry studies on isolated tau from inclusion bodies show k48 and k63 ubiquitin linkages on tau , which are the recognition signals for degradation via the ups [ 112114 ] .
it has also been shown that the 20s proteasome co - precipitated with tau aggregates and that the amount of pulled down tau aggregates was higher in samples with low proteasome activity , suggesting an inhibitory interaction between tau aggregates and proteasome activity .
in addition , in vitro studies show that tau aggregates isolated from human ad brain directly inhibit the proteasome .
in contrast , non - aggregated tau isolated from ad brain or from control brain did not interfere with proteasome activity .
these data show that different aggregation states of tau modify its turnover via the proteasome . particularly intriguing
it has been shown that tau co - immunoprecipitates with chip , an e3 ubiquitin ligase , that ubiquitinates tau for subsequent degradation via the proteasome and soluble phosphorylated tau accumulates in brains of chip knockout mice [ 93 , 116 , 117 ] . in ad ,
the mechanism of stabilization and accumulation of hyper - phosphorylated tau may involve inhibition of tau interaction with chip .
in addition to phosphorylation , tau is also post - translationally acetylated and this acetylation impairs proteosomal degradation and enhances accumulation of tau .
moreover , changes in the combination of proteasome subunits have been reported in ad brain , resulting in an altered proteasome activity .
taken together , these data clearly indicate that proteasome activity is necessary for tau turnover and that aggregated tau inhibits proteasomal function .
ubiquitin immunoreactivity accumulates in aggregates in ad brains and it has also been shown that some of these structures enclose ubiquitin - b mutant protein ( ubb+1 ) .
overexpression of this mutant , which contains a c - terminal amino acid extension , leads to an impairment of the proteasome and induces neuronal cell death [ 120 , 121 ] .
oxidative stress is one factor that leads to protein damage and subsequently to protein ubiquitination and degradation via the proteasome .
it is interesting to note that the ubiquitin carboxy - terminal hydrolase l1 ( uch - l1 ) is oxidized in ad patients and is down - regulated in specific brain regions of early ad cases [ 122 , 123 ] .
uch - l1 functions as a deubiquitination enzyme ( dub ) that hydrolyses ubiquitin from polyubiquitinated proteins to liberate and stabilize ubiquitin monomers and it promotes proteasomal degradation .
interestingly , when overexpressed , uch - l1 reverses behavioral deficits in ad model mice consistent with an impairment of the ups in ad [ 124 , 125 ] .
several lines of evidence implicate a tight regulation of the ups and a strong interaction with autophagic pathways to maintain proteostasis .
for example , tau seems to be a substrate of the ups and of the autophagic system in vivo .
recently , it has been shown that truncated tau ( tauc ) is rapidly degraded via the autophagic system whereas non - truncated tau is favorably degraded via the ups .
in addition , tau gets targeted for degradation through hsp70 regardless of its phosphorylation state involving the molecular chaperone machinery , which in turn interacts with the ups - related proteins bag1 and chip , both known to be involved in tau degradation .
taken together , these data indicate that decreased ups function may be involved ( or even partially causative ) for ad pathogenesis .
this view is further strengthened by recent genetic evidence showing a positive association between ad and several single nucleotide polymorphisms in an ubiquitin - like protein called ubiquilin 1 ( ubqln1 ) .
ubqln1 is capable of preventing the aggregation of app both in vitro and in vivo [ 127 , 128 ] .
interestingly , mutations in another member of the ubiquilin family , ubiquilin 2 ( ubql2 ) , are associated with the motor neuron disease als , which supports a general role of the ups in neurodegeneration .
in fact , already in 1967 suzuki and terry reported dystrophic neurite swellings filled with vacuolar structures positive for acid phosphatase .
these structures have been shown to be unique for ad and ultrastructural imaging of ad brains revealed that they consist of lysosomes and a bulk of autophagic vacuoles ( avs ) [ 131 , 132 ] .
these histological changes reflect either an increased synthesis of components of the lysosomal system , a disturbed clearance of avs , or both , as the accumulation of vesicles can be initiated by blocking lysosomal activity in primary neuronal cell lines [ 133135 ] .
a general disturbance of neurite transport mechanism is not causative for the accumulation of avs as the transport of organelles such as mitochondria appears unaltered under conditions of lysosomal inhibition .
neuronal tissue might be extraordinarily susceptible for disturbances within the autophagic - lysosomal system , because this network enables neurons to perform highly specific functions such as vesicle release and synaptic transmission .
furthermore , post - mitotic neurons have to cope with elevated levels of stress and damaged organelles as well as misfolded or aggregated proteins during their lifetime , cellular aging and disease progression , which highlight the importance of an effective degradation system in this particular cell type . besides changes of the autophagic system on a histological level , also molecular regulators of autophagic degradation are altered during ad progression .
genes that represent negative regulators of autophagy are repressed , whereas positive modulators are more likely to be up - regulated in the brain of ad patients . however ,
enhanced expression of autophagy activators does not result in a persistent increase of autophagic activity as the autophagic efficacy declines during the course of the disease , resulting in an accumulation of undegraded proteins .
the most prominent physiological modulator of mtor signaling is caloric restriction ( cr ) meaning the energy content of food is reduced without compromising the supply of essential nutrients .
attenuation of ad as well as a prolonged lifespan in healthy humans mediated by cr might not only be due to reduced metabolic toxicity and decreased vulnerability to metabolic diseases like diabetes , but may also rely on cytoprotective effects of mtor - mediated autophagic activity [ 141 , 142 ] .
interestingly , a42 , but not a40 , affects mtor signaling and is as well affected by mtor modulation itself .
the discussion , if a silences or enhances mtor signaling is still ongoing : some studies show inhibitory effects of a on mtor signaling , while others show an activation [ 143146 ] . in a murine neuroblastoma cell line ( n2a ) and
hippocampal slice cultures exposed to a42 , in appswe / ps1 , a double - transgenic mouse model for ad , and in lymphocytes of ad patients , the phosphorylation of mtor as well as the phosphorylation of its substrate p70s6-kinase is reduced [ 144 , 145 ] .
furthermore , in the tg2576 app - overexpressing mice , the a-mediated reduction of mtor signaling has been shown to be accompanied by a decline in synaptic activity that can be rescued by mtor up - regulation .
in contrast , cells which produce high levels of a oligomers show an enhanced mtor activity which can be counteracted by decreasing a formation or administration of rapamycin .
similar results have been seen in triple transgenic ad mice ( 3tg - ad mice ) , combining a and tau - pathology of ad , which show a reduction in tau phosphorylation and improved learning and memory after rapamycin treatment .
interestingly , the effects on mtor signaling are only observed in brain areas such as hippocampus and cortex which are also affected in brains of ad patients , but not in others .
the beneficial effect of rapamycin treatment and , therefore , autophagy stimulation on learning and memory is also reported for a further ad mouse model .
these animals show a recovery of the autophagic flux by enhanced autophagic activity in the hippocampus after administration of rapamycin .
interestingly , already in 1992 , haass and colleagues reported a lysosomal pathway that is involved in app processing and potentially responsible for the generation of amyloid - bearing fragments in ad via the generation of an extensive array of app c - terminal fragments ( app - ctfs ) which are found in lysosomes .
later , alterations in the metabolism of app have been shown in models for glycosphingolipid storage disease , where a mtor - independent increase in the autophagic vacuole - associated protein lc3-ii occurs , indicating an impaired lysosomal flux .
this suggests an anti - amyloidogenic role of lysosomal proteolysis in post - secretase app - ctf catabolism , which does not directly involve macroautophagy .
the accumulation of sphingolipids , which characterizes lysosomal lipid storage disorders , has also been shown to decrease the lysosome - dependent degradation of app - ctfs and to stimulate -secretase activity .
thus , sphingolipids might trigger increased generation of a via impairment of the autophagic lysosomal system and contribute to neurodegeneration in sporadic ad . pharmacological enhancement of autophagy or induction of autophagy via starvation greatly decreased the levels of a peptides and app - ctfs in a -secretase independent manner . consequently , after inhibition of autophagy , a significant accumulation of a peptides and app - ctfs has been observed .
the upcoming data support the involvement of autophagy in the clearance of a and app - ctfs .
the autophagy receptor p62/sqstm1 shows a reduced expression in ad patients , as well as in 3tg - ad - expressing mice .
this might be caused by oxidative stress and subsequent dna damage of the p62/sqstm1 promoter , which leads to reduced p62/sqstm1 transcription [ 151 , 152 ] . because guanine is the nucleobase which is most susceptible to oxidation , the p62/sqstm1 promoter is exceptionally prone to oxidative stress , as it is rich of gc regions [ 153 , 154 ] .
interestingly , tau is degraded via the autophagic - lysosomal system , whereby tau aggregates are cleared via macroautophagy .
as tau possesses two putative cma - targeting motifs , soluble tau is most likely degraded via cma .
hyper - phosphorylated tau accumulates in p62/sqstm1-knockout mice , indicating an interplay of oxidative stress and the clearance of protein aggregates in ad .
another important modulator of autophagic activity is beclin 1 which is a hub - protein - forming multiprotein complexes that possess different functions according to their composition [ 157160 ] .
beclin 1 is down - regulated in the brain of ad patients even in cases of mild cognitive impairment .
in addition , mutant app - overexpressing mice with a heterozygous deletion of beclin 1 show enhanced microglial activation and an accumulation of lysosomes .
moreover , the knockdown of beclin 1 in cultured cells increases the levels of app and its cleavage products which are accompanied by impaired autophagic flux .
however , the overexpression of mutant app has no effect on beclin 1 expression in cells and mice [ 161 , 162 ] .
recently , it has been shown that ps1 is important for a normal autophagic - lysosomal function independent of a .
ps1 has been found to be important for the acidification of the lysosomal compartment and thus to be crucial for lysosomal proteolysis and autophagic function . in neurons lacking ps1 ,
the subunit v01a of the vacuolar atpase is not correctly delivered to the lysosomes and not properly integrated into the lysosomal membrane .
this subunit is needed for correct function of the proton pump , which is responsible for lysosomal acidification .
-secretase lacking ps1 is not able to sufficiently facilitate n - glycosylation of the v01a subunit in the er , which is crucial for efficient transport to the lysosomes and assembly of the functional proton pump . as a result , v01a is entrapped in the er and cells loose lysosomal function .
in addition , an accumulation of avs is seen in ps1 hypomorphic mice and the pharmacological block of lysosomal acidification leads to similar accumulation of lysosomes and avs in primary neuronal cell lines .
the first abnormalities of the lysosomal - endosomal system occur prior to other hallmarks of the disease such as amyloid deposits in the neocortex and persist until massive impairments of the autophagic system emerge in the later stages of the disease . taken together
, all these findings suggest an important role for autophagy in the pathogenesis of ad .
there is quite some evidence that in ad , the proteostasis network is impaired . based on pathological analysis , ad mouse models , in vitro and cellular investigations a molecular link between chaperones , the ubiquitin proteasome system , autophagy pathways and pathogenetic mechanisms of ad can be suggested .
deregulation and changes of chaperones and proteasome activity might have serious implications for aging as well as for age - associated diseases .
autophagy pathways are key mechanisms and of vital importance for cellular function and , especially , for cell survival under adverse conditions .
consequently , an effect of proteostasis control on neurodegeneration or , vice versa , of neurodegeneration on proteostasis is reasonable . as with other pathomechanisms that are investigated in the search for the cause of ad , it is still open whether an impairment of proteostasis is an upstream or downstream event during ad onset and progression .
experimental approaches employing mouse models clearly demonstrate that stabilization or induction of proteostasis can be neuroprotective . whether this can be translated into the human condition and , most importantly ,
whether supporting proteome integrity can be a real target for pharmacological intervention for the prevention and treatment of ad is currently still open .
also , one has to consider that , e.g. the stimulation of general autophagy may in the long term lead to the stimulation of proliferation of non - neuronal cells , eventually resulting in tumor formation . indeed , a permanent autophagy induction is known as one cellular mechanism that promotes the escape of cells from proliferation control . as beneficial
it may be to support autophagy in post - mitotic neurons that are confronted with disturbed proteostasis and an impairment of proteasome function , this stimulation should target specific autophagy pathways , such as selective chaperone - mediated macroautophagy involved in the degradation of disease - associated protein aggregation .
although there are obviously still many questions to be answered to understand the role of proteostasis in ad ( and also in other age - associated neurodegenerative disorders ) , it is a big step forward in ad research to consider a possible role of pathogenetic pathways that are not directly linked to the usual suspects a and tau
. a better understanding of how the proteostasis network is regulated might help to identify targets which lead to prevention of the deleterious loss of neuronal cells and tissue in ad .
|
alzheimer s disease ( ad ) is one key medical challenge of the aging society and despite a great amount of effort and a huge collection of acquired data on molecular mechanisms that are associated with the onset and progression of this devastating disorder , no causal therapy is in sight .
the two main hypotheses of ad , the amyloid cascade hypothesis and the tau hypothesis , are still in the focus of ad research . with aging
as the accepted main risk factor of the most important non familial and late onset sporadic forms of ad , it is now mandatory to discuss more intensively aspects of cellular aging and aging biochemistry and its impact on neurodegeneration .
since aging is accompanied by changes in cellular protein homeostasis and an increasing demand for protein degradation , aspects of protein folding , misfolding , refolding and , importantly , protein degradation need to be linked to ad pathogenesis .
this is the purpose of this short review .
|
Introduction
Maintenance of Proteostasis via Molecular Chaperones: the First Line of Defense Against Protein Misfolding
Molecular Chaperones Get in Touch with the Protein Degradation Machineries
Proteostasis and AD
Chaperones and AD
UbiquitinProteasome System and AD
Autophagy and AD
Outlook
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folic acid modification of magnetic nanoparticles could be used to facilitate uptake to specific cancer cells for cancer therapy and diagnosis .
our results showed that the uptake of folic - acid modified nanoparticles by 5rp7 cancer cells was also much higher than that of 3t3 cells .
this modification can be used for successful targeting of cancer cells expressing the folate receptor .
many conventional cancer chemotherapies are ineffective because of an inability to reach the tumor site in effective concentrations.1 there is little doubt that nanoparticles offer new opportunities in many fields.2 nanotechnology is expected to revolutionize medicine .
nanostructures can play a major role in medicine , especially in cancer diagnosis and therapy.3 magnetic nanoparticles have been investigated for various biomedical applications , nanoparticles , and prospected in diagnostic research for magnetic resonance eg , fe3o4 imaging and application of nanotechnologies in medicine.4 magnetic nanoparticles could enhance therapeutic effects and reduce side effects of drugs when used in combination with conventional cancer treatment.5 the combination of fe3o4 magnetic nanoparticles with different chemotherapeutics may provide new strategies in the treatment of specific cancer cells.6 moreover , fe3o4 nanoparticles are the only magnetic nanomaterials approved for clinical use by the us food and drug administration , and the preparation method is relatively simple.7 we aimed to determine whether the anticancer effects of methacrylamido - folic acid ( ma - fol ) would have improved anticancer activity if incorporated into magnetic nanoparticles .
we demonstrated that magnetic fe3o4 nanoparticles coupled with folic acid can inhibit tumor proliferation and induce apoptosis of cancer cells in a dose- and time - dependent manner .
the folate receptor is significantly overexpressed on the surface of human cancer cells.8,9 folate receptor - mediated drug delivery is based on conjugation with folic acid , which is internalized by folate receptor - mediated endocytosis .
folic acid has been immobilized on superparamagnetic particles,10 polymer nanoparticles,11 and incorporated into dendrimer - based therapeutic nanodevices12 for selective targeting of tumor cells .
folate receptors exhibit limited expression on healthy cells , but are often present in large numbers on cancer cells.13 folic acid receptors are overexpressed by epithelial cancers in the ovary , mammary gland , colon , lung , prostate , nose , throat , and brain,14 so represent an important target for tumor - specific delivery of anticancer drugs .
apoptosis , or programmed cell death , is an active process characterized by cytoplasmic shrinkage , chromatin condensation , nuclear fragmentation , and activation of caspases.13 in addition , phosphatidylserine is exposed on the external surface of the cell in the early phase of apoptosis , and this exposure precedes membrane damage and dna fragmentation.15 on the other hand , necrosis is passive , and is characterized by cell swelling , rupture of the plasma membrane , and cell lysis , with leakage of cytoplasmic components , such as lactate dehydrogenase.13 in the present study , folic acid was coupled on the surface of fe3o4 for selective binding to cancer cells and immobilized on the surfaces of magnetic nanoparticles , to disperse particles and improve their cell internalization and target cancer cells , respectively .
further , the apoptotic effects of ma - fol - modified fe3o4 nanoparticles were determined in a 5rp7 ( h - ras - transformed rat embryonic fibroblasts ) and in a nih/3t3 control cell line ( normal mouse embryonic fibroblasts ) by flow cytometry and transmission electron microscopy ( tem ) .
nanoparticles are generally internalized into cells via fluid - phase endocytosis,16,17 receptor - mediated endocytosis , or phagocytosis .
one strategy to realize efficient and specific cellular uptake of nanoparticles is to modify the nanoparticle surface with a ligand that is efficiently taken up by target cells via receptor - mediated endocytosis.18 the objective of this research was to assess the potential effects of fe3o4 magnetic nanoparticles modified with ma - fol on 5rp7 cancer cells and nih/3t3 cells .
ph was set to 910 by addition of 1 m naoh solution to obtain anionic folate in solution .
a solution of methacryloyl benzotriazole19,20 in dioxane ( 15 ml ) was added to the folate solution .
completion of the reaction was monitored by thin layer chromatography , which showed free 1h - benzotriazole spot .
reaction mixture was extracted with ethyl acetate ( 3 20 ml ) to remove 1h - benzotriazole .
water was evaporated under vacuum to get ma - fol as yellow microcrystals in an 85% yield .
h nuclear magnetic resonance ( nmr , 500 mhz , dmso - d6 ) was as follows : 8.56 ( s , 1 h ) , 7.59 ( d , jhh = 8.80 hz , 1 h ) , 6.65 ( d , jhh = 8.80 hz , 1 h ) , 5.565.53 ( m , 1 h ) , 5.035.00 ( m , 1 h ) , 4.45 ( s , 2 h ) , 4.05 ( dd , 3 jhh = 5.05 , 7.0 hz , 1 h ) , 2.252.15 ( m , 2 h ) , 2.101.90 ( m , 2 h ) , 1.96 ( s , 3 h ) ppm .
c nmr ( 125 mhz , dmso - d6 ) : 174.0 , 173.9 , 167.2 , 167 , 164.9 , 164.2 , 153.1 , 151.9 , 151.4 , 149.4 , 141.4 , 129.5 , 126.7 , 118.7 , 112.8 , 110.6 , 53.9 , 45.7 , 30.1 , 27.9 , 18.7 ppm .
superparamagnetic iron oxides ( size 2050 nm ) , were obtained from sigma - aldrich chemical co ( st . louis , mo ) .
the surfaces of 0.1 g iron nanoparticles were modified in 2.5 ml toluene , adding 0.5 ml trimethoxysilyl propyl methacrylate .
these silylated superparamagnetic iron oxide nanoparticles were mixed with a 5 mg / ml molality of 1 ml ma - fol in 1 ml dimethyl sulfoxide ( sigma - aldrich chemical co ) .
5rp7 ( h - ras - transformed rat embryonic fibroblasts ) and nih/3t3 ( a control cell line of normal mouse embryonic fibroblasts ) were used in these experiments .
cells were routinely cultured at 37c in a humidified atmosphere with 5% co2 in 25 cm flasks containing 5 ml of dulbecco s modified eagle s medium , supplemented with 10% fetal bovine serum , 2 mm l - glutamine , 50 iu / ml penicillin , and 50 mg / ml streptomycin .
, the cells were washed twice with phosphate - buffered saline and incubated with trypsin - ethylenediamine tetra - acetic acid solution ( 0.25% trypsin , 1 mm ethylenediamine tetra - acetic acid ) for 2 minutes at 37c to detach the cells , and the complete media were then added into the flask at room temperature to inhibit the effect of trypsin .
the cells were washed twice by centrifugation and resuspended in the complete media for reseeding and g rowing in new culture flasks .
cell viability was determined through staining with trypan blue , and cells were counted using a hemocytometer . to study the cellular uptake of the nanoparticles by flow cytometry and tem ,
ma - fol - modified magnetic nanoparticles were added to the cell culture media at concentrations of 2.5 g / ml , 4.5 g / ml , and 9 g / ml .
the cells were cultured and then reseeded with the nano - particle - dispersed culture media . after 24 and 48 hours of incubation at 37c
, the cells were washed twice with phosphate - buffered saline , detached using trypsin - ethyl - enediamine tetra - acetic acid solution , and resuspended in culture media . in control cultures ,
the cells were placed in 5 ml of medium without magnetic nanoparticles at the same cell density .
annexin v - fluorescein isothiocyanate ( fitc ) is a sensitive probe for identifying apoptotic cells .
it binds to negatively charged phospholipid surfaces with a higher specificity for phosphatidylserine , a membrane phospholipid , than for most other phospholipids.21 in apoptotic cells , phosphatidylserine is translocated from the inner leaflet of the plasma membrane to the outer leaflet , thereby exposing phosphatidylserine to the exter nal environment.22 a facsaria ( bd cor poration , bedford , ma ) flow cytometer was used for analysis of the cells . a recent report nanoparticles by cells suggested that the uptake of fe3o4 could be quantitatively measured using flow cytometric light scatter.6 annexin v binding was performed using an annexinv - fitc kit ( bd corporation ) as described by the manufacturer .
cells were washed twice with cold phosphate - buffered saline and were then resuspended in 1 binding buffer at a concentration of 1 10 cells / ml , after which 100 l of solution ( 1 10 cells ) was transferred to a 5 ml culture tube .
annexin v - fitc 5 l and propidium iodide 5 l were added , and the cells were then incubated for 15 minutes at room temperature in the dark , after which 400 l of 1 binding buffer was added to each tube and analyzed in the facsaria .
uptake of nanoparticles modified with folic acid by nih/3t3 and 5rp7 cancer cells , as well as nanoparticle size and morphology , were determined using a tem ( fei tecnai biotwin ) .
cells were deposited on formvar - coated 200300 mesh copper grids and dried , fixed with 2.5% glutaraldehyde in 0.1 m phosphate buffer ( ph 7.4 ) , and left in phosphate - buffered saline overnight at 4c . after being embedded in agar and after fixation in 2% osmium tetroxide ,
the cells were dehydrated in graded ethanol , ie , 70 , 90 , 96 , and 100% .
they were thin - sectioned using a diamond knife to a maximum thickness of 100 nm .
ph was set to 910 by addition of 1 m naoh solution to obtain anionic folate in solution .
a solution of methacryloyl benzotriazole19,20 in dioxane ( 15 ml ) was added to the folate solution .
completion of the reaction was monitored by thin layer chromatography , which showed free 1h - benzotriazole spot .
reaction mixture was extracted with ethyl acetate ( 3 20 ml ) to remove 1h - benzotriazole .
water was evaporated under vacuum to get ma - fol as yellow microcrystals in an 85% yield .
h nuclear magnetic resonance ( nmr , 500 mhz , dmso - d6 ) was as follows : 8.56 ( s , 1 h ) , 7.59 ( d , jhh = 8.80 hz , 1 h ) , 6.65 ( d , jhh = 8.80 hz , 1 h ) , 5.565.53 ( m , 1 h ) , 5.035.00 ( m , 1 h ) , 4.45 ( s , 2 h ) , 4.05 ( dd , 3 jhh = 5.05 , 7.0 hz , 1 h ) , 2.252.15 ( m , 2 h ) , 2.101.90 ( m , 2 h ) , 1.96 ( s , 3 h ) ppm .
c nmr ( 125 mhz , dmso - d6 ) : 174.0 , 173.9 , 167.2 , 167 , 164.9 , 164.2 , 153.1 , 151.9 , 151.4 , 149.4 , 141.4 , 129.5 , 126.7 , 118.7 , 112.8 , 110.6 , 53.9 , 45.7 , 30.1 , 27.9 , 18.7 ppm .
superparamagnetic iron oxides ( size 2050 nm ) , were obtained from sigma - aldrich chemical co ( st . louis , mo ) .
the surfaces of 0.1 g iron nanoparticles were modified in 2.5 ml toluene , adding 0.5 ml trimethoxysilyl propyl methacrylate .
these silylated superparamagnetic iron oxide nanoparticles were mixed with a 5 mg / ml molality of 1 ml ma - fol in 1 ml dimethyl sulfoxide ( sigma - aldrich chemical co ) .
5rp7 ( h - ras - transformed rat embryonic fibroblasts ) and nih/3t3 ( a control cell line of normal mouse embryonic fibroblasts ) were used in these experiments .
cells were routinely cultured at 37c in a humidified atmosphere with 5% co2 in 25 cm flasks containing 5 ml of dulbecco s modified eagle s medium , supplemented with 10% fetal bovine serum , 2 mm l - glutamine , 50 iu / ml penicillin , and 50 mg / ml streptomycin .
, the cells were washed twice with phosphate - buffered saline and incubated with trypsin - ethylenediamine tetra - acetic acid solution ( 0.25% trypsin , 1 mm ethylenediamine tetra - acetic acid ) for 2 minutes at 37c to detach the cells , and the complete media were then added into the flask at room temperature to inhibit the effect of trypsin .
the cells were washed twice by centrifugation and resuspended in the complete media for reseeding and g rowing in new culture flasks .
cell viability was determined through staining with trypan blue , and cells were counted using a hemocytometer . to study the cellular uptake of the nanoparticles by flow cytometry and tem
, ma - fol - modified magnetic nanoparticles were added to the cell culture media at concentrations of 2.5 g / ml , 4.5 g / ml , and 9 g / ml .
the cells were cultured and then reseeded with the nano - particle - dispersed culture media .
after 24 and 48 hours of incubation at 37c , the cells were washed twice with phosphate - buffered saline , detached using trypsin - ethyl - enediamine tetra - acetic acid solution , and resuspended in culture media . in control cultures ,
the cells were placed in 5 ml of medium without magnetic nanoparticles at the same cell density .
annexin v - fluorescein isothiocyanate ( fitc ) is a sensitive probe for identifying apoptotic cells .
it binds to negatively charged phospholipid surfaces with a higher specificity for phosphatidylserine , a membrane phospholipid , than for most other phospholipids.21 in apoptotic cells , phosphatidylserine is translocated from the inner leaflet of the plasma membrane to the outer leaflet , thereby exposing phosphatidylserine to the exter nal environment.22 a facsaria ( bd cor poration , bedford , ma ) flow cytometer was used for analysis of the cells
. a recent report nanoparticles by cells suggested that the uptake of fe3o4 could be quantitatively measured using flow cytometric light scatter.6 annexin v binding was performed using an annexinv - fitc kit ( bd corporation ) as described by the manufacturer .
cells were washed twice with cold phosphate - buffered saline and were then resuspended in 1 binding buffer at a concentration of 1 10 cells / ml , after which 100 l of solution ( 1 10 cells ) was transferred to a 5 ml culture tube .
annexin v - fitc 5 l and propidium iodide 5 l were added , and the cells were then incubated for 15 minutes at room temperature in the dark , after which 400 l of 1 binding buffer was added to each tube and analyzed in the facsaria .
uptake of nanoparticles modified with folic acid by nih/3t3 and 5rp7 cancer cells , as well as nanoparticle size and morphology , were determined using a tem ( fei tecnai biotwin ) .
cells were deposited on formvar - coated 200300 mesh copper grids and dried , fixed with 2.5% glutaraldehyde in 0.1 m phosphate buffer ( ph 7.4 ) , and left in phosphate - buffered saline overnight at 4c . after being embedded in agar and after fixation in 2% osmium tetroxide ,
the cells were dehydrated in graded ethanol , ie , 70 , 90 , 96 , and 100% .
they were thin - sectioned using a diamond knife to a maximum thickness of 100 nm .
a logical method to promote internalization of nanoparticles is to modify their surface with a ligand such as folic acid which can be efficiently taken up by cells through receptor - mediated endocytosis.23 folic acid binds to the folate receptors at cell surfaces with very high affinity and is internalized by receptor - mediated endocytosis.24,26 more importantly , folic acid is stable , poorly immunogenic , and has the ability to target cancer cells preferentially because the folate receptor is frequently overexpressed on the surface of cancer cells .
flow cytometry is a process whereby physical or biochemical parameters of single biological cells or particles are measured as the cells move through a fluidic channel.27 the annexin v binding assay provides a very specific , rapid , and reliable technique to detect apoptosis by flow cytometry or by fluorescence microscopy.28,29 fitc is a very useful fluorescent moiety that can be used to label essentially any protein .
fitc - conjugated annexin v is used to detect apoptotic cells in a diverse range of cell types and in response to many different proapoptotic stimuli.28,29 iron nanoparticles were successfully modified with ma - fol as described in the methods section .
ma - fol was used to target preferentially cancer cells with folate receptors expressed on their surfaces , and to facilitate the nanoparticles to transit across the cell membrane .
annexin v - fitc allows detection of cell surface changes that occur early during apoptosis , because annexin v binds to phosphatidylserine which becomes exposed on the outer surface of the plasma membranes by flipping from the inner side of the membrane.22,30,31 exposure of phosphatidylserine during apoptosis precedes nuclear changes .
this change in membrane surface is important for macrophages to recognize cells undergoing apoptosis.30,32 annexin v , an anticoagulant protein , binds to phosphatidylserine with high affinity .
thus , by staining cells with annexin v - fitc and additionally with dna - specific fluorochrome , eg , propidium iodide , it is possible to identify live cells , early apoptotic cells , as well as late apoptotic and necrotic cells , by flow cytometry.22,33 subsequently , annexin v - fitc binds to cells expressing phosphatidylserine , an early marker of apoptosis on the cell surface .
target cells are gated upon as propidium iodide - negative and quantified with respect to their annexin v positivity .
the shift from annexin v to annexin v is a discrete event , such that all target cells fall within discernible populations with respect to annexin v. dead or dying cells are then stained with annexin v - fitc.34 annexin v has been used to detect apoptotic cells in a wide variety of cell types .
externalization of phosphatidylserine has been demonstrated in plasma membrane permeability changes , as measured by propidium iodide uptake and annexin v binding , and precedes the morphological features of apoptosis as assessed by flow cytometric analysis of cell shrinkage.29,34 the sensitivity and early kinetics of annexin v binding make it an ideal marker for cell death in a flow cytometric assay.34 analysis of annexin v / propidium iodide - stained cells by flow cytometry allows quantitation of the fraction of cells that are annexin v - negative and propidium iodide - negative ( double negative ) , annexin v - positive and propidium iodide - negative ( single positive ) , or annexin v - positive and propidium iodide - positive ( double positive).28 a number of detectors are aimed at the point where the stream passes through the light beam , ie , one in line with the light beam ( forward scatter ) and several perpendicular to it ( side scatter ) and one or more fluorescent detectors . forward
scatter correlates with the cell volume , and side scatter depends on the inner complexity of the particle ( ie , shape of the nucleus , amount and type of cytoplasmic granules , or membrane roughness).35 data analysis was performed with diva software ( bd facs diva software 6.0 ; bd corporation , bedford , ma ) .
apoptosis was quantitatively confirmed by analyzing the percentage of early apoptotic cells using annexin v - fitc / propidium iodide double staining .
a marker of early apoptosis , measured by annexin - v , is phosphatidylserine , which is released as a result of redistribution of the plasma membrane of the cells.22 three main populations of cells were distributed in dot - plots for viable cells q3 ( annexin v - negative / propidium iodide - negative ) , early apoptotic cells q4 ( annexin v - positive / propidium iodide - negative ) , and late apoptotic and necrotic cells q2
the cytotoxicity of folic acid - modified nanoparticles was determined by exposing normal cells and cancer cells to various concentrations in dulbecco s modified eagle s medium for 24 and 48 hours .
the results and respective percentages of cells in apoptotic regions for both nih/3t3 and 5rp7 cells are given in figures 1 and 2 .
the results indicate that there was no significant loss of cell survival if the incubated concentration of ma - fol - modified nanoparticles was 4.5 g / ml or below in cancer cells .
as the concentration increased to 9 g / ml , the rate of viability was close to that of normal cells .
these results confirm that the cells are saturated with ma - fol - modified nanoparticles at a concentration of 4.5 g / ml . up to this concentration
figure 2 shows cell death due to the ma - fol - modified magnetic nanoparticles at 4.5 g / ml for 24 hours in cancer cells .
when cells were exposed to a nanoparticle concentration of 4.5 g / ml for 24 hours , cell survival decreased to 83% . when incubation time was extended to 48 hours
as shown in figure 2 , folic acid - modified nanoparticles caused worse damage to cancer cells than to normal cells .
the population of annexin v - positive live cells increased in a time - dependent manner , and most ( 12.9% ) of the cells became annexin v - positive at 48 hours ( figure 2 ) .
when cancer cells were incubated with modified magnetic ma - fol nanoparticles for 48 hours ( 9 g / ml ) , the population of annexin v - positive live cells was found to be 7.3% ( figures 2a and 2b ) .
cells in the late stages of apoptosis are located in the bottom right quadrant of the dot - plot as double positive annexin v - fitc- and propidium iodide - binding cells because , at this stage , the cell membranes are damaged and propidium iodide has penetrated into the cell . in the later stages of apoptosis , propidium iodide enters the cell , leading to a double positive population.28 cells undergoing necrotic cell death also stain as double positive for annexin v and propidium iodide , as discussed earlier .
flow cytometry by this double staining method enables clear detection of three populations of cells ( viable , apoptotic , and necrotic ) .
thus , the early apoptotic cells bind only to annexin - v fitc , and late apoptotic cells to both annexin v - fitc and propidium iodide , and viable cells do not take up any of the dye . the annexin v - fitc
/ propidium iodide population was considered to reflect normal healthy cells , whereas annexin v - fitc - positive / propidium iodide - negative cells were taken to show early apoptosis .
annexin v - fitc -positive / propidium iodide - negative cells were in late apoptosis or necrosis.35 our flow cytometric analysis has shown that48 hours of treatment with ma - fol - modified magnetic nanoparticles caused apoptosis in a 5rp7 cell line in a concentration - dependent manner .
ma - fol - modified magnetic nanoparticles at a dose of 4.5 g / ml showed 12.9% cells in the apoptotic zone in the case of nih/3t3 cells , and the same concentration produced apoptosis in 4.7% of cells in dot - plot analysis .
late apoptosis and necrosis increased steadily following the increase at a concentration of 4.5 g / ml . flow cytometric determination of cell cycle phase distribution has further confirmed that folic acid - modified magnetic nanoparticles cause cell cycle deformation in a cancer cell line .
the results of this study confirm that cancer cells have more folate receptors , and that a 4.5 g / ml concentration of folic acid - modified magnetic nanoparticles causes apoptosis in 5rp7 cells .
the uptake of folic acid - modified nanoparticles by cancer cells was also much higher than that of normal cells .
this indicates that folic acid modification not only facilitates the nanoparticles to target specific cells , but also increases the yield of cell internalization .
the interaction between folic acid and folate receptors expressed on the surface of cancer cells might have contributed to the improvement of nanoparticle uptake , based on receptor - mediated endocytosis.37 the schemes for the modification of nanoparticles display tem images ( figure 3 ) of ma - fol - modified with fe3o4 nanoparticles for this purpose , firstly , magnetic nanoparticles were imaged .
figure 4 shows the tem images of nih/3t3 cells not treated with ma - fol - modified magnetic nanoparticles .
figure 5 shows the uptake of folic acid - modified nanoparticles into normal cells at 24 hours and 48 hours .
the viability of normal cells after each incubation time with the nanoparticles was close to that of control cells , and was in the 95%96% range in flow cytometric analysis .
the uptake of magnetic nanoparticles by cancerous rat 5rp7 fibroblasts is shown in figure 7 .
after 48 hours of culture in media containing folic acid - modified nanoparticles , the morphology and viability of normal fibroblast cells containing the modified nanoparticles were close to that of the control cells , suggesting biocompatibility of the nanoparticles .
the tem and flow cytometry results showed that folic acid - modified nanoparticles were internalized into both normal cells and cancer cells .
the immobilization of folic acid on the nanoparticles was demonstrated by increasing the amount of nanoparticle uptake into cancer cells in comparison with normal cells .
this suggests that the modification of magnetic nanoparticles with ma - fol could be used to resist nonspecific uptake and thus avoid their recognition by macrophage cells , and simultaneously facilitate nanoparticle uptake to specific cancer cells for cancer therapy . in flow cytometry results , the population of annexin v - positive live cells in the cancer cell line increased in a time - dependent manner , and most ( 12.9% ) of the cells became annexin v - positive at 48 hours at a concentration of 4.5 g / ml .
the results indicate that there was no significant loss of cell survival if the incubated concentration of ma - fol modified nanoparticles is 4.5 g / ml or below in cancer cells .
as the concentration increased to 9 g / ml , the rate of viability was close to that of normal cells .
these results confirm that cells saturated with folic acid - modified nanoparticles are detected in the 4.5 g / ml concentration .
up to this concentration , magnetic nanoparticles were aggregated and had difficulty penetrating the cells . in tem analysis ,
the concentration of 4.5 g / ml was used to compare the cancer cells and normal cells at 24 and 48 hours . in normal cells , organelles and cell structures
were not affected by the presence of ma - fol - modified nanoparticles inside the cells ( figure 5 ) . however , cancer cells treated with the 4.5 g / ml concentration of ma - fol - modified nanoparticles underwent apoptosis .
a representative picture of intracellular nanoconjugate uptake by nih/3t3 cells during a 24-hour and 48-hour treatment is shown in figures 5 and 6 .
the amount of intracellular uptake of the nanoparticles can be seen in normal cells , whereas in the 5rp7 cell line the amount of the nanoconjugate was increased and cell modifications were damaged , figure 7 clearly illustrates the internalization of ma - fol - modified nanoparticles and their localization near the nucleus and a great number of apoptotic particles .
this picture shows clearly that nanoconjugate uptake was maximum , with 5rp7 cells having maximum receptor expression , and hence the most intense color of the pellet , whereas the uptake gradually decreased with reduction in folate receptor expression and , accordingly , the intensity of the color of the pellet in the case of nih/3t3 .
the picture confirms our hypothesis about the targeting efficacy of nanoconjugates containing folic acid , ie , that a positive correlation exists between maximum folate receptor expression and maximum uptake of nanoconjugates .
the 5rp7 cells with 90% receptor expression showed maximum uptake , and the nih/3t3 cells showed the least .
this is further substantiated by tem and flow cytometry analysis , confirming that folic acid - modified magnetic nano - particles can be used as a targeting agent .
ma - fol - modified magnetic nanoparticles represent a potential novel delivery system for compounds with anticancer activity because the folate receptor is frequently overexpressed in cancer cells .
moreover , it may increase our ability to target drugs to tumor cells , protect the drug from in vivo degradation , and reduce drug toxicity .
this modification containing folic acid can be used for successful targeting of tumor cells expressing the folate receptor .
future studies will focus on determining the stability and targeting efficacy of this modification in vivo .
this study has important implications in cancer cell imaging , tumor ablation , and drug delivery because of its targeting efficacy .
furthermore , it needs to be investigated whether nanoparticles could cause long - term changes in systemic activity .
however , the effect and usage of such combinations need to be investigated further in in vivo experiments .
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background : magnetic nanoparticles show great promise for use as tools in a wide variety of biomedical applications .
the purpose of this study was to investigate the potential effects of methacrylamido - folic acid ( ma - fol)-modified magnetic nanoparticles on 5rp7 ( h - ras - transformed rat embryonic fibroblasts ) and nih/3t3 ( normal mouse embryonic fibroblasts).methods : the cytotoxicity and viability of 5rp7 and nih/3t3 cells were detected .
the percentage of cells undergoing apoptosis was analyzed by flow cytometry using annexin v - fluorescein isothiocyanate staining .
nanoparticle internalization into 5rp7 and nih/3t3 cells was visualized by transmission electron microscopy.conclusion:in this study , folic acid coupled to the surface of iron oxide for selective binding to cancer cells and immobilized the surfaces of magnetic nanoparticles
. this complex improves cell internalization and targeting of cancer cells .
we detected increased apoptosis using flow cytometry and transmission electron microscopy.results:folic acid modification of magnetic nanoparticles could be used to facilitate uptake to specific cancer cells for cancer therapy and diagnosis .
our results showed that the uptake of folic - acid modified nanoparticles by 5rp7 cancer cells was also much higher than that of 3t3 cells .
this modification can be used for successful targeting of cancer cells expressing the folate receptor .
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Results:
Introduction
Materials and methods
Synthesis and characterization of magnetic nanoparticles with Ma-Fol
Cell culture
Flow cytometry
Transmission electron microscopic analysis
Results and discussion
Conclusion
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in modern society , health is a n important component , which requires sustained development ( 1 ) . in recent decades
, a person s health has become one of the most important human rights among social groups throughout the world ( 2 ) .
the meaning of health has been extended beyond borders , and different definitions have emerged , owing to diverse geographic regions and cultural views in societies ( 3 ) .
the definition of health may vary by cultural background , and may influence health practices and compliance to medical treatment ( 4 ) .
individuals perceive health on the basis of their experiences of ill health ( 5 ) .
so , understanding health as a subjective experience can enable health care providers to manage the care giving procedure based on their clients needs .
furthermore , one of the most important aspects with regard to health perception , manifests when patients suffer from a chronic disease , through which their perception of health is affected ( 5 , 6 ) .
diabetes is incurable , and has the third highest mortality rate on the planet ( 8) . the probability of the incidence and prevalence of this disease has increased recently ( 9 ) . in 2004 , it was estimated that by 2030 , this number will have grown to 366 million ( 10 ) and in 2010 , the number of people afflicted by diabetes was estimated to increase up to 439 million by 2030 ( 8) .
current estimates indicate that the number of diabetic patients in iran is around 3.5 million out of a total population of just over 68 million ( 11 ) .
previous studies have demonstrated that chronic illnesses such as diabetes can influence a person s perception of health ( 12 ) .
in addition , the attitudes and beliefs regarding health undoubtedly play an important role in promoting healthy behaviour ( 13 ) .
therefore , the understanding of diabetic patients perceptions can help the development of precautionary programmes for diabetics ( 14 ) .
although the concept of health is gaining more importance and it has constantly been improved and changed by the passage of time ( 15 , 16 ) , few studies are reported to have focused on health and illness , particularly with regard to diabetic patients belonging to different ethnic groups ( 17 ) .
health is a relative concept ; nonetheless , several criteria have been proposed for exploring it through various ethnic and cultural groups ( 2 , 18 ) .
prior studies have shown lack of awareness about the concept of health among iranian kurdish diabetic patients .
kurds are an ethnic group living in the middle east , particularly in iran , who are estimated to be the third largest ethnic group , constituting 9 percent of the total population ( 19 ) .
consequently , investigating the perception of health - related meaning among this ethnic group can increase the knowledge about the nature of health and its effects .
therefore , this study explored the meaning of health as perceived by iranian kurdish diabetic patients in order to develop an appropriate multidisciplinary outlook toward this ethnicity of patients .
considering the aim of this research , a qualitative approach with a conventional content analysis approach was used .
content analysis is a unique qualitative method , which encompasses a host of analytic approaches that offer flexibility to a researcher , and are of theoretical and substantive interest with regards to the case being studied ( 20 , 21 ) .
the research was conducted over a ten - month period during 2014 , in the diabetes care centre of tohid hospital at the kurdistan university of medical sciences in sanandaj , in the center of the kurdistan province in western - iran .
a purposive sampling method was used to select the candidates for the study from among the diabetic patients who were admitted to the centre .
the inclusion criteria for the participants were : having kurdish ethnicity irrespective of gender , having diabetes type 2 , while at the same time having a lapse of at least five years since the diagnosis of diabetes , the absence of psychiatric disorders , and finally , having the ability to communicate .
the sample size was determined only when the interviewing stage reached saturation ( 22 ) .
the sample was restricted to 20 because no new data emerged after the twentieth interview .
therefore , the researcher was completely saturated with data to achieve a new understanding and insight about the phenomenon . in - depth , face - to - face , semi - structured interviews were used for data collection .
the participants were encouraged to present their ideas extensively and provide further explanations and details if their narratives were unclear . moreover ,
an interview guideline was defined to direct the interviews toward the aims of the study .
the interviews began with a general question about health and its perception , and proceeded to seek a deep understanding of participants perception .
examples of questions asked during the interview were as follows : when do you feel healthy ?
further , the patients were asked to describe all the factors that could affect their health .
twenty interviews were conducted in kurdish language ; each interview was tape - recorded with the participants permission .
each interview spanned 4090 minutes ( average duration : 60 minutes ) . each interview was transcribed and then used as a guide for the other interviews .
two of the author s colleagues checked the accuracy of the transcriptions by matching the corresponding transcripts with the tape - recorded version of the interviews .
the kurdish transcripts were analysed and only the verbatim quotations intended for publication were translated into english .
the researchers attempted to translate the participants descriptions in a manner in which they closely corresponded to their meaning in kurdish , while making them grammatically correct for english readers . through these inquiries ,
the researchers tried to use a set of measures for increasing the rigor of the data and thereby increase the scientific precision of the results , including : the allocation of a proper place and sufficient time for data collection , due communication with participants , using the supplementary views of colleagues , reviewing the participants hand writing and examining the data by all engaged researchers .
this study was approved by the research committee of the kurdistan university of medical sciences , no.572.14 .
this study was approved by the code of ethics of the ethics committee of kurdistan university of medical sciences , no : muk.rec.1387.572.14 .
all participants were volunteers and written consent was obtained from each of them in which the voluntary nature of the participation was mentioned .
the participants were assured that they could leave the study at any time even after they had signed the consent form .
they were assured that their care would not be affected if they chose not to participate in the study .
they were also assured of the data confidentiality ; this meant that their names and other significant details , that might reveal their identity , would not be published in the study report .
all the participants names were changed into codes during the transcription of the interviews , data was locked in separate locations and the coded information was used for data analysis and discussions . in this study ,
the conventional content analysis was used for data analysis ; this method is generally adopted with a study design which aims to describe a particular phenomenon .
this type of design is usually appropriate when the existing theory or research literature on a phenomenon is limited ( 20 ) .
the researchers defined the categories on the basis of the type of data and avoided using predetermined categories .
this method is also described as inductive category development ( 23 ) , where researchers do not use predetermined data groups .
researchers analysed all the data in order to gain a new insight and expand their knowledge about it . by analyzing the literature that they had collected on the participants , and reading the transcriptions of all the participants interviews word by word ,
one of the advantages of this approach is that the results of the participants responses in the study can be obtained without imposing preconceived categories or theoretical perspectives ( 20 , 24 ) .
considering the aim of this research , a qualitative approach with a conventional content analysis approach was used .
content analysis is a unique qualitative method , which encompasses a host of analytic approaches that offer flexibility to a researcher , and are of theoretical and substantive interest with regards to the case being studied ( 20 , 21 ) .
the research was conducted over a ten - month period during 2014 , in the diabetes care centre of tohid hospital at the kurdistan university of medical sciences in sanandaj , in the center of the kurdistan province in western - iran .
a purposive sampling method was used to select the candidates for the study from among the diabetic patients who were admitted to the centre .
the inclusion criteria for the participants were : having kurdish ethnicity irrespective of gender , having diabetes type 2 , while at the same time having a lapse of at least five years since the diagnosis of diabetes , the absence of psychiatric disorders , and finally , having the ability to communicate .
the sample size was determined only when the interviewing stage reached saturation ( 22 ) .
the sample was restricted to 20 because no new data emerged after the twentieth interview .
therefore , the researcher was completely saturated with data to achieve a new understanding and insight about the phenomenon .
in - depth , face - to - face , semi - structured interviews were used for data collection .
the participants were encouraged to present their ideas extensively and provide further explanations and details if their narratives were unclear . moreover ,
an interview guideline was defined to direct the interviews toward the aims of the study .
the interviews began with a general question about health and its perception , and proceeded to seek a deep understanding of participants perception .
examples of questions asked during the interview were as follows : when do you feel healthy ?
further , the patients were asked to describe all the factors that could affect their health .
twenty interviews were conducted in kurdish language ; each interview was tape - recorded with the participants permission .
two of the author s colleagues checked the accuracy of the transcriptions by matching the corresponding transcripts with the tape - recorded version of the interviews .
the kurdish transcripts were analysed and only the verbatim quotations intended for publication were translated into english .
the researchers attempted to translate the participants descriptions in a manner in which they closely corresponded to their meaning in kurdish , while making them grammatically correct for english readers .
through these inquiries , the researchers tried to use a set of measures for increasing the rigor of the data and thereby increase the scientific precision of the results , including : the allocation of a proper place and sufficient time for data collection , due communication with participants , using the supplementary views of colleagues , reviewing the participants hand writing and examining the data by all engaged researchers .
this study was approved by the research committee of the kurdistan university of medical sciences , no.572.14 .
this study was approved by the code of ethics of the ethics committee of kurdistan university of medical sciences , no : muk.rec.1387.572.14 .
all participants were volunteers and written consent was obtained from each of them in which the voluntary nature of the participation was mentioned .
the participants were assured that they could leave the study at any time even after they had signed the consent form .
they were assured that their care would not be affected if they chose not to participate in the study .
they were also assured of the data confidentiality ; this meant that their names and other significant details , that might reveal their identity , would not be published in the study report .
all the participants names were changed into codes during the transcription of the interviews , data was locked in separate locations and the coded information was used for data analysis and discussions .
in this study , the conventional content analysis was used for data analysis ; this method is generally adopted with a study design which aims to describe a particular phenomenon .
this type of design is usually appropriate when the existing theory or research literature on a phenomenon is limited ( 20 ) .
the researchers defined the categories on the basis of the type of data and avoided using predetermined categories .
this method is also described as inductive category development ( 23 ) , where researchers do not use predetermined data groups .
researchers analysed all the data in order to gain a new insight and expand their knowledge about it . by analyzing the literature that they had collected on the participants , and reading the transcriptions of all the participants interviews word by word ,
one of the advantages of this approach is that the results of the participants responses in the study can be obtained without imposing preconceived categories or theoretical perspectives ( 20 , 24 ) .
twenty diabetic patients participated in this study ; the participants included seven men with an age range from 53 to 80 years ( average age : 68 years ) and thirteen women with an age range from 32 to 55 ( average age : 40 years ) .
meanwhile , the average number of years which had lapsed since diagnosis of diabetes was seven years with a range of 517 years . based on the thoughts and feelings of the participants , the following three main themes emerged : the syndrome of the healthy body and the happy heart life without compulsory limitations the syndrome of the healthy body and the happy heart is an important theme that emerged in our study .
this theme is a kurdish idiom concerning health . according to this theme ( bivy bi ) , health marks the presence of particular characteristics , which is an established symbol in the history of kurdish literature .
it refers to the ancient kurdish description about health as being associated with not only physical well - being but also vivacity , satisfaction , and calmness of the mind .
the syndrome of the healthy body and the happy heart is an accepted concept among the laymen and is considered as an ideal and a prayer for every person .
similarly , the participants wished good health to each other with the idea that health implies a healthy body and a contented heart .
one of the participants in the study said , a person with a healthy and contented heart is well - behaved and kind to others ( p1 ) .
the participants always used local terminology to describe their ideas of health , and their descriptions and perception of health indicated that health is tantamount to worshipping god , for them .
another participant said , health reflects a healthy and happy heart ; physical well - being holds less importance as compared to the human spirit , and , both , the heart and the spirit need to be healthy
the participants mentioned that health incorporates several specific factors like calmness and happiness without any worries ; sadness , or any family crises .
health means that you have achieved peace in your family and home , and as a member of your family , you have exhibited good behaviour .
health means that the child and his or her parents share a cordial relationship , and life is a gift from god that ensures calmness and safety ( p 11 ) .
another participant said , health means that we do not have any problems or complaints of illness
( p6 ) , and , health brings calmness , and it is like praying to god without having to think about stressful matters (p2 ) , and , when you are healthy , you do nt have any complaints and anxiety , and so the few problems that people have to face seem petty when they have the support of their families .
so , the complaints gradually go away ( p 18 ) all the participants mentioned that life without any compulsory limitations is one of the important dimensions of health and that they wished they could eat all kinds of food and participate in any occasion and be like others .
one of the women ( 43 years old ) said , when i eat rice , i need more water , and then i know that i am not healthy but fatigued , and i feel like travelling to the mountains and running away for a longer time , and then i suffer from sleeplessness and my body feels weak (
sometimes , i want to eat some comestibles that our family bought between us , but i can not do that , and this is so hard for me
when you can participate in a ceremony or accompany someone , do anything that you want , or eat anything that you like , you feel a sense of freedom from others and you can go anywhere without worrying about your medicines , this freedom is very fantastic
, spirituality is subjective ; the individual and personal concept that each person defines is based on his or her understanding and on the evidence that spirituality improves mental and physical health .
participants emphasis on achieving exalted spirituality in their lives was tantamount to health for them .
most of the participants primarily belonged to the category that perceived health as exalted spirituality , and believed in satisfying self and others and trusting and honoring god . a woman ( 52 years old ) said , some of the people whose heart is near to god have good health , because honoring god helps to heal them , and when the spirit is healthy , the body is healthy . and
, a healthy spirit in a healthy body , which believes in prayer and spirituality , finds calmness and peace
( p 18 ) , and , healthy patients who have the virtue to trust in god and are honest , do nt lose their spirituality , religion or prophet (p 11 ) another participant said , when i pray to god , i think about how fortunate i am to be born in this world , and then i am happy (p 6 ) .
the syndrome of the healthy body and the happy heart is an important theme that emerged in our study .
this theme is a kurdish idiom concerning health . according to this theme ( bivy bi ) , health marks the presence of particular characteristics , which is an established symbol in the history of kurdish literature .
it refers to the ancient kurdish description about health as being associated with not only physical well - being but also vivacity , satisfaction , and calmness of the mind .
the syndrome of the healthy body and the happy heart is an accepted concept among the laymen and is considered as an ideal and a prayer for every person .
similarly , the participants wished good health to each other with the idea that health implies a healthy body and a contented heart .
one of the participants in the study said , a person with a healthy and contented heart is well - behaved and kind to others ( p1 ) .
the participants always used local terminology to describe their ideas of health , and their descriptions and perception of health indicated that health is tantamount to worshipping god , for them .
another participant said , health reflects a healthy and happy heart ; physical well - being holds less importance as compared to the human spirit , and , both , the heart and the spirit need to be healthy
the participants mentioned that health incorporates several specific factors like calmness and happiness without any worries ; sadness , or any family crises .
health means that you have achieved peace in your family and home , and as a member of your family , you have exhibited good behaviour .
health means that the child and his or her parents share a cordial relationship , and life is a gift from god that ensures calmness and safety ( p 11 ) .
another participant said , health means that we do not have any problems or complaints of illness
( p6 ) , and , health brings calmness , and it is like praying to god without having to think about stressful matters (p2 ) , and , when you are healthy , you do nt have any complaints and anxiety , and so the few problems that people have to face seem petty when they have the support of their families .
all the participants mentioned that life without any compulsory limitations is one of the important dimensions of health and that they wished they could eat all kinds of food and participate in any occasion and be like others .
one of the women ( 43 years old ) said , when i eat rice , i need more water , and then i know that i am not healthy but fatigued , and i feel like travelling to the mountains and running away for a longer time , and then i suffer from sleeplessness and my body feels weak ( p 13 ) .
sometimes , i want to eat some comestibles that our family bought between us , but i can not do that , and this is so hard for me
for some participants , the limitations had led to the feeling that they were different from others
when you can participate in a ceremony or accompany someone , do anything that you want , or eat anything that you like , you feel a sense of freedom from others and you can go anywhere without worrying about your medicines , this freedom is very fantastic
in general , spirituality is subjective ; the individual and personal concept that each person defines is based on his or her understanding and on the evidence that spirituality improves mental and physical health .
participants emphasis on achieving exalted spirituality in their lives was tantamount to health for them .
most of the participants primarily belonged to the category that perceived health as exalted spirituality , and believed in satisfying self and others and trusting and honoring god . a woman ( 52 years old )
said , some of the people whose heart is near to god have good health , because honoring god helps to heal them , and when the spirit is healthy , the body is healthy . and , a healthy spirit in a healthy body , which believes in prayer and spirituality , finds calmness and peace
( p 18 ) , and , healthy patients who have the virtue to trust in god and are honest , do nt lose their spirituality , religion or prophet (p 11 ) another participant said , when i pray to god , i think about how fortunate i am to be born in this world , and then i am happy (p 6 ) .
as the results for this group of diabetic patients indicated , health is composed of the syndrome of the healthy body and the happy heart , life without compulsory limitations , and exalted spirituality .
many studies have shown that health perception is a reflection on , not only health but also health as a culture .
for all patients , health perception and points of view are the important factors that affect health behaviour and determine any related behaviour ( 25 , 26 ) . an individual may be ill and yet may feel healthy ; therefore , many people can compare their health status during suffering from illness with that in the past ( 22 , 27 ) .
the themes that emerged in our study showed these instances briefly . in this study , participants talked about the effects of diabetes on physical and psychological health , which indicated that our results were similar to those of other studies ( 28 , 29 ) .
the most important theme of this study was the syndrome of the healthy body and the happy heart .
the patients always used local terminology for health description , and these terminologies have extended meanings . in this context , health is considered as a feeling that can affect all components of the human body , including their physical , psychological , social , emotional , and spiritual facets .
this view about health is mainly accepted about diabetes because diabetes affects all aspects of a patient s life ( 11 , 28 , 29 ) . in this study , participants admitted that , not only physical health but also vivacity , satisfaction , and calmness of mind , good relationship with family and good economic status are all considered as aspects of health . hence , health care professionals should strongly consider the issue that health implies lack of having any problems , complaints , illnesses , etc .
furthermore , physical illness and its adverse effects are important topics in the health experiences of diabetic patients ; however , when these patients were healthy , they said that without peace of mind one could not feel healthy .
moreover , in a study about health in spanish immigrants , ailinger and causey found that some concepts of health are related to the patients psychological point of view and peace of mind , calmness in family , absence of complaints , and so on ( 30 ) .
assessed the effectiveness of self - efficacy and family inter - relationships among young turkish patients afflicted with diabetes type i ( 29 ) .
their study showed that calmness of mind and some other related factors such as relationship among family members , conflicts or compatibility in the family , and emotional situations can affect self - efficacy , and accordingly , these factors affect their overall health .
hence , considering the important effect of calmness of mind on diabetic patients health perception and points of view , we , as health care professionals , ought to be obliged to associate a disturbed mind with bad health .
overall , by using these implications as the bases of patients culture , we can improve health programmes for all patients .
one of the most plausible themes in our interviews was health as life without compulsory limitations .
miklaucich studied the limitations of life as meaningful health experiences in patients with coronary angina ( 31 ) .
abazari et al . also demonstrated that diabetic patients have dietary restrictions as well as limitations with regards to bearing children ( 18 ) .
thomson & gifford indicated that trying to keep a balance was the preferred meaning of health for diabetic participants ( 32 ) .
it is more important for the professional health care teams to help diabetic patients feel that they are not different from others and in this regard , consultation with dieticians can help to obviate some part of this limitation of life .
studies also show that having a regular activity program and regular follow- up consultation with other health care professionals can help diabetic patients to expel limitations ( 33 , 34 ) . in this article
, all participants talked about the importance of the spiritual dimension of health and achieving exalted spirituality .
some other studies showed that spirituality is one of the significant factors that can affect health ; hence , spirituality plays a very important role in gaining adaptation to a lifestyle , with regards to chronic diseases , and helps patients to manage their health - related problems ( 35 ) .
this theme was plausible in traditional societies ; hence , nursing professionals must pay more attention to this influential factor .
patients can adopt a spiritual approach by becoming a member of a religious group , praying , and meditating .
exalted spirituality , in fact , helps patients to cope with the disease , and feel better about their lives ( 36 , 37 ) .
he showed that praying helps patients to not only connect with the higher power , but also to relate to each other .
their act of praying is a response to their anxieties about themselves or their families .
walton showed that women with chronic diseases , used prayer to calm themselves and connect with others to support each other and cope with diseases ( 39 ) .
therefore , health care professionals should be aware of the effects of spirituality on patients having a chronic disease .
the results of our study primarily indicated that the factors that affect health perception include complaining about the illness , side effects and outcomes of the disease , and the extent of the complaints that make the patient feel sick .
patients with chronic diseases encountered some social and emotional problems due to changes in their lifestyle ( 36 ) .
diabetes affects patients quality of life , and especially their perception and attitude toward health ( 12 ) .
it fully depends on how patients and health professionals define health ( 40 , 41 ) . due to chronic and acute complaints of diabetic patients ,
we need to create a programme aiming to reduce these adverse effects and promote self - care for diabetic patients . especially , a program must be developed based on paying attention to body and mind simultaneously , trying to remove the limitation in life of patients by considering the extent of the limitations and limitations that exist for the patients which compel them to believe that the disease has been brought about just for them .
this program should not affect or limit the beliefs and spiritual activity of patients because a person s previous experiences influence the perception of present time , which also impact on their future .
besides , having been brought up in a different society and within a different ethnic group , influences a person s health - related behavior .
so , health care providers must manage care situations by enhancing health experience to enable patients lives with the least limitations , and giving them an opportunity to preserve exalted spirituality .
studying health perception among patients with chronic diseases is an important factor that facilitates the development of appropriate programmes , to improve health levels among such patients . in this regard ,
the professional health - care provider team ought to consider health perception among patients , and then they apply this knowledge for setting up suitable health improvement programs .
nurses need to pay more attention to diabetic patients limitations and facilitate a programme to improve their health in conjunction with other health care teams .
although nursing and other health care professionals have the responsibility to help those patients achieve peace of mind , the patients families should also play an active role in the health care programme and treatment process .
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introductionhealth is an exclusive and subjective phenomenon , and one of the most important situations with regard to perception of health , arises when patients suffer from a chronic disease.this study was conducted within the qualitative research framework and aimed to explore the meanings of health as perceived by a group of iranian diabetic patients.methodsa descriptive qualitative analysis design was used .
data were collected through semi - structured interviews with 20 participants among diabetic patients , who were admitted to the diabetes care centre of tohid hospital of the kurdistan university of medical sciences , sanandaj , iran during a ten - month period in 2014 .
interviews were transcribed and analysed through conventional content analysis.resultsbased on the findings of the study , three major health - related themes emerged : 1 ) the syndrome of the healthy body and the happy heart ( physical well - being vivacity , satisfaction , and calmness of the mind ) , 2 ) life without compulsory limitations ( lack of dietary limitations , no activity limitations , lack of social limitations ) , and 3 ) exalted spirituality ( satisfying self and others , trusting god , remembering god).conclusionhealth care providers should consider the meaning of health in special groups , chiefly in patients with chronic diseases .
it facilitates the development of appropriate programmes to improve desirable health levels among diabetic patients .
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1. Introduction
2. Material and Methods
2.1. Design
2.2. Participants
2.3. Data collection
2.4. Rigor
2.5. Ethical Considerations
2.6. Data Analysis
3. Results
3.1. The syndrome of the healthy body and the happy heart
3.2. Life without compulsory limitations
3.3. Exalted spirituality
4. Discussion
5. Conclusions
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the study included 340 whites from italy with type 2 diabetes ( according to american diabetes association 2003 criteria ) and coronary artery disease ( cad ) , as indicated by previous myocardial infarction ( mi ) or > 50% stenosis of at least one major vessel at coronary angiography , or both .
these individuals were cases of the cross - sectional case - control gargano heart study ( ghs ) ( 30 ) and were consecutively recruited at the scientific institute casa sollievo della sofferenza from 2001 to 2008 and monitored until the end of 2009 .
ten patients became untraceable before the first follow - up visit ; therefore , data were available for 330 patients .
they all had been diagnosed with an acute mi according to the european society of cardiology and american heart consensus guidelines .
exclusion criteria were the presence of malignancies and a medical record of diabetes , although 22 study participants ( 15.6% ) were found to have subclinical diabetes after an oral glucose tolerance test . because recruitment is still in progress
, only patients who were recruited up to 2007 , and as such underwent at least one follow - up visit , were included in this study .
two patients became untraceable before the first follow - up ; therefore , data were available for 141 patients .
this study comprises 283 whites with end - stage renal disease ( esrd ) , with 231 requiring hemodialysis and 52 receiving long - term ambulatory peritoneal dialysis ( 34 ) .
exclusion criteria were dialysis for less than 6 months , left ventricular ejection fraction < 35% , history of circulatory congestion , and hospitalization for intercurrent illness , including major infections . no dropouts were observed .
blood samples for dna extraction were unavailable for 17 subjects ; thus , 266 patients were included .
of these , 43 ( 16.2% ) had diabetes , a proportion similar to that reported by a nationwide epidemiologic study of kidney disease in italy ( 35 ) .
the end point considered in all three samples was a major cardiovascular event , defined as nonfatal stroke , nonfatal mi , or cardiovascular death .
information on the occurrence of nonfatal events was sought yearly from study participants and confirmed by a review of hospital records if cardiovascular events were reported . if patients did not report to a scheduled visit , information on the occurrence of cardiovascular events
was obtained by telephone interview , from their primary care physicians , or from death certificates .
deaths were ascribed to cvd according to the international classification of diseases codes : 410.0410.9 , 415.1 , 427.4427.5 , 428.0428.9 , 433.1 , 434.1 , 444.2 , 444.8 ( 9th edition ) or i21.0i21.9 , i46.1 , i49.0 , i50.1 , i62.9 , i63.0i63.9 , i71.3 ( 10th edition ) .
a total of 339 white patients with type 2 diabetes who had survived an mi were studied .
they are part of two ongoing investigations on the genetics of cad in type 2 diabetes ( 30,36,37 ) . of these ,
casa sollievo della sofferenza in san giovanni rotondo ( gargano , center east coast of italy ) , as cases of the cross - sectional case - control ghs ( 29 ) .
most of these patients ( n = 160 ) were further studied for incident major cardiovascular events in the prospective ghs ( see above ) .
another 170 were recruited in boston from the beth israel deaconess medical center ( bidmc ) and the joslin clinic ( which serves as the bidmc diabetes clinic ) as part of an ongoing investigation on the genetics of cad in type 2 diabetes that has been described previously ( 37 ) .
all subjects underwent a clinical examination and a standardized interview ( at the time of recruitment and at each subsequent time point , if applicable ) , which was identical in all three prospective samples .
a fasting blood sample ( collected between 8:00 a.m. and 9:00 a.m. ) was obtained from the prospective study participants and the cross - sectional study participants recruited in italy .
a random blood sample was obtained from the cross - sectional study participants recruited in boston .
bmi was calculated by dividing the weight ( in kilograms ) by the square of height ( in meters ) .
presence of hypertension was defined as a systolic blood pressure 130 mmhg or diastolic blood pressure 85 mmhg , or both , or the presence of antihypertensive treatment .
current and former smokers were considered as one group and compared with those who never smoked .
all study protocols were approved by the local institutional review boards and performed according to the helsinki declaration .
genotyping of the enpp1 k121q polymorphism ( rs1044498 ) was performed by taqman allele discrimination ( assay c_16190162_10 ; applied biosystems , foster city , ca ) on the ht7900 platform ( applied biosystems ) .
patients characteristics are reported as mean and sd for continuous variables and as frequencies and percentages for categoric variables .
comparisons between genotype groups were performed by pearson or mann - whitney u tests for continuous or categoric variables , respectively .
because of the low number of qq individuals , only the dominant genetic model was tested by comparing individuals carrying the k121/q121 or the q121/q121 genotype ( q121 ) ( i.e. , kq heterozygotes + qq homozygotes ) to k121 homozygotes ( kk ) . in prospective studies ,
a time - to - event analysis was conducted by means of cox proportional hazards regression models using the breslow approach in the case of ties and reported as hazard ratios ( hrs ) along with their 95% ci . the time to event
was defined as the time between enrollment date and the date of the first cardiovascular event . for censored subjects ,
the time variable was defined as the time between the enrollment date and the date of the last available clinical data .
the assumption of proportionality of the hazards was tested by using scaled schoenfeld residuals . in cross - sectional studies ,
the association between enpp1 q121 variant and age at mi was analyzed by multiple linear regression analysis , and results are given as regression coefficients .
pooled data analyses were performed in an individual patient data meta - analysis fashion ( 38 ) ( i.e. , adjusting for study sample ) after excluding genotype - by - sample interactions .
genotype - by - obesity interaction was tested by adding a cross - product term to the regression model .
the discriminatory power of prediction models was assessed by estimating the survival c - index ( 39 ) and by measuring the integrated discrimination improvement ( idi ) ( 40 ) .
all analyses were performed using sas 9.1 software ( sas institute , cary , nc ) .
the study included 340 whites from italy with type 2 diabetes ( according to american diabetes association 2003 criteria ) and coronary artery disease ( cad ) , as indicated by previous myocardial infarction ( mi ) or > 50% stenosis of at least one major vessel at coronary angiography , or both .
these individuals were cases of the cross - sectional case - control gargano heart study ( ghs ) ( 30 ) and were consecutively recruited at the scientific institute casa sollievo della sofferenza from 2001 to 2008 and monitored until the end of 2009 .
ten patients became untraceable before the first follow - up visit ; therefore , data were available for 330 patients .
they all had been diagnosed with an acute mi according to the european society of cardiology and american heart consensus guidelines .
exclusion criteria were the presence of malignancies and a medical record of diabetes , although 22 study participants ( 15.6% ) were found to have subclinical diabetes after an oral glucose tolerance test . because recruitment is still in progress
, only patients who were recruited up to 2007 , and as such underwent at least one follow - up visit , were included in this study .
two patients became untraceable before the first follow - up ; therefore , data were available for 141 patients .
this study comprises 283 whites with end - stage renal disease ( esrd ) , with 231 requiring hemodialysis and 52 receiving long - term ambulatory peritoneal dialysis ( 34 ) .
exclusion criteria were dialysis for less than 6 months , left ventricular ejection fraction < 35% , history of circulatory congestion , and hospitalization for intercurrent illness , including major infections . no dropouts were observed .
blood samples for dna extraction were unavailable for 17 subjects ; thus , 266 patients were included .
of these , 43 ( 16.2% ) had diabetes , a proportion similar to that reported by a nationwide epidemiologic study of kidney disease in italy ( 35 ) .
the end point considered in all three samples was a major cardiovascular event , defined as nonfatal stroke , nonfatal mi , or cardiovascular death .
information on the occurrence of nonfatal events was sought yearly from study participants and confirmed by a review of hospital records if cardiovascular events were reported . if patients did not report to a scheduled visit , information on the occurrence of cardiovascular events
was obtained by telephone interview , from their primary care physicians , or from death certificates .
deaths were ascribed to cvd according to the international classification of diseases codes : 410.0410.9 , 415.1 , 427.4427.5 , 428.0428.9 , 433.1 , 434.1 , 444.2 , 444.8 ( 9th edition ) or i21.0i21.9 , i46.1 , i49.0 , i50.1 , i62.9 , i63.0i63.9 , i71.3 ( 10th edition ) .
a total of 339 white patients with type 2 diabetes who had survived an mi were studied .
they are part of two ongoing investigations on the genetics of cad in type 2 diabetes ( 30,36,37 ) . of these ,
casa sollievo della sofferenza in san giovanni rotondo ( gargano , center east coast of italy ) , as cases of the cross - sectional case - control ghs ( 29 ) .
most of these patients ( n = 160 ) were further studied for incident major cardiovascular events in the prospective ghs ( see above ) .
another 170 were recruited in boston from the beth israel deaconess medical center ( bidmc ) and the joslin clinic ( which serves as the bidmc diabetes clinic ) as part of an ongoing investigation on the genetics of cad in type 2 diabetes that has been described previously ( 37 ) .
the study included 340 whites from italy with type 2 diabetes ( according to american diabetes association 2003 criteria ) and coronary artery disease ( cad ) , as indicated by previous myocardial infarction ( mi ) or > 50% stenosis of at least one major vessel at coronary angiography , or both .
these individuals were cases of the cross - sectional case - control gargano heart study ( ghs ) ( 30 ) and were consecutively recruited at the scientific institute casa sollievo della sofferenza from 2001 to 2008 and monitored until the end of 2009 .
ten patients became untraceable before the first follow - up visit ; therefore , data were available for 330 patients .
they all had been diagnosed with an acute mi according to the european society of cardiology and american heart consensus guidelines .
exclusion criteria were the presence of malignancies and a medical record of diabetes , although 22 study participants ( 15.6% ) were found to have subclinical diabetes after an oral glucose tolerance test . because recruitment is still in progress
, only patients who were recruited up to 2007 , and as such underwent at least one follow - up visit , were included in this study .
two patients became untraceable before the first follow - up ; therefore , data were available for 141 patients .
this study comprises 283 whites with end - stage renal disease ( esrd ) , with 231 requiring hemodialysis and 52 receiving long - term ambulatory peritoneal dialysis ( 34 ) .
exclusion criteria were dialysis for less than 6 months , left ventricular ejection fraction < 35% , history of circulatory congestion , and hospitalization for intercurrent illness , including major infections . no dropouts were observed .
blood samples for dna extraction were unavailable for 17 subjects ; thus , 266 patients were included .
of these , 43 ( 16.2% ) had diabetes , a proportion similar to that reported by a nationwide epidemiologic study of kidney disease in italy ( 35 ) .
the end point considered in all three samples was a major cardiovascular event , defined as nonfatal stroke , nonfatal mi , or cardiovascular death .
information on the occurrence of nonfatal events was sought yearly from study participants and confirmed by a review of hospital records if cardiovascular events were reported . if patients did not report to a scheduled visit , information on the occurrence of cardiovascular events
was obtained by telephone interview , from their primary care physicians , or from death certificates .
deaths were ascribed to cvd according to the international classification of diseases codes : 410.0410.9 , 415.1 , 427.4427.5 , 428.0428.9 , 433.1 , 434.1 , 444.2 , 444.8 ( 9th edition ) or i21.0i21.9 , i46.1 , i49.0 , i50.1 , i62.9 , i63.0i63.9 , i71.3 ( 10th edition ) .
a total of 339 white patients with type 2 diabetes who had survived an mi were studied .
they are part of two ongoing investigations on the genetics of cad in type 2 diabetes ( 30,36,37 ) . of these ,
casa sollievo della sofferenza in san giovanni rotondo ( gargano , center east coast of italy ) , as cases of the cross - sectional case - control ghs ( 29 ) .
most of these patients ( n = 160 ) were further studied for incident major cardiovascular events in the prospective ghs ( see above ) .
another 170 were recruited in boston from the beth israel deaconess medical center ( bidmc ) and the joslin clinic ( which serves as the bidmc diabetes clinic ) as part of an ongoing investigation on the genetics of cad in type 2 diabetes that has been described previously ( 37 ) .
all subjects underwent a clinical examination and a standardized interview ( at the time of recruitment and at each subsequent time point , if applicable ) , which was identical in all three prospective samples .
a fasting blood sample ( collected between 8:00 a.m. and 9:00 a.m. ) was obtained from the prospective study participants and the cross - sectional study participants recruited in italy .
a random blood sample was obtained from the cross - sectional study participants recruited in boston .
bmi was calculated by dividing the weight ( in kilograms ) by the square of height ( in meters ) .
presence of hypertension was defined as a systolic blood pressure 130 mmhg or diastolic blood pressure 85 mmhg , or both , or the presence of antihypertensive treatment .
current and former smokers were considered as one group and compared with those who never smoked .
all study protocols were approved by the local institutional review boards and performed according to the helsinki declaration . written informed consent was obtained from each study participant .
genotyping of the enpp1 k121q polymorphism ( rs1044498 ) was performed by taqman allele discrimination ( assay c_16190162_10 ; applied biosystems , foster city , ca ) on the ht7900 platform ( applied biosystems ) .
patients characteristics are reported as mean and sd for continuous variables and as frequencies and percentages for categoric variables .
comparisons between genotype groups were performed by pearson or mann - whitney u tests for continuous or categoric variables , respectively .
deviations from hardy - weinberg equilibrium were investigated by exact test . because of the low number of qq individuals , only the dominant genetic model was tested by comparing individuals carrying the k121/q121 or the q121/q121 genotype ( q121 ) ( i.e. , kq heterozygotes + qq homozygotes ) to k121 homozygotes ( kk ) . in prospective studies , a time - to - event analysis was conducted by means of cox proportional hazards regression models using the breslow approach in the case of ties and reported as hazard ratios ( hrs ) along with their 95% ci . the time to event
was defined as the time between enrollment date and the date of the first cardiovascular event . for censored subjects ,
the time variable was defined as the time between the enrollment date and the date of the last available clinical data .
the assumption of proportionality of the hazards was tested by using scaled schoenfeld residuals . in cross - sectional studies ,
the association between enpp1 q121 variant and age at mi was analyzed by multiple linear regression analysis , and results are given as regression coefficients .
pooled data analyses were performed in an individual patient data meta - analysis fashion ( 38 ) ( i.e. , adjusting for study sample ) after excluding genotype - by - sample interactions .
genotype - by - obesity interaction was tested by adding a cross - product term to the regression model .
the discriminatory power of prediction models was assessed by estimating the survival c - index ( 39 ) and by measuring the integrated discrimination improvement ( idi ) ( 40 ) . a value of p < 0.05 was considered significant .
all analyses were performed using sas 9.1 software ( sas institute , cary , nc ) .
we studied three cohorts of subjects who were at very high risk of major cardiovascular events : the ghs individuals with type 2 diabetes and previously diagnosed cad ; the tor vergata atherosclerosis study ( tvas)individuals from the general population who had experienced an mi ; and the cardiovascular risk extended evaluation in dialysis ( creed)individuals with esrd who required dialysis .
no significant differences in baseline characteristics across genotype groups were observed in any of the three studies .
clinical features of very high - risk individuals in the three prospective studies data are expressed as absolute numbers , percentage , or mean ( sd ) .
the average mean ( sd ) follow - up was 37.1 ( 19.4 ) months ( range 191 ) in the ghs , 30.6 ( 11.3 ) months ( range 137 ) in the tvas , and 36.3 ( 22.0 ) months ( range , 169 ) in the creed . during follow - up , 43 major cardiovascular events occurred in the ghs , 39 in the tvas , and 94 in the creed , resulting in respective incidence rates of 4.2 , 10.8 , and 11.7 per 100 person - years ( table 2 ) . in all studies , incidence rates per 100 person - years were numerically higher in q121 carriers than in kk homozygotes : 5.4 vs. 3.6 in the ghs , 19.2 vs. 8.1 in the tvas , and 14.1 vs. 10.8 in the creed ( table 2 ) .
the difference was significant in the tvas ( p = 0.025 ) and in a pooled analysis of the three studies ( p = 0.005 ) .
no difference in the magnitude of the genetic effect was observed among studies ( p = 0.32 for interaction ) .
incidence of major cardiovascular events in ghs , tvas , and creeds * per 100 person - years .
the hr of cardiovascular events for q121 carriers versus kk homozygotes was 1.47 ( 95% ci 0.802.70 , p = 0.21 ) in the ghs , 2.31 ( 95% ci 1.224.34 , p = 0.01 ) in the tvas , and 1.36 ( 95% ci 0.882.10 , p = 0.16 ) in the creed ( fig .
1a , b , and c , respectively ; p = 0.32 for gene - by - sample interaction ) . in a pooled analysis ( i.e. , individual patient data meta - analysis ) of the three studies , which included 737 subjects with 176 events ,
the hr for q121 carriers versus kk homozygotes was 1.56 ( 95% ci 1.152.12 , p = 0.004 ; fig .
hr 1.04 [ 95% ci 1.031.06 ] , p < 0.0001 ) , diabetes ( 2.23 [ 95% ci 1.503.31 ] , p < 0.0001 ) , and smoking status ( 1.71 [ 95% ci 1.242.36 ] , p = 0.001 ) were additional predictors of incident events .
bmi ( hr 1.03 [ 95% ci 0.991.06 ] , p = 0.08 ) , hypertension ( 1.50 [ 95% ci 0.992.27 ] , p = 0.054 ) , and sex ( 1.30 [ 95% ci 0.951.79 ] , p = 0.10 ) also tended to be associated with increased rate of events , although these did not reach statistical significance .
the increased risk of events associated with the q121 variant remained significant ( hr 1.55 [ 95% ci 1.142.12 ] , p = 0.005 ) after adjusting for bmi and diabetes , both of which had been reported to be associated with the q121 variant ( 3840 ) , as well as after further adjustment for age , sex , hypertension , and smoking status ( hr 1.45 [ 95% ci 1.052.00 ] , p = 0.022 ) .
kaplan - meier survival curves are shown for major cardiovascular events in ghs ( a ) , tvas ( b ) , and creed ( c ) .
the addition of the k121q genotype did not improve the risk discrimination provided by the predictive model that included age , sex , bmi , smoking status , hypertension and diabetes , as indicated by the survival c - index , which went from 0.704 to 0.713 ( p = 0.94 ) , or by the idi , with 0.42% improvement ( p = 0.16 ) .
given the previous evidence for an enpp1-by - obesity interaction in the modulation of traits related to insulin resistance ( 2527,29,30,3941 ) , we investigated this hypothesis in our study . in the ghs , which entirely consisted of patients with type 2 diabetes , we indeed observed a significant interaction between the q121 variant and obesity .
an association between the variant and risk of events was present among the 159 subjects who had a bmi 30 kg / m ( hr 3.56 [ 95% ci 1.2110.5 ] , p = 0.02 ) , but not among the 171 individuals who had a bmi < 30 kg / m ( 0.91 [ 95% ci 0.402.06 ] , p = 0.82 ; p = 0.039 for interaction ) . no evidence of gene - by - obesity interaction was instead observed in the tvas ( p = 0.53 ) and the creed ( p = 0.41 ) .
because approximately 85% of these two cohorts consisted of nondiabetic individuals , we hypothesized that the genotype - by - obesity interaction might be specific to diabetes .
indeed , a pattern consistent with such an effect was also observed in these two studies when the analysis was restricted to individuals with diabetes , even though the small sample size prevented statistical significance ( data not shown ) .
thus , we further investigated the q121-by - obesity interaction in a pooled analysis of the three studies after stratification by diabetes status . in the diabetic stratum ( n = 395 ) , the q121 variant was associated with an increased risk of incident events among the 177 obese ( fig .
2b ) individuals , with adjusted hrs of 5.94 ( 95% ci 1.8818.78 , p = 0.002 ) vs. 0.62 ( 95% ci 0.321.24 , p = 0.18 ) .
the interaction between the q121 variant and obesity was significant ( p = 0.003 ) .
by contrast , no evidence of interaction was observed in the nondiabetic stratum ( n = 344 , p = 0.26 ) , with adjusted hrs of 0.82 ( 95% ci 0.223.11 , p = 0.77 ) in the 39 obese individuals and 1.85 ( 95% ci 1.182.90 , p = 0.008 ) in the 305 nonobese subjects .
survival curves for major cardiovascular events in obese ( a ) and nonobese ( b ) patients with type 2 diabetes .
curves are estimates generated by cox regression in the pooled analysis of the three prospective studies . among obese diabetic individuals ,
the addition of the k121q genotype to the multivariable model produced a slight improvement from 0.802 to 0.831 in risk discrimination when this was assessed by the survival c - index ( p = 0.81 ) .
a much larger effect , approaching statistical significance , was observed when the improvement was assessed by idi with a 4.56% improvement ( 95% ci 0.27 to 9.42 , p = 0.09 ) . to seek replication of the gene - by - obesity interaction observed in patients with diabetes
, we analyzed the association between the q121 variant and age at mi in two cross - sectional samples of individuals with type 2 diabetes who had had a previous mi .
one sample was from the gargano area in italy , the other was from boston .
salient clinical features of the study subjects are summarized in table 3 . because no significant genotype - by - sample interaction was observed in the association with age at mi ( p = 0.11 ) , pooled analyses were performed by adjusting for study sample .
to make the analysis comparable to that of prospective studies , sex , smoking status , hypertension , and bmi , but not age ( due to its collinearity with age at mi and diabetes because all study participants were diabetic )
64 q121 carriers had had the mi almost 3 years earlier than the 124 kk homozygotes , at 54.5 ( 9.6 ) vs. 57.2 ( 8.9 ) years of age ( p = 0.035 ) .
in contrast , no significant difference in age at mi was observed among nonobese subjects : 59.2 ( 10.5 ) in 41 q121 carriers versus 57.2 ( 10.4 ) in 110 kk homozygotes ( p = 0.16 ; p = 0.025 value for q121-by - obesity interaction ) .
clinical features of patients with type 2 diabetes who survived an mi in the two cross - sectional studies data are expressed as number , percentage , or mean ( sd ) .
virtually identical results were obtained when duration of diabetes was also added to the multivariate model , with q121 carriers having had an mi at a significantly younger age than kk individuals among obese ( p = 0.039 ) but not among nonobese ( p = 0.20 ) individuals ( p = 0.032 for interaction ) .
in addition , when bmi was considered as a continuous trait , it was inversely related to the age at mi among q121 carriers ( = 0.44 [ 95% ci 0.75 to 0.12 ] , p = 0.008 ; fig .
3a ) , but not among kk individuals ( = 0.17 [ 95% ci 0.41 to 0.07 ] , p = 0.16 ; p = 0.069 ; for q121-by - bmi interaction ; fig .
3b ) . linear regression between bmi and age at mi in diabetic patients with q121 ( a ) and kk ( b ) genotypes .
data are obtained by individual data meta - analysis of the two cross - sectional studies from gargano and boston .
we studied three cohorts of subjects who were at very high risk of major cardiovascular events : the ghs individuals with type 2 diabetes and previously diagnosed cad ; the tor vergata atherosclerosis study ( tvas)individuals from the general population who had experienced an mi ; and the cardiovascular risk extended evaluation in dialysis ( creed)individuals with esrd who required dialysis .
no significant differences in baseline characteristics across genotype groups were observed in any of the three studies .
clinical features of very high - risk individuals in the three prospective studies data are expressed as absolute numbers , percentage , or mean ( sd ) .
the average mean ( sd ) follow - up was 37.1 ( 19.4 ) months ( range 191 ) in the ghs , 30.6 ( 11.3 ) months ( range 137 ) in the tvas , and 36.3 ( 22.0 ) months ( range , 169 ) in the creed . during follow - up , 43 major cardiovascular events occurred in the ghs , 39 in the tvas , and 94 in the creed , resulting in respective incidence rates of 4.2 , 10.8 , and 11.7 per 100 person - years ( table 2 ) . in all studies , incidence rates per 100 person - years were numerically higher in q121 carriers than in kk homozygotes : 5.4 vs. 3.6 in the ghs , 19.2 vs. 8.1 in the tvas , and 14.1 vs. 10.8 in the creed ( table 2 ) .
the difference was significant in the tvas ( p = 0.025 ) and in a pooled analysis of the three studies ( p = 0.005 ) .
no difference in the magnitude of the genetic effect was observed among studies ( p = 0.32 for interaction ) .
incidence of major cardiovascular events in ghs , tvas , and creeds * per 100 person - years .
the hr of cardiovascular events for q121 carriers versus kk homozygotes was 1.47 ( 95% ci 0.802.70 , p = 0.21 ) in the ghs , 2.31 ( 95% ci 1.224.34 , p = 0.01 ) in the tvas , and 1.36 ( 95% ci 0.882.10 , p = 0.16 ) in the creed ( fig .
1a , b , and c , respectively ; p = 0.32 for gene - by - sample interaction ) . in a pooled analysis ( i.e. , individual patient data meta - analysis ) of the three studies , which included 737 subjects with 176 events ,
the hr for q121 carriers versus kk homozygotes was 1.56 ( 95% ci 1.152.12 , p = 0.004 ; fig .
age at study entry ( hr 1.04 [ 95% ci 1.031.06 ] , p < 0.0001 ) , diabetes ( 2.23 [ 95% ci 1.503.31 ] , p < 0.0001 ) , and smoking status ( 1.71 [ 95% ci 1.242.36 ] , p = 0.001 ) were additional predictors of incident events .
bmi ( hr 1.03 [ 95% ci 0.991.06 ] , p = 0.08 ) , hypertension ( 1.50 [ 95% ci 0.992.27 ] , p = 0.054 ) , and sex ( 1.30 [ 95% ci 0.951.79 ] , p = 0.10 ) also tended to be associated with increased rate of events , although these did not reach statistical significance .
the increased risk of events associated with the q121 variant remained significant ( hr 1.55 [ 95% ci 1.142.12 ] , p = 0.005 ) after adjusting for bmi and diabetes , both of which had been reported to be associated with the q121 variant ( 3840 ) , as well as after further adjustment for age , sex , hypertension , and smoking status ( hr 1.45 [ 95% ci 1.052.00 ] , p = 0.022 ) .
kaplan - meier survival curves are shown for major cardiovascular events in ghs ( a ) , tvas ( b ) , and creed ( c ) .
the addition of the k121q genotype did not improve the risk discrimination provided by the predictive model that included age , sex , bmi , smoking status , hypertension and diabetes , as indicated by the survival c - index , which went from 0.704 to 0.713 ( p = 0.94 ) , or by the idi , with 0.42% improvement ( p = 0.16 ) .
given the previous evidence for an enpp1-by - obesity interaction in the modulation of traits related to insulin resistance ( 2527,29,30,3941 ) , we investigated this hypothesis in our study . in the ghs , which entirely consisted of patients with type 2 diabetes , we indeed observed a significant interaction between the q121 variant and obesity .
an association between the variant and risk of events was present among the 159 subjects who had a bmi 30 kg / m ( hr 3.56 [ 95% ci 1.2110.5 ] , p = 0.02 ) , but not among the 171 individuals who had a bmi < 30 kg / m ( 0.91 [ 95% ci 0.402.06 ] , p = 0.82 ; p = 0.039 for interaction ) .
no evidence of gene - by - obesity interaction was instead observed in the tvas ( p = 0.53 ) and the creed ( p = 0.41 ) . because approximately 85% of these two cohorts consisted of nondiabetic individuals
, we hypothesized that the genotype - by - obesity interaction might be specific to diabetes .
indeed , a pattern consistent with such an effect was also observed in these two studies when the analysis was restricted to individuals with diabetes , even though the small sample size prevented statistical significance ( data not shown ) .
thus , we further investigated the q121-by - obesity interaction in a pooled analysis of the three studies after stratification by diabetes status . in the diabetic stratum ( n = 395 ) , the q121 variant was associated with an increased risk of incident events among the 177 obese ( fig .
2b ) individuals , with adjusted hrs of 5.94 ( 95% ci 1.8818.78 , p = 0.002 ) vs. 0.62 ( 95% ci 0.321.24 , p = 0.18 ) . the interaction between the q121 variant and obesity was significant ( p = 0.003 ) .
by contrast , no evidence of interaction was observed in the nondiabetic stratum ( n = 344 , p = 0.26 ) , with adjusted hrs of 0.82 ( 95% ci 0.223.11 , p = 0.77 ) in the 39 obese individuals and 1.85 ( 95% ci 1.182.90 , p = 0.008 ) in the 305 nonobese subjects .
survival curves for major cardiovascular events in obese ( a ) and nonobese ( b ) patients with type 2 diabetes .
curves are estimates generated by cox regression in the pooled analysis of the three prospective studies . among obese diabetic individuals ,
the addition of the k121q genotype to the multivariable model produced a slight improvement from 0.802 to 0.831 in risk discrimination when this was assessed by the survival c - index ( p = 0.81 ) .
a much larger effect , approaching statistical significance , was observed when the improvement was assessed by idi with a 4.56% improvement ( 95% ci 0.27 to 9.42 , p = 0.09 ) .
to seek replication of the gene - by - obesity interaction observed in patients with diabetes , we analyzed the association between the q121 variant and age at mi in two cross - sectional samples of individuals with type 2 diabetes who had had a previous mi .
one sample was from the gargano area in italy , the other was from boston .
salient clinical features of the study subjects are summarized in table 3 . because no significant genotype - by - sample interaction was observed in the association with age at mi ( p = 0.11 ) , pooled analyses were performed by adjusting for study sample .
to make the analysis comparable to that of prospective studies , sex , smoking status , hypertension , and bmi , but not age ( due to its collinearity with age at mi and diabetes because all study participants were diabetic ) were included as covariates . among obese subjects ,
64 q121 carriers had had the mi almost 3 years earlier than the 124 kk homozygotes , at 54.5 ( 9.6 ) vs. 57.2 ( 8.9 ) years of age ( p = 0.035 ) .
in contrast , no significant difference in age at mi was observed among nonobese subjects : 59.2 ( 10.5 ) in 41 q121 carriers versus 57.2 ( 10.4 ) in 110 kk homozygotes ( p = 0.16 ; p = 0.025 value for q121-by - obesity interaction ) .
clinical features of patients with type 2 diabetes who survived an mi in the two cross - sectional studies data are expressed as number , percentage , or mean ( sd ) .
virtually identical results were obtained when duration of diabetes was also added to the multivariate model , with q121 carriers having had an mi at a significantly younger age than kk individuals among obese ( p = 0.039 ) but not among nonobese ( p = 0.20 ) individuals ( p = 0.032 for interaction ) .
in addition , when bmi was considered as a continuous trait , it was inversely related to the age at mi among q121 carriers ( = 0.44 [ 95% ci 0.75 to 0.12 ] , p = 0.008 ; fig .
3a ) , but not among kk individuals ( = 0.17 [ 95% ci 0.41 to 0.07 ] , p = 0.16 ; p = 0.069 ; for q121-by - bmi interaction ; fig .
3b ) . linear regression between bmi and age at mi in diabetic patients with q121 ( a ) and kk ( b ) genotypes .
data are obtained by individual data meta - analysis of the two cross - sectional studies from gargano and boston .
our results indicate that the enpp1 k121q polymorphism predicts acceleration of major cardiovascular events in very high - risk patients .
the increased risk conferred by the q121 variant is independent of that of age , sex , bmi , diabetes , and cigarette smoking .
our findings are in agreement with a previous cross - sectional study of 445 mi survivors from central europe ( 29 ) .
by contrast , case - control genome - wide association studies reported that a single nucleotide polymorphism ( snp , rs7767502 ) , which is in perfect linkage disequilibrium with the enpp1 k121q polymorphism , was not associated with cad ( 610 ) .
several differences between our study and the genome - wide association studies , such as the prospective versus cross - sectional designs , the different end points under investigation , and the different baseline cardiovascular risk , with only the patients enrolled in our study being very high - risk as per selection criteria , might be responsible for this apparent discordance .
an additional important result of our study is that the effect of the q121 variant was modulated by obesity in diabetic patients among whom the risk of incident events was five times higher in q121 than in kk genotype carriers .
although not the aim of our study , one can infer that obese individuals ( fig .
2a ) as a whole tend to have a lower risk of future cardiovascular events than nonobese patients ( fig . 2b ; adjusted hr 0.68 [ 95% ci 0.4111.124 ] , p = 0.13 ) .
this paradoxic protective effect of obesity resembles that observed in patients with cad ( 41 ) , esrd ( 42 ) , heart failure ( 43 ) , and older age ( 44 ) , all conditions heavily over - represented in our samples . in this context , the q121 variant seems to eliminate the paradoxic protective effect of obesity .
an important finding was that the q121 variant - by - obesity interaction observed in the prospective study was replicated in a cross - sectional study on age at mi in diabetic patients .
information on the k121q genotypes tended to improve risk prediction in these patients when the improvement was measured by the idi , the approach that is currently favored to evaluate predictive ability increase conferred by a new marker when added to a well - performing model ( 39 ) .
thus , pending further validation in larger studies , one can hypothesize clinical implementation of the q121 variant as a marker of early cardiovascular events among obese diabetic patients .
given the increasing incidence worldwide of both obesity and diabetes ( 24 ) and the poor ability to stratify cardiovascular risk among diabetic patients , a large sector of society would be likely to benefit in the future from the availability of such a test .
the synergistic effect of the genetic marker and obesity in the modulation of cardiovascular risk resembles results repeatedly reported in the risk modulation of insulin resistance and related traits ( 2527,30,33,4549 ) .
placed in a broader perspective , this is an excellent example of genetic heterogeneity ( i.e. , different genetic effects being at play in different population subgroups ) and clearly illustrates how accounting for such heterogeneity may be critical to dissect the genetic architecture of multifactorial diseases .
understanding the mechanisms through which the q121 variant is associated with cvd is beyond the scope of this study .
however , one can speculate that the q121 variant exacerbates cardiovascular risk by inducing systemic insulin resistance ( 22,2527 ) and proatherogenic phenotypes ( 24,29,30,33 )
. it may also act by way of a direct detrimental effect on insulin - dependent endothelial function , as suggested by the observation that human endothelial cells carrying the q121 variant show impaired insulin receptor signaling and , most importantly , reduced release of nitric oxide ( 24 ) , a potent vasodilator whose deficiency is an established early step in the pathway development of atherosclerosis ( 50 ) .
one can hypothesize that the interaction between the q121 variant and obesity is sustained by the different sites of action on the insulin - signaling pathway .
although enpp1 acts at the insulin receptor level ( 21,23 ) , obesity acts by different mechanisms , mostly at a postreceptor level ( 51 ) .
it is , therefore , possible that postreceptor insulin - signaling abnormalities are necessary for the q121 variant to be fully effective in inducing insulin resistance and , eventually , related clinical outcomes .
the three cohorts of very high - risk individuals that we studied were quite different from each other : one comprised only patients with type 2 diabetes and cad , another included patients with a previous mi who did not have frank type 2 diabetes , and the third included only patients with esrds . despite such apparent phenotypic heterogeneity , the effect of the q121 variant was not heterogeneous across the three studies .
not only did this allow us to analyze the three cohorts together , increasing statistical power , but it also suggests that our findings may be generalizable to all high - risk patients , irrespective of their background clinical characteristics .
whether the predictive role of the q121 variant extends to situations characterized by a more moderate cardiovascular risk remains to be determined .
we acknowledge that , mainly because of the relatively small size of our samples , the significance level of our findings is still compatible with a false - positive result .
however , this seems unlikely given that the association between q121 was not heterogeneous across the three cohorts and , importantly , was further confirmed in cross - sectional studies as far as the interaction with obesity in diabetic patients is concerned .
we also acknowledge that due to the relatively small sample size of the studies that we analyzed , we can not exclude that the gene - by - obesity interaction that we observed among diabetic patients also occurs among nondiabetic individuals , as is the case for the modulation of insulin resistance ( 2527 ) .
therefore , our findings need further replication in larger samples before they can be considered as established . finally , because this study was entirely performed in individuals of european ancestry , we do not know whether our findings can be extended to populations of different race . in conclusion ,
pending confirmation in further larger studies , the q121 variant has the potential to become a clinical tool for identifying those very high - risk patients who are especially prone to major cardiovascular events and need , therefore , to be targeted with specific and even more aggressive preventive strategies .
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objectiveinsulin resistance ( ir ) and cardiovascular disease may share a common genetic background .
we investigated the role of ir - associated enpp1 k121q polymorphism ( rs1044498 ) on cardiovascular disease in high - risk individuals.research design and methodsa prospective study ( average follow - up , 37 months ) was conducted for major cardiovascular events ( myocardial infarction [ mi ] , stroke , cardiovascular death ) from the gargano heart study ( ghs ; n = 330 with type 2 diabetes and coronary artery disease ) , the tor vergata atherosclerosis study ( tvas ; n = 141 who had mi ) , and the cardiovascular risk extended evaluation in dialysis ( creed ) database ( n = 266 with end - stage renal disease )
. age at mi was investigated in cross - sectional studies of 339 type 2 diabetic patients ( n = 169 from italy , n = 170 from the u.s.).resultsincidence of cardiovascular events per 100 person -- years was 4.2 in ghs , 10.8 in tvas , and 11.7 in creed . hazard ratios ( hrs ) for kq+qq versus individuals carrying the k121/k121 genotype ( kk ) individuals were 1.47 ( 95% ci 0.802.70 ) in ghs , 2.31 ( 95% ci 1.224.34 ) in tvas , and 1.36 ( 95% ci 0.882.10 ) in creed , and 1.56 ( 95% ci 1.152.12 ) in the three cohorts combined . in the 395 diabetic patients , the q121 variant predicted cardiovascular events among obese but not among nonobese individuals
( hr 5.94 vs. 0.62 , p = 0.003 for interaction ) .
a similar synergism was observed in cross - sectional studies , with age at mi being 3 years younger in q121 carriers than in kk homozygotes among obese but not among nonobese patients ( p = 0.035 for interaction).conclusionsthe enpp1 k121q polymorphism is an independent predictor of major cardiovascular events in high - risk individuals . in type 2 diabetes , this effect is exacerbated by obesity .
future larger studies are needed to confirm our finding .
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RESEARCH DESIGN AND METHODS
Study participants
Prospective studies.
Cross-sectional study of age at MI.
Data collection.
Genotyping.
Statistical analysis.
RESULTS
Characteristics of cohort members at baseline.
None
Interaction between Q121 variant and obesity in predicting major cardiovascular events.
Interaction between Q121 variant and obesity on age at MI in cross-sectional studies.
DISCUSSION
|
alzheimer 's disease ( ad ) is a common neurodegenerative disease in the central nervous system .
the pathological features of ad include senile plaques formed by extracellular amyloid ( a ) aggregation , neurofibrillary tangles formed by abnormal accumulation of hyperphosphorylated tau protein , synaptic impairments , and neuronal loss in the cerebral cortex and hippocampus .
a aggregation in the brain is central to the pathological changes of ad and leads to a series of pathological events , which further promote a aggregation , resulting in cascade amplification .
the endoplasmic reticulum ( er ) , a dynamic membranous organelle , is involved in protein synthesis , posttranslational modification , folding , calcium storage , lipid metabolism , and steroid hormone synthesis .
specific stress conditions such as hyperglycemia , hyperlipidemia , hypoxia , chemical toxicants , and genetic mutations can induce the accumulation of unfolded or misfolded proteins in the er and changes in er functions , resulting in endoplasmic reticulum stress ( ers ) . in turn , ers stimulates the unfolded protein response ( upr ) and restores cellular homeostasis while sustained ers leads to cell apoptosis .
many studies have shown that ers is involved in the development and progression of neurodegenerative diseases such as ad , parkinson 's disease , huntington 's disease , and amyotrophic lateral sclerosis although the underlying mechanisms still remain blurred .
the activation of the upr pathway requires the activation of three sensor proteins : inositol - requiring enzyme 1 ( ire-1 ) , activating transcription factor 6 ( atf6 ) , and protein kinase rna - like er kinase .
these sensors serve to promote the expression of chaperone protein , glucose - regulated protein ( grp ) 78 , to repair misfolded or unfolded proteins , to accelerate er - associated protein degradation ( erad ) , and to aggravate the phosphorylation of eukaryotic translation initiation factor 2a .
sustained ers induces the activation of the er - specific apoptosis pathway by promoting the expression of the transcriptional activator ccaat / enhancer - binding protein homologous protein ( chop ) and caspase-12 activation .
activated ire-1 interacts with tumor necrosis factor receptor - associated factor 2 ( traf2 ) and apoptosis signal - regulating kinase 1 ( ask1 ) via the cytosolic enzyme domain to form ire-1-traf2-ask1 complexes , which in turn stimulate the c - jun amino - terminal kinase ( jnk ) pathway to promote cell apoptosis , thus preventing the damaging impact of misfolded and secreted proteins on tissues and organisms .
genetic mutations in ad lead to a overexpression in the brain and subsequent neurotoxicity , which leads to pathogenesis .
the five familial alzheimer 's disease ( 5fad ) mice were established by overexpressing the five familial - inherited ad mutant genes ( app k670n / m671l [ sweden]+i716v [ florida ] + v717i [ london]+ps1 m146l+l286v ) under the control of the neuron - specific thy-1 promoter .
this mouse model is characterized by many pathological features similar to ad , including amyloid plaque deposition , gliosis , neuronal degeneration , neuronal loss , and cognitive deficits at the age of 45 months .
more importantly , this mouse model has been documented to show the earliest signs of intracellular a aggregation in neurons in 1.5-month - old mice , which indicates these mice as a suitable candidate to investigate the early events of ad . in this study
, we investigated the time - ordered changes of pro - apoptotic and anti - apoptotic factors to determine the role of the upr signaling pathway in cognitive decline of 5fad mice
. the outcome may clarify the role of ers in the pathological progression of ad .
the 5fad app / ps1 transgenic b6/sjl mice with five familial inherited ad mutant genes ( app k670n / m671l [ sweden]+i716v [ florida]+v717i [ london]+ps1 m146l+l286v ) under the control of the neuron - specific thy-1 promoter and wild - type ( wt ) b6/sjl mice with the identical genetic background were provided by prof .
marry jo ladu ( department of anatomy and cell biology , university of illinois at chicago , usa ) .
experimental animals were housed in a pathogen - free colony ( ivc system , tecniplast , italy ) and allowed free access to food and water .
they were raised , 45 animals / cage , under 12-h light/12-h dark conditions at 22c25c with a humidity of 5060% . all protocols and procedures used in this study
were approved by the institutional animal care and use committee at fujian medical university in compliance with the us national institutes of health
the 5fad transgenic and wt mice at 2 , 7 , or 12 months of age were used for subsequent experiments with 1014 animals per group ( n = 12 in each 2-month - old group , n = 10 in each 7-month - old group , n = 10 in the 12-month - old wt group , and n = 14 in the 12-month - old 5fad group ) .
the swimming trace was monitored by camera and analyzed with smart 2.0 software ( panlab , barcelona , spain ) .
the escape latency(s ) and number of crossings over a hidden platform in 60 s were recorded .
mice were anesthetized using 10% chloral hydrate by intraperitoneal injection ( 3 ml / kg ) .
then , left ventricular perfusion was performed and brain tissues were isolated quickly on ice .
brain tissues were cut along the central sagittal suture , and the left hemisphere was fixed in 4%
paraformaldehyde/0.1 mol / l phosphate - buffered saline ( ph 7.4 ) at 4c for 24 h , followed by dehydration in 30% sucrose buffer for 4872 h. the brain tissues were then embedded and cut into 30 m cortical slices using a freezing microtome ( cm1850 , leica , wetzlar , germany ) and stored at 20c .
immunohistochemistry was performed as follows : brain slices were washed with tris - buffered saline ( tbs ) and treated with 10% hydrogen peroxide at room temperature for 10 min to diminish endogenous catalase activity .
the brain slices were then blocked with a buffer ( containing 5% goat serum [ gs ] , 0.25% bovine serum albumin [ bsa ] , 0.3% triton x-100 , tbs ) at room temperature for 1 h. primary antibodies 6e10 ( 1:8000 , covance , princeton , nj , usa ) and neun ( 1:4000 , abcam , cambridge , uk ) were diluted in a buffer ( containing 2% gs , 0.25% bsa , 0.3% triton x-100 , tbs ) and incubated at 4c overnight .
biotin - labeled secondary antibodies anti - mouse igg ( 1:600 ) and anti - rabbit igg ( 1:400 ) ( vector laboratories , burlingame , ca , usa ) were added subsequently and incubated at room temperature for 1.5 h. 3,3-diaminobenzidine staining was employed , and slices were air - dried at room temperature overnight .
the slices were then hydrated for 5 min , dehydrated using an ethanol gradient , treated with xylene , and finally mounted with neutral balsam .
the slices were imaged using a microscopy ( leica dm 4000b , germany ) , and image acquisition was performed with image - pro express 5.1 image analysis software ( media cybernetics , rockville , md , usa ) . for quantitative analysis , 6 mice were randomly selected from each group and 3 consecutive sections of each mouse were measured .
the prefrontal cortex region was selected as regions of interest , and the identical area within the measuring frame in a 10 objective lens was labeled .
the clear brown cellular boundaries were considered positive although positive cells outside the frame were rejected .
cells that were lightly stained or had irregular shapes were excluded from quantification .
brain slices were washed with tbs buffer and blocked with a specific buffer ( containing 5% donkey serum [ ds ] , 0.25% bsa , 0.3% triton x-100 , tbs ) at room temperature for 1 h. the primary antibodies ( diluted in 2% ds , 0.25% bsa , 0.3% triton x-100 , tbs ) used were : 6e10 , grp 78 ( 1:50 , santa cruz , ca , usa ) , chop ( 1:50 , santa cruz ) , glial fibrillary acidic protein ( gfap ) ( 1:4000 , millipore , boston , usa ) , and -iii - tubulin ( 1:4000 , abcam ) .
the 6e10 was co - incubated with grp 78 , chop , gfap , and tubulin primary antibodies .
alexa fluor 488- or 594-conjugated donkey anti - mouse or anti - rabbit igg ( 1:1500 , invitrogen , carlsbad , ca , usa ) were then added and incubated at room temperature in the dark for 1.5 h. the 4,6-diamidino-2-phenylindole ( dapi ) ( diluted by 1:8000 in h2o ) was then added and incubated for 5 min . prolong gold antifade reagent ( invitrogen ) was used for slice mounting , and slices were imaged using confocal microscopy ( leica tcs sp5 , leica microsystems wetzlar gmbh , germany ) .
triton x-100 , 50 mmol / l sodium fluoride , 2 mmol / l sodium orthovanadate , 10 mmol / l -sodium glycerophosphate , 10 mmol / l sodium pyrophosphate , 1% protease inhibitor cocktail [ p8340 , sigma - aldrich , st .
louis , mo , usa ] dissolved in tbs buffer , ph 7.4 ) at a ratio of 1:10 ( mg / ml ) .
the mixture was incubated on ice for 30 min , and lysis was performed using the ultrasound method .
supernatants were collected and proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis ( sds - page ) .
subsequently , the proteins were transferred to polyvinylidene fluoride ( pvdf ) membranes and blocked with 3% nonfat milk or 5% bsa in tbst buffer at room temperature for 2 h. the membranes were incubated at 4c overnight with the following primary antibodies : grp 78 ( 1:200 ) , chop ( 1:500 ) , sapk / jnk ( 1:1000 , cell signaling , boston , usa ) , p - sapk / jnk ( thr183/tyr185 ) ( 1:1000 , cell signaling ) , caspase-12 ( 1:500 , cell signaling ) , cleaved caspase-3 ( 1:500 , cell signaling ) , syvn1 ( 1:2000 , abcam ) , -actin ( 1:2000 , abcam ) , and -iii tubulin ( 1:100,000 ) .
horseradish peroxidase - labeled goat anti - rabbit or anti - mouse iggs ( 1:2000 ) were subsequently added and incubated at 37c for 1.5 h. imagej software ( national institutes of health , bethesda , ma , usa ) was used for quantitative analysis .
quantitative data were expressed as the mean standard error ( se ) and analyzed with the graphpad prism 6.01 software package ( graphpad , san diego , ca , usa ) .
escape latency in the behavior analysis was performed using a repeated - measure multiway analysis of variance ( anova ) while other data were evaluated using two - way anova . if the main effects and the interaction were significant , the genotypes were compared by student 's t - test , or multiple conditions among the same genotype were evaluated with one - way anova .
the 5fad app / ps1 transgenic b6/sjl mice with five familial inherited ad mutant genes ( app k670n / m671l [ sweden]+i716v [ florida]+v717i [ london]+ps1 m146l+l286v ) under the control of the neuron - specific thy-1 promoter and wild - type ( wt ) b6/sjl mice with the identical genetic background were provided by prof .
marry jo ladu ( department of anatomy and cell biology , university of illinois at chicago , usa ) .
experimental animals were housed in a pathogen - free colony ( ivc system , tecniplast , italy ) and allowed free access to food and water .
they were raised , 45 animals / cage , under 12-h light/12-h dark conditions at 22c25c with a humidity of 5060% . all protocols and procedures used in this study
were approved by the institutional animal care and use committee at fujian medical university in compliance with the us national institutes of health
the 5fad transgenic and wt mice at 2 , 7 , or 12 months of age were used for subsequent experiments with 1014 animals per group ( n = 12 in each 2-month - old group , n = 10 in each 7-month - old group , n = 10 in the 12-month - old wt group , and n = 14 in the 12-month - old 5fad group ) .
morris water maze tests were performed as described previously . the swimming behavior of mice was evaluated four times a day for 5 days .
the swimming trace was monitored by camera and analyzed with smart 2.0 software ( panlab , barcelona , spain ) .
the escape latency(s ) and number of crossings over a hidden platform in 60 s were recorded .
mice were anesthetized using 10% chloral hydrate by intraperitoneal injection ( 3 ml / kg ) . then , left ventricular perfusion was performed and brain tissues were isolated quickly on ice .
brain tissues were cut along the central sagittal suture , and the left hemisphere was fixed in 4%
paraformaldehyde/0.1 mol / l phosphate - buffered saline ( ph 7.4 ) at 4c for 24 h , followed by dehydration in 30% sucrose buffer for 4872 h. the brain tissues were then embedded and cut into 30 m cortical slices using a freezing microtome ( cm1850 , leica , wetzlar , germany ) and stored at 20c .
immunohistochemistry was performed as follows : brain slices were washed with tris - buffered saline ( tbs ) and treated with 10% hydrogen peroxide at room temperature for 10 min to diminish endogenous catalase activity .
the brain slices were then blocked with a buffer ( containing 5% goat serum [ gs ] , 0.25% bovine serum albumin [ bsa ] , 0.3% triton x-100 , tbs ) at room temperature for 1 h. primary antibodies 6e10 ( 1:8000 , covance , princeton , nj , usa ) and neun ( 1:4000 , abcam , cambridge , uk ) were diluted in a buffer ( containing 2% gs , 0.25% bsa , 0.3% triton x-100 , tbs ) and incubated at 4c overnight .
biotin - labeled secondary antibodies anti - mouse igg ( 1:600 ) and anti - rabbit igg ( 1:400 ) ( vector laboratories , burlingame , ca , usa ) were added subsequently and incubated at room temperature for 1.5 h. 3,3-diaminobenzidine staining was employed , and slices were air - dried at room temperature overnight .
the slices were then hydrated for 5 min , dehydrated using an ethanol gradient , treated with xylene , and finally mounted with neutral balsam .
the slices were imaged using a microscopy ( leica dm 4000b , germany ) , and image acquisition was performed with image - pro express 5.1 image analysis software ( media cybernetics , rockville , md , usa ) . for quantitative analysis ,
6 mice were randomly selected from each group and 3 consecutive sections of each mouse were measured .
the prefrontal cortex region was selected as regions of interest , and the identical area within the measuring frame in a 10 objective lens was labeled .
the clear brown cellular boundaries were considered positive although positive cells outside the frame were rejected . cells that were lightly stained or had irregular shapes were excluded from quantification .
brain slices were washed with tbs buffer and blocked with a specific buffer ( containing 5% donkey serum [ ds ] , 0.25% bsa , 0.3% triton x-100 , tbs ) at room temperature for 1 h. the primary antibodies ( diluted in 2% ds , 0.25% bsa , 0.3% triton x-100 , tbs ) used were : 6e10 , grp 78 ( 1:50 , santa cruz , ca , usa ) , chop ( 1:50 , santa cruz ) , glial fibrillary acidic protein ( gfap ) ( 1:4000 , millipore , boston , usa ) , and -iii - tubulin ( 1:4000 , abcam ) .
the 6e10 was co - incubated with grp 78 , chop , gfap , and tubulin primary antibodies .
alexa fluor 488- or 594-conjugated donkey anti - mouse or anti - rabbit igg ( 1:1500 , invitrogen , carlsbad , ca , usa ) were then added and incubated at room temperature in the dark for 1.5 h. the 4,6-diamidino-2-phenylindole ( dapi ) ( diluted by 1:8000 in h2o ) was then added and incubated for 5 min . prolong gold antifade reagent ( invitrogen ) was used for slice mounting , and slices were imaged using confocal microscopy ( leica tcs sp5 , leica microsystems wetzlar gmbh , germany ) .
isolated mouse cortical tissues were added into tissue lysates ( 1% triton x-100 , 50 mmol / l sodium fluoride , 2 mmol / l sodium orthovanadate , 10 mmol / l -sodium glycerophosphate , 10 mmol / l sodium pyrophosphate , 1% protease inhibitor cocktail [ p8340 , sigma - aldrich , st .
louis , mo , usa ] dissolved in tbs buffer , ph 7.4 ) at a ratio of 1:10 ( mg / ml ) .
the mixture was incubated on ice for 30 min , and lysis was performed using the ultrasound method .
supernatants were collected and proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis ( sds - page ) .
subsequently , the proteins were transferred to polyvinylidene fluoride ( pvdf ) membranes and blocked with 3% nonfat milk or 5% bsa in tbst buffer at room temperature for 2 h. the membranes were incubated at 4c overnight with the following primary antibodies : grp 78 ( 1:200 ) , chop ( 1:500 ) , sapk / jnk ( 1:1000 , cell signaling , boston , usa ) , p - sapk / jnk ( thr183/tyr185 ) ( 1:1000 , cell signaling ) , caspase-12 ( 1:500 , cell signaling ) , cleaved caspase-3 ( 1:500 , cell signaling ) , syvn1 ( 1:2000 , abcam ) , -actin ( 1:2000 , abcam ) , and -iii tubulin ( 1:100,000 ) .
horseradish peroxidase - labeled goat anti - rabbit or anti - mouse iggs ( 1:2000 ) were subsequently added and incubated at 37c for 1.5 h. imagej software ( national institutes of health , bethesda , ma , usa ) was used for quantitative analysis .
quantitative data were expressed as the mean standard error ( se ) and analyzed with the graphpad prism 6.01 software package ( graphpad , san diego , ca , usa ) .
escape latency in the behavior analysis was performed using a repeated - measure multiway analysis of variance ( anova ) while other data were evaluated using two - way anova . if the main effects and the interaction were significant , the genotypes were compared by student 's t - test , or multiple conditions among the same genotype were evaluated with one - way anova .
to investigate cognitive changes , we tested mice 's behavioral performance in the morris water maze .
we found that compared with age - matched wt mice , 7- and 12-month - old 5fad mice displayed a prolonged escape latency ( f = 14.710 , p < 0.01 for 7-month - old ; f = 5.939 , p < 0.05 for 12-month - old ) , indicating an obvious decline in learning ability and memory retention . in the probe trial ,
when the platform was removed , the 7- and 12-month - old 5fad mice crossed significantly less over the location of the removed platform than age - matched wt mice ( t = 2.331 , p < 0.05 for 7-month - old ; t = 2.075 , p < 0.05 for 12-month - old ) [ figure 1a ] .
immunohistochemical analysis showed that a was mainly localized in pyramidal neurons situated deep within the fifth layer of the cortex in 2-month - old mice .
a accumulation was increased in the brain tissues of 7-month - old mice and amyloid plaque deposition appeared around neurons secreting a. in 12-month - old mice , a large amount of plaque deposition was observed in the brain tissues [ figure 1b ] .
furthermore , we examined cleaved caspase-3 ( the activated form ) , an executor molecule , in the apoptotic cascade by western blots .
levels of cleaved caspase-3 in 5fad mice were increased in a time - dependent manner .
cleaved caspase-3 levels of 5fad mice were higher than those of wt mice at all - time points .
the increase was significant at the age of 12 months ( n = 8 , t = 2.504 , p < 0.05 ) [ figure 1c ] .
the number of neuron - positive staining in 12-month - old 5fad decreased in the fifth layer of the cortex when compared with that of the age - matched wt mice , indicating neuronal loss in 5fad mice ( n = 6 , t = 2.987 , p < 0.05 ) [ figure 1d and 1e ] . the declined cognition in 7-month - old 5fad mice and the loss of neurons in the frontal cortex of the 12-month - old ones .
escape latency and the number of crossings over the platform in 7- and 12-month old 5fad mice ( n = 1014 , *
p < 0.05 , p < 0.01 vs. age - matched wild - type mice ) . ( b ) 6e10 staining in 2- , 7- , and 12-month - old 5fad mice and wild - type mice . scale bar = 100 m .
( c ) western blots analysis showed that activated caspase-3 increased in 5fad mice at 12 months of age ( n = 8 , * p < 0.05 vs. wild - type mice ) .
( d ) the neurons within the fifth layer of the cortex was quantified ( n = 6 , * p < 0.05 vs. wild - type mice ) .
( e ) neun staining for neural nuclei in the frontal cortical slices from 2- , 7- , and 12-month - old mice .
scale bar = 100 m ; 5fad : transgenic mice with five familiar alzheimer 's disease ; wt : wild - type mice ; a : amyloid ; v : the fifth layer of the frontal cortex .
grp 78 is a chaperone specifically localized in the er , whose expression increases in response to ers and serves as an er - specific marker . in wild mice ,
grp 78 expression increased insignificantly from the age of 27 months . in 5fad mice ,
grp 78 expression decreased from the age of 2 to 12 months ( n = 6 , t = 5.629 , p < 0.01 for 12 vs. 2 months ) .
interestingly , at the age of 2 months , grp 78 expression in 5fad mice was significantly higher than that in age - matched wt mice ( n = 6 , t = 2.549 , p < 0.05 ) [ figure 2a ] .
grp 78 expression ( stained green ) in wt mice increased gradually from the age of 212 months . in 2-month - old 5fad mice ,
grp 78 expression was higher than that of age - matched wt mice in the fifth layer of the cortex [ figure 2c ] .
furthermore , grp 78 staining was localized mainly in the staining of -iii tubulin ( red ) , not in the staining of gfap ( red ) . meanwhile , the distribution of a and grp 78 staining ( red ) was localized totally with the 6e10 staining ( green ) , as shown in figure 2d .
these data indicate that grp 78 is expressed mainly in neurons , not in astrocytes .
significant upregulation of anti - apoptotic factors , grp 78 and syvn1 , in 2-month - old 5fad mice .
( a ) grp 78 expression of 5fad mice and age - matched wt mice at 2 , 7 , and 12 months old ( n = 6 , * p < 0.05 vs. 2-month - old wt mice ; p < 0.01 vs. 2-month - old 5fad mice ) . ( b ) syvn1 expression of 5fad mice and age - matched wt mice at 2 , 7 , and 12 months old ( n = 5 , * p < 0.05 vs. 2-month - old wt mice ) .
( c ) confocal images of grp 78 ( green ) , 6e10 ( a , red ) and nuclei ( dapi , blue ) in the cortical slices of 2- , 7- and 12-month - old mice by immunofluorescence staining .
( d ) confocal images of grp 78 ( green ) , gfap ( red ) or -iii - tubulin ( red ) in the frontal cortical slices of 7-month - old 5fad mice by immunofluorescent staining .
the bottom row of ( d ) indicates the immunofluorescent staining of grp 78 ( red ) and 6e10 ( green , targeting a ) .
5fad : transgenic mice with five familiar alzheimer 's disease ; wt : wild - type mice ; grp 78 : glucose - regulated protein 78 ; syvn1 : ubiquitin ligase synovial apoptosis inhibitor 1 ; a : amyloid ; 6e10 : the antibody targeting a ; gfap : glial fibrillary acidic protein ; dapi : 6-diamidino-2-phenylindole .
syvn1 , a component of the erad pathway , is involved in the degradation of abnormal proteins to reduce ers - induced apoptosis . at the age of 7 months and 12 months ,
however , at the age of 2 months , syvn1 expression in 5fad mice was obviously higher than that in age - matched wt mice ( n = 5 , t = 2.000 , p < 0.05 ) [ figure 2b ] .
although chop is rarely expressed in normal neurons , its expression can be increased by ers to promote er - specific apoptosis . in the current study ,
western blots analysis revealed that chop expression increased with age in both wt mice and 5fad mice ( n = 7 , t = 2.806 , p < 0.05 for 12-month - old wt mice vs. 2-month - old wt mice ) .
of note , compared with age - matched wt mice , chop expression increased significantly in 2-month - old 5fad mice ( n = 7 , t = 2.322 , p
the chop expression was further detected by immunofluorescence and the chop staining ( red ) showed the same changing trend as that of the western blots assay [ figure 3d ] .
punctate distribution of chop in the cytoplasm and nucleus indicated chop activation , which serves as a transcription factor .
furthermore , chop staining ( green ) was localized mainly in the staining of -iii tubulin ( red ) , not in the staining of gfap ( red ) .
meanwhile , chop staining ( red ) was localized totally with the 6e10 staining ( green ) , as shown in figure 3e .
these data indicate that chop is predominantly expressed in 6e10-positive neurons , not in astrocytes .
the cleaved caspase-12 expression was gradually increased with age in both wt mice ( n = 6 ; t = 6.280 , p < 0.01 and t = 2.625 , p < 0.05 , for 7- and 12-month - old mice vs. 2-month - old ones , respectively ) and 5fad mice ( n = 6 , t = 3.320 , p < 0.05 , t = 3.427 , p < 0.05 for 7- and 12-month - old mice vs. 2-month - old ones , respectively ) . of note ,
caspase-12 activation was higher in 5fad mice at 2- , 7- , and 12-month - old when compared with the activation status observed in wt mice .
this increase was significant at 2 and 7 months of age ( n = 6 , t = 5.365 , p < 0.01 for 2-month - old mice ; t = 2.869 , p < 0.05 for 7-month - old mice ) [ figure 3b ] .
significant increase of pro - apoptotic factors , chop and cleaved caspase-12 in 5fad mice .
( a ) western blots analyses revealed that chop protein increased with age in 5fad mice ( n = 7 , * p < 0.05 vs. 2-month wt mice ) .
( b ) western blots analysis showed that caspase-12 activation increased with age in 5fad mice ( n = 6 , * p < 0.05 , p < 0.01 vs. age - matched wt mice ; p <
0.05,p < 0.01 vs. 2-month wt mice ; p < 0.05 vs. 2-month 5fad mice ) .
( c ) western blots analysis showed the expression of p - jnk / jnk .
( d ) confocal images of chop ( red ) , 6e10 ( a , green ) and nuclei ( dapi , blue ) in the cortical slices of 2- , 7- , and 12-month - old mice by immunofluorescence staining .
( e ) confocal images of chop ( green ) , gfap ( red ) or -iii - tubulin ( red ) in the frontal cortical slices of 7-month - old 5fad mice by immunofluorescent staining .
the bottom row of ( e ) indicates immunofluorescent staining of chop ( red ) and 6e10 ( green , targeting a ) .
5fad : transgenic mice with five familiar alzheimer 's disease ; wt : wild - type mice ; chop : ccaat / enhancer - binding protein homologous protein ; jnk : c - jun amino - terminal kinase ; a : amyloid ; 6e10 : the antibody targeting a ; gfap : glial fibrillary acidic protein ; dapi : 6-diamidino-2-phenylindole . a recent study has reported that the p38 pathway is simultaneously activated to promote chop transcription to induce cell apoptosis . however , it remains unknown if jnk signaling is activated to trigger chop .
the present data displayed that p - jnk / jnk increased mildly in 5fad mice at 2- and 12-month - old when in comparison with the levels detected in wt mice although the increase was not statistically different .
these observations indicate that cell apoptosis in 5fad mice is not dependent on the jnk pathway [ figure 3c ] . taken together
, these data suggest that ers - specific chop and cleaved caspase-12 are sustainably upregulated in 5fad mice .
to investigate cognitive changes , we tested mice 's behavioral performance in the morris water maze .
we found that compared with age - matched wt mice , 7- and 12-month - old 5fad mice displayed a prolonged escape latency ( f = 14.710 , p < 0.01 for 7-month - old ; f = 5.939 , p < 0.05 for 12-month - old ) , indicating an obvious decline in learning ability and memory retention . in the probe trial ,
when the platform was removed , the 7- and 12-month - old 5fad mice crossed significantly less over the location of the removed platform than age - matched wt mice ( t = 2.331 , p < 0.05 for 7-month - old ; t = 2.075 , p < 0.05 for 12-month - old ) [ figure 1a ] .
immunohistochemical analysis showed that a was mainly localized in pyramidal neurons situated deep within the fifth layer of the cortex in 2-month - old mice .
a accumulation was increased in the brain tissues of 7-month - old mice and amyloid plaque deposition appeared around neurons secreting a. in 12-month - old mice , a large amount of plaque deposition was observed in the brain tissues [ figure 1b ] .
furthermore , we examined cleaved caspase-3 ( the activated form ) , an executor molecule , in the apoptotic cascade by western blots .
levels of cleaved caspase-3 in 5fad mice were increased in a time - dependent manner .
cleaved caspase-3 levels of 5fad mice were higher than those of wt mice at all - time points .
the increase was significant at the age of 12 months ( n = 8 , t = 2.504 , p < 0.05 ) [ figure 1c ] .
the number of neuron - positive staining in 12-month - old 5fad decreased in the fifth layer of the cortex when compared with that of the age - matched wt mice , indicating neuronal loss in 5fad mice ( n = 6 , t = 2.987 , p < 0.05 ) [ figure 1d and 1e ] . the declined cognition in 7-month - old 5fad mice and the loss of neurons in the frontal cortex of the 12-month - old ones .
escape latency and the number of crossings over the platform in 7- and 12-month old 5fad mice ( n = 1014 , *
p < 0.05 , p < 0.01 vs. age - matched wild - type mice ) . ( b ) 6e10 staining in 2- , 7- , and 12-month - old 5fad mice and wild - type mice . scale bar = 100 m .
( c ) western blots analysis showed that activated caspase-3 increased in 5fad mice at 12 months of age ( n = 8 , * p < 0.05 vs. wild - type mice ) .
( d ) the neurons within the fifth layer of the cortex was quantified ( n = 6 , * p < 0.05 vs. wild - type mice ) .
( e ) neun staining for neural nuclei in the frontal cortical slices from 2- , 7- , and 12-month - old mice .
scale bar = 100 m ; 5fad : transgenic mice with five familiar alzheimer 's disease ; wt : wild - type mice ; a : amyloid ; v : the fifth layer of the frontal cortex .
grp 78 is a chaperone specifically localized in the er , whose expression increases in response to ers and serves as an er - specific marker . in wild mice ,
grp 78 expression increased insignificantly from the age of 27 months . in 5fad mice ,
grp 78 expression decreased from the age of 2 to 12 months ( n = 6 , t = 5.629 , p < 0.01 for 12 vs. 2 months ) .
interestingly , at the age of 2 months , grp 78 expression in 5fad mice was significantly higher than that in age - matched wt mice ( n = 6 , t = 2.549 , p < 0.05 ) [ figure 2a ] .
grp 78 expression ( stained green ) in wt mice increased gradually from the age of 212 months . in 2-month - old 5fad mice ,
grp 78 expression was higher than that of age - matched wt mice in the fifth layer of the cortex [ figure 2c ] .
furthermore , grp 78 staining was localized mainly in the staining of -iii tubulin ( red ) , not in the staining of gfap ( red ) . meanwhile ,
the distribution of a and grp 78 staining ( red ) was localized totally with the 6e10 staining ( green ) , as shown in figure 2d .
these data indicate that grp 78 is expressed mainly in neurons , not in astrocytes .
significant upregulation of anti - apoptotic factors , grp 78 and syvn1 , in 2-month - old 5fad mice . ( a ) grp 78 expression of 5fad mice and age - matched wt mice at 2 , 7 , and 12 months old ( n = 6 , * p < 0.05 vs. 2-month - old wt mice ; p < 0.01 vs. 2-month - old 5fad mice ) .
( b ) syvn1 expression of 5fad mice and age - matched wt mice at 2 , 7 , and 12 months old ( n = 5 , * p < 0.05 vs. 2-month - old wt mice ) .
( c ) confocal images of grp 78 ( green ) , 6e10 ( a , red ) and nuclei ( dapi , blue ) in the cortical slices of 2- , 7- and 12-month - old mice by immunofluorescence staining .
( d ) confocal images of grp 78 ( green ) , gfap ( red ) or -iii - tubulin ( red ) in the frontal cortical slices of 7-month - old 5fad mice by immunofluorescent staining .
the bottom row of ( d ) indicates the immunofluorescent staining of grp 78 ( red ) and 6e10 ( green , targeting a ) .
5fad : transgenic mice with five familiar alzheimer 's disease ; wt : wild - type mice ; grp 78 : glucose - regulated protein 78 ; syvn1 : ubiquitin ligase synovial apoptosis inhibitor 1 ; a : amyloid ; 6e10 : the antibody targeting a ; gfap : glial fibrillary acidic protein ; dapi : 6-diamidino-2-phenylindole .
syvn1 , a component of the erad pathway , is involved in the degradation of abnormal proteins to reduce ers - induced apoptosis . at the age of 7 months and 12 months ,
however , at the age of 2 months , syvn1 expression in 5fad mice was obviously higher than that in age - matched wt mice ( n = 5 , t = 2.000 , p < 0.05 ) [ figure 2b ] .
although chop is rarely expressed in normal neurons , its expression can be increased by ers to promote er - specific apoptosis . in the current study ,
western blots analysis revealed that chop expression increased with age in both wt mice and 5fad mice ( n = 7 , t = 2.806 , p < 0.05 for 12-month - old wt mice vs. 2-month - old wt mice ) .
of note , compared with age - matched wt mice , chop expression increased significantly in 2-month - old 5fad mice ( n = 7 , t = 2.322 , p < 0.05 ) [ figure 3a ] .
the chop expression was further detected by immunofluorescence and the chop staining ( red ) showed the same changing trend as that of the western blots assay [ figure 3d ] .
punctate distribution of chop in the cytoplasm and nucleus indicated chop activation , which serves as a transcription factor .
furthermore , chop staining ( green ) was localized mainly in the staining of -iii tubulin ( red ) , not in the staining of gfap ( red ) .
meanwhile , chop staining ( red ) was localized totally with the 6e10 staining ( green ) , as shown in figure 3e .
these data indicate that chop is predominantly expressed in 6e10-positive neurons , not in astrocytes .
the cleaved caspase-12 expression was gradually increased with age in both wt mice ( n = 6 ; t = 6.280 , p < 0.01 and t = 2.625 , p < 0.05 , for 7- and 12-month - old mice vs. 2-month - old ones , respectively ) and 5fad mice ( n = 6 , t = 3.320 , p < 0.05 , t = 3.427 , p < 0.05 for 7- and 12-month - old mice vs. 2-month - old ones , respectively ) . of note ,
caspase-12 activation was higher in 5fad mice at 2- , 7- , and 12-month - old when compared with the activation status observed in wt mice .
this increase was significant at 2 and 7 months of age ( n = 6 , t = 5.365 , p
< 0.01 for 2-month - old mice ; t = 2.869 , p < 0.05 for 7-month - old mice ) [ figure 3b ] . significant increase of pro - apoptotic factors , chop and cleaved caspase-12 in 5fad mice .
( a ) western blots analyses revealed that chop protein increased with age in 5fad mice ( n = 7 , * p < 0.05 vs. 2-month wt mice ) .
( b ) western blots analysis showed that caspase-12 activation increased with age in 5fad mice ( n = 6 , * p < 0.05 , p < 0.01 vs. age - matched wt mice ; p <
0.05,p < 0.01 vs. 2-month wt mice ; p < 0.05 vs. 2-month 5fad mice ) .
( c ) western blots analysis showed the expression of p - jnk / jnk .
( d ) confocal images of chop ( red ) , 6e10 ( a , green ) and nuclei ( dapi , blue ) in the cortical slices of 2- , 7- , and 12-month - old mice by immunofluorescence staining .
( e ) confocal images of chop ( green ) , gfap ( red ) or -iii - tubulin ( red ) in the frontal cortical slices of 7-month - old 5fad mice by immunofluorescent staining .
the bottom row of ( e ) indicates immunofluorescent staining of chop ( red ) and 6e10 ( green , targeting a ) .
5fad : transgenic mice with five familiar alzheimer 's disease ; wt : wild - type mice ; chop : ccaat / enhancer - binding protein homologous protein ; jnk : c - jun amino - terminal kinase ; a : amyloid ; 6e10 : the antibody targeting a ; gfap : glial fibrillary acidic protein ; dapi : 6-diamidino-2-phenylindole . a recent study has reported that the p38 pathway is simultaneously activated to promote chop transcription to induce cell apoptosis .
the present data displayed that p - jnk / jnk increased mildly in 5fad mice at 2- and 12-month - old when in comparison with the levels detected in wt mice although the increase was not statistically different .
these observations indicate that cell apoptosis in 5fad mice is not dependent on the jnk pathway [ figure 3c ] . taken together
, these data suggest that ers - specific chop and cleaved caspase-12 are sustainably upregulated in 5fad mice .
in 5fad mice , we observed that neuronal loss and cleaved caspase-3 increase at the age of 12 months , cognitive deficit at the age of 7 months and 12 months .
interestingly , at the age of 2-month - old 5fad mice , the related factors involved in the ers - associated upr pathway , including chop , cleaved caspase-12 , grp 78 , and syvn1 , were significantly increased compared with those in age - matched wt mice .
these findings suggest that 2-month - old 5fad mice exhibit enhanced ers - associated upr pathway , consistent with intracellular a aggregation in neurons .
oakley et al . reported , in 5fad brain , accumulated intraneuronal a42 starting at 1.5 months of age , the presence of cerebral amyloid plaques and gliosis at 2 months of age , decreased synaptic markers including synaptophysin and postsynaptic density 95 ( psd95 ) accompanied by cognition deficit at 4 months of age , and pyramidal neuron loss in cortical layer 5 at 9 months of age .
consistently , in our study , the deposit of intra- and extra - neuronal a increased with age in 5fad brain ; neuronal loss in cortical layer 5 was observed at 12 months of age ; however , before significant neuron loss , impaired memory of 5fad mice at 7 months of age was monitored in the morris water maze .
our previous study has also shown that cognitive impairment present in 5fad mice was accompanied by structural degradation of synapses and decreased expression of synaptophysin and psd95 in the brain at the age of 67 months . under physiological conditions , approximately one - third of proteins in the er are assembled abnormally and do not form mature proteins .
aberrant proteins are bound by chaperone proteins , such as grp 78 , to be repaired or transported into the er - associated ubiquitin - proteasome system for degradation via the erad pathway , which serves as an accurate quality control system .
erad promotes the interaction between ubiquitin and proteins waiting for degradation via e1 , e2 , and e3 .
an impaired proteasome system leads to accumulation of aberrant proteins and increased risk of cell death .
sustained ers induces the activation of the er - specific apoptosis pathway by promoting the expression of chop and caspase-12 activation .
ers characterized by increased ers - specific apoptosis is obvious in postmortem examinations of brain tissues of ad patients . in vitro experiments have also confirmed that a induces the cellular ers response directly , indicating that abnormal aggregation of a is responsible for ers induction .
other studies have shown that astrocytes and macrophages also induce ers . however , in our study , the co - immunostaining of grp 78 and chop with astrocytic or neuronal markers showed that ers in cortical tissues occurred mainly in neurons .
therefore , the ers - associated proteins examined here represent neuronal ers and reveal the impact of the upr on neuronal functions under a-induced stress conditions . in this study , we selected mice at 2 , 7 , and 12 months of age to investigate the changes of ers - associated proteins in 5fad mice and wt mice .
our results showed that the expression of grp 78 and syvn1 changed without statistical significance but displayed an early - increase and subsequent - decline tendency from 2 to 12 months in wt mice .
interestingly , at the age of 2 months , the expression of grp 78 and syvn1 in 5fad brains was significantly higher than that in wt mice ; however , at the age of 12 months , grp 78 level in 5fad brains significantly declined when compared with that of 2-month - old 5fad mice .
meanwhile , the expression of chop and cleaved caspase-12 increased continuously with age , either in 5fad mice or in wt mice . of note , the level of chop and cleaved caspase-12 in 5fad mice was obviously higher than that in wt mice .
altogether , we speculate that a in 2-month - old 5fad brains lead to higher ers level than that of wt mice , which produces a cellular protective effect , on the one hand , up - regulating the expression of er chaperones and related degradation proteins mainly via pire1a - xbp1s and atf6a pathways at the early stage of ad to eliminate a ; on the other hand , inducing increased expression of downstream signaling pathways molecules , especially pro - apoptotic proteins ( cleaved caspase-12 and chop ) .
interestingly , no significant differences in pjnk / jnk levels were found between 5fad mice and wt mice at different periods , which indicates that the pjnk / jnk pathway is not involved in a-induced ers . under sustained stress conditions caused by the a-associated toxic effects ,
the protective function of the upr declines and the pro - apoptotic functions are enhanced gradually .
the pro - apoptotic functions of the upr may lead to functional impairment of neurons and even death [ figure 4 ] .
schematic drawing shows that activated upr pathways of ers induced by intraneuronal -amyloid in 5fad mice .
a in 2-month - old 5fad brains lead to higher ers level than that of wt mice , which produces a cellular protective effect , on the one hand , up - regulating the expression of er chaperones and related degradation proteins mainly via p - ire-1-xbp1s and atf6 pathways at the early stage of ad to eliminate a ; on the other hand , inducing increased expression of downstream signaling pathways molecules , especially pro - apoptotic proteins ( cleaved caspase-12 and chop ) . under sustained stress conditions caused by the a-associated toxic effects ,
the protective function of the upr declines and the pro - apoptotic functions are enhanced gradually .
the pro - apoptotic functions of the upr may lead to functional impairment of neurons and even death .
upr : unfolded protein response ; ers : endoplasmic reticulum stress ; a : amyloid ; 5fad : transgenic mice with five familiar alzheimer 's disease ; p - perk : phosphorylated protein kinase rna - like er kinase ; p - eif2 : phosphorylated eukaryotic translation initiation factor 2 ; atf4 : activating transcription factor 4 ; chop : ccaat / enhancer - binding protein homologous protein ; p - ire-1 : phosphorylated inositol - requiring enzyme 1 ; xbp1s : spliced x box - binding protein 1 ; atf6 : activating transcription factor 6 ; grp 78 : glucose - regulated protein 78 ; erad : endoplasmic reticulum - associated protein degradation ; syvn1 : ubiquitin ligase synovial apoptosis inhibitor 1 ; traf2 : tumor necrosis factor - receptor - associated factor 2 ; ask1 : apoptosis signal - regulating kinase 1 ; jnk : c - jun amino - terminal kinase . in conclusion ,
current data reveal that intracellular a aggregation induces obvious ers in neurons at the early stage of 5fad brains .
imbalanced regulation of grp 78 or syvn1 and chop or cleaved caspase-12 may be involved in the resistance to the restoration of er homeostasis in a-secreting neurons . if the development of ers can be delayed or reduced appropriately or the protective functions of the upr can be enhanced exogenously , the capacity of the er to tolerate abnormal proteins can be increased to delay cell damage and disease progression .
targeting at ers to control the pathophysiological changes at the early stage of ad may be a better strategy for the prevention and treatment of ad .
this work was supported by grants from the national natural science foundation of china ( no .
xiao - dong pan ) and the national and fujian province 's key clinical specialty discipline construction programs .
this work was supported by grants from the national natural science foundation of china ( no .
xiao - dong pan ) and the national and fujian province 's key clinical specialty discipline construction programs .
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background : amyloid ( a ) deposits and the endoplasmic reticulum stress ( ers ) are both well established in the development and progression of alzheimer 's disease ( ad ) .
however , the mechanism and role of a-induced ers in ad - associated pathological progression remain to be elucidated.methods:the five familial ad ( 5fad ) mice and wild - type ( wt ) mice aged 2 , 7 , and 12 months were used in the present study .
morris water maze test was used to evaluate their cognitive performance .
immunofluorescence and western blot analyses were used to examine the dynamic changes of pro - apoptotic ( ccaat / enhancer - binding protein homologous protein [ chop ] and cleaved caspase-12 ) and anti - apoptotic factors ( chaperone glucose - regulated protein [ grp ] 78 and endoplasmic reticulum - associated protein degradation - associated ubiquitin ligase synovial apoptosis inhibitor 1 [ syvn1 ] ) in the ers - associated unfolded protein response ( upr ) pathway.results:compared with age - matched wt mice , 5fad mice showed higher cleaved caspase-3 , lower neuron - positive staining at the age of 12 months , but earlier cognitive deficit at the age of 7 months ( all p < 0.05 ) .
interestingly , for 2-month - old 5fad mice , the related proteins involved in the ers - associated upr pathway , including chop , cleaved caspase-12 , grp 78 , and syvn1 , were significantly increased when compared with those in age - matched wt mice ( all p < 0.05 ) .
moreover , ers occurred mainly in neurons , not in astrocytes.conclusions:these findings suggest that compared with those of age - matched wt mice , ers - associated pro - apoptotic and anti - apoptotic proteins are upregulated in 2-month - old 5fad mice , consistent with intracellular a aggregation in neurons .
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Animal feeding and grouping
Behavior analysis
Immunohistochemistry
Immunocytochemistry
Western blots
Statistical analysis
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Increased amyloid induces the declined cognition in 7-month-old five familial Alzheimer's disease mice and the loss of neurons in the frontal cortex of the 12-month-old ones
Anti-apoptotic factors, glucose-regulated protein 78 and SYVN1, are significantly upregulated in 2-month-old five familial Alzheimer's disease mice
Pro-apoptotic factors, CHOP and cleaved caspase-12, are significantly increased in five familial Alzheimer's disease mice
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Financial support and sponsorship
Conflicts of interest
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cvds are the leading causes of death in diabetic patients and the substantial rise in diabetes prevalence will ultimately lead to an increase in the burden of diabetes - related cvd in coming decades . among the cardiovascular diseases ,
ischemic heart disease is the most frequent [ 5 , 6 ] and is associated with the highest morbidity and mortality in patients with type 2 dm . despite the advances in the treatment of coronary artery disease ( cad ) , morbidity and mortality are still high , especially in patients with type 2 dm .
moreover , despite the evolution in the treatment of hyperglycemia , patients with both cad and diabetes have worse clinical outcomes , irrespective of the treatment applied .
although dm alters the healthy functioning of arteries and blood compounds , some clinical studies suggest that myocardial responses to ischemic insults may be deficient in diabetic patients [ 11 , 12 ] , leading to higher myocardial damage and risk of complications .
thus , recent studies have focused on the understanding of such myocardial responses , aiming at discovering the mechanisms of myocardial protection to achieve less aggression and a better prognosis .
a substantial effort has been put forth to investigate any promising cardioprotective strategy to effectively reduce myocardial infarct size .
and this matter is of particular importance to diabetic cad patients . by 1986 , in a landmark experimental study , murry and colleagues demonstrated that a short antecedent period of ischemia could result in a great reduction in myocardial infarct size .
for the first time , a method for reducing myocardial cell death other than reperfusion had been discovered .
since its discovery , this phenomenon , termed ischemic preconditioning ( ip ) has been extensively studied .
currently , ip is the intrinsic myocardial mechanism that provides the greatest protection regarding the reduction in myocardial ischemic damage .
it has demonstrated a 75 % reduction in the infarcted area in animal models that have undergone the preconditioning protocol .
ip is assumed as a mechanism of myocardial protection in which brief episodes of sublethal myocardial ischemia followed by reperfusion trigger multiple intracellular pathways that ultimately result in greater myocardial resistance to a subsequent intense ischemic injury .
experimentally , this phenomenon was demonstrated by the reduction in the infarcted area by short episodes of ischemia prior to a pronounced ischemic insult . although ip was initially thought to result from the opening of collateral vessels and higher coronary flow , some interesting studies have shown that the phenomenon occurs irrespective of coronary flow changes [ 17 , 18 ] .
such studies also demonstrated that ip can be observed by sequential exercise tests ( sets ) , in which the improvement in ischemic parameters in the second of 2 sets were confirmed by invasive measurements of myocardial oxygen consumption .
moreover , the clinical observation that patients with chronic ischemic heart disease frequently describe attenuation or even cessation of angina symptoms if they rest and restart the exercise is termed warm - up or walk - through angina . this phenomenon has been related to ip and documented in studies using sets [ 19 , 20 ] . following the demonstration of ip , the cellular mechanisms underlying the phenomenon began to be investigated .
although some cellular pathways have not been fully elucidated , there is a growing understanding of some phenomena and currently it is assumed that ip may be modulated by external factors like age , diseases [ 2224 ] , as well as some specific classes of medications [ 2527 ] .
currently , the opening of k - atp channels plays a fundamental role in the cellular cascades of ip .
it is also assumed that medications that bind to this channel may interfere with the ip mechanism .
thus , some classes of medications like the oral hypoglycemic agents may also block such channels in extrapancreatic sites , such as in the heart .
it has been shown by some authors that these medications may cause the blockage of myocardial ip . however , despite the fact that some oral hypoglycemic agents may interfere with ip , it is still uncertain whether the intrinsic , complex , intracellular alterations of diabetes itself may affect the cellular mechanisms of this cardioprotective phenomenon .
some of these studies have shown that diabetes does not interfere with ip [ 31 , 32 ] , but others have shown negative influences [ 33 , 34 ] .
the great variability in these results is especially due to differences in study protocols , most notably the variability in animal models studied , in the protocols to induce diabetes , the duration of the disease , as well as the variability in myocardial injury protocols .
on the other hand , studies in humans are scarce and their results have also been conflicting [ 12 , 13 , 35 ] .
thus , in this study , we aimed at identifying ip in symptomatic multivessel cad patients and compared the results among patients with and without type 2 dm .
this was a prospective study that included patients with symptomatic cad followed by the medicine , angioplasty , or surgery study ( mass ) research group .
briefly , patients were enrolled who had angina symptoms , multivessel cad , preserved left systolic ventricular function , and a recent , positive ischemic treadmill stress test .
the diagnosis of type 2 dm was based on american diabetes association guidelines diagnostic criteria .
multivessel cad was confirmed by the finding on cineangiocoronariography of atherosclerotic stenosis of at least 70 % obstruction in 2 or more coronary artery territories .
the systolic ventricular function was measured by transthoracic echocardiography and was considered preserved if ejection fraction was 0.50 .
treadmill exercise tests were considered positive for myocardial ischemia if a horizontal or downsloping st - segment deviation was 1.0 mm , associated or not with thoracic discomfort .
exclusion criteria were single - vessel cad , left main cad , impaired systolic ventricular function ( defined as an ejection fraction < 0.50 ) , recent and negative treadmill exercise test , high - risk positive treadmill exercise test , limiting angina symptoms , an acute coronary syndrome in the prior 3 months , electrocardiographic signs that could make difficult the interpretation of ischemic changes ( left bundle - branch block , st - segment deviation ) , arrhythmias like atrial fibrillation or flutter , severe valvular disease , cardiomyopathies , or patient refusal to participate in this study .
after cardiologic evaluation , all patients were instructed to stop medications with cardiovascular properties 5 days before sets .
diabetic patients were also instructed to stop antidiabetes medications 5 days before the tests .
patients were also recommended to not perform physical activities during the period without the use of medications and were under appropriate nutritional control .
a telephone contact was available 24 h a day in case of questions or worsening symptoms . before treadmill test initiation ,
all patients underwent 2 sets , symptom limited , with a 30-min interval between them .
tests were conducted during the same period each day , 1 h after lunchtime , on the same treadmill , ( mat 2100 treadmill and a fukuda denshi ml8000 stress test system ( fukuda denshi ; bunkyo - ku , tokyo , japan ) . a 12-lead electrocardiogram , heart rate , and arterial blood pressure were obtained with the patient in the standing position at baseline .
a 12-lead electrocardiogram was also obtained at each 1.0-min interval during exercise , at peak exercise , each minute up to 5 min during the recovery phase , at the onset of 1.0 mm st - segment depression , during arrhythmias , and when it was clinically relevant .
the level of the st - segment deviation was based on visual assessment of the 0.08 s after the j point by 2 independent cardiologists . in case of disagreement
only the horizontal or downsloping st - segment deviations were considered for the time to onset of 1.0-mm st - segment depression evaluation ( t-1.0 mm ) .
criteria for interrupting the exercise test were st - segment depression 3.0 mm , st segment elevation 2.0 mm , maximum age - related heart rate , severe arterial hypotension or hypertension , severe chest pain , physical exhaustion , and sustained arrhythmias .
the following parameters were systematically measured : resting heart rate and arterial blood pressure , heart rate and arterial blood pressure at peak exercise , t-1.0 mm in seconds , rate pressure product ( rpp ) at the onset of t-1.0 mm , and exercise duration in seconds .
the improvement in ischemic parameters ( t-1.0 mm and rpp ) in the second exercise test compared to the first one indicated the presence of ip .
other parameters recorded during sets were the total exercise time , the occurrence and density of supraventricular and ventricular arrhythmias , and st - segment deviation morphology , during exercise and recovery phases .
both arrhythmias and st - segment morphology were graded according to their density and severity .
according to the levels of fasting glycemia and glycosylated hemoglobin from baseline , patients were separated into quartiles . for fasting glycemia ,
the quartiles were defined as quartile 1 : < 90 mg / dl , quartile 2 : 90100 mg / dl , quartile 3 : 101125 mg / dl , and quartile 4 : 126 mg / dl . for glycosylated hemoglobin ,
the quartiles were defined as quartile 1 : < 5.7 % , quartile 2 : 5.76.3 % , quartile 3 : 6.46.9 % , and quartile 4 : 7.0 % .
briefly , it was determined by the analysis of studies with similar methodologies that included only diabetic patients and studies that included only nondiabetic patients . by the differences in t-1.0 mm in diabetic and nondiabetic patients , and accounting for an expected loss of 30 % of patients who do not demonstrate the ip phenomenon ( estimated based on the experience of our research group )
, 70 patients with diabetes and 70 without diabetes should be included to test the null hypothesis that the population means were similar ( power 0.9 and alpha error 0.05 ) .
continuous variables were compared using an unpaired student s t test or mann whitney test , when appropriate .
data are expressed as mean standard deviation or as absolute frequencies and percentages .
all tests were 2-sided , and a value of p < 0.05 was considered significant .
this was a prospective study that included patients with symptomatic cad followed by the medicine , angioplasty , or surgery study ( mass ) research group .
briefly , patients were enrolled who had angina symptoms , multivessel cad , preserved left systolic ventricular function , and a recent , positive ischemic treadmill stress test .
the diagnosis of type 2 dm was based on american diabetes association guidelines diagnostic criteria .
multivessel cad was confirmed by the finding on cineangiocoronariography of atherosclerotic stenosis of at least 70 % obstruction in 2 or more coronary artery territories .
the systolic ventricular function was measured by transthoracic echocardiography and was considered preserved if ejection fraction was 0.50 .
treadmill exercise tests were considered positive for myocardial ischemia if a horizontal or downsloping st - segment deviation was 1.0 mm , associated or not with thoracic discomfort .
exclusion criteria were single - vessel cad , left main cad , impaired systolic ventricular function ( defined as an ejection fraction < 0.50 ) , recent and negative treadmill exercise test , high - risk positive treadmill exercise test , limiting angina symptoms , an acute coronary syndrome in the prior 3 months , electrocardiographic signs that could make difficult the interpretation of ischemic changes ( left bundle - branch block , st - segment deviation ) , arrhythmias like atrial fibrillation or flutter , severe valvular disease , cardiomyopathies , or patient refusal to participate in this study .
after cardiologic evaluation , all patients were instructed to stop medications with cardiovascular properties 5 days before sets .
diabetic patients were also instructed to stop antidiabetes medications 5 days before the tests .
patients were also recommended to not perform physical activities during the period without the use of medications and were under appropriate nutritional control .
a telephone contact was available 24 h a day in case of questions or worsening symptoms . before treadmill test initiation ,
all patients underwent 2 sets , symptom limited , with a 30-min interval between them . the modified bruce protocol was applied .
tests were conducted during the same period each day , 1 h after lunchtime , on the same treadmill , ( mat 2100 treadmill and a fukuda denshi ml8000 stress test system ( fukuda denshi ; bunkyo - ku , tokyo , japan ) . a 12-lead electrocardiogram , heart rate , and arterial blood pressure were obtained with the patient in the standing position at baseline .
a 12-lead electrocardiogram was also obtained at each 1.0-min interval during exercise , at peak exercise , each minute up to 5 min during the recovery phase , at the onset of 1.0 mm st - segment depression , during arrhythmias , and when it was clinically relevant .
the level of the st - segment deviation was based on visual assessment of the 0.08 s after the j point by 2 independent cardiologists . in case of disagreement ,
only the horizontal or downsloping st - segment deviations were considered for the time to onset of 1.0-mm st - segment depression evaluation ( t-1.0 mm ) .
criteria for interrupting the exercise test were st - segment depression 3.0 mm , st segment elevation 2.0 mm , maximum age - related heart rate , severe arterial hypotension or hypertension , severe chest pain , physical exhaustion , and sustained arrhythmias .
the following parameters were systematically measured : resting heart rate and arterial blood pressure , heart rate and arterial blood pressure at peak exercise , t-1.0 mm in seconds , rate pressure product ( rpp ) at the onset of t-1.0 mm , and exercise duration in seconds .
the improvement in ischemic parameters ( t-1.0 mm and rpp ) in the second exercise test compared to the first one indicated the presence of ip .
other parameters recorded during sets were the total exercise time , the occurrence and density of supraventricular and ventricular arrhythmias , and st - segment deviation morphology , during exercise and recovery phases .
both arrhythmias and st - segment morphology were graded according to their density and severity . according to the levels of fasting glycemia and glycosylated hemoglobin from baseline , patients were separated into quartiles . for fasting glycemia , the quartiles were defined as quartile 1 : < 90 mg / dl , quartile 2 : 90100 mg / dl , quartile 3 : 101125 mg / dl , and quartile 4 : 126 mg / dl . for glycosylated hemoglobin ,
the quartiles were defined as quartile 1 : < 5.7 % , quartile 2 : 5.76.3 % , quartile 3 : 6.46.9 % , and quartile 4 : 7.0 % .
the sample size calculation has been previously published elsewhere . briefly , it was determined by the analysis of studies with similar methodologies that included only diabetic patients and studies that included only nondiabetic patients . by the differences in t-1.0 mm in diabetic and nondiabetic patients , and accounting for an expected loss of 30 % of patients who do not demonstrate the ip phenomenon ( estimated based on the experience of our research group ) , 70 patients with diabetes and 70 without diabetes should be included to test the null hypothesis that the population means were similar ( power 0.9 and alpha error 0.05 ) .
continuous variables were compared using an unpaired student s t test or mann whitney test , when appropriate .
data are expressed as mean standard deviation or as absolute frequencies and percentages .
all tests were 2-sided , and a value of p < 0.05 was considered significant .
of 2,140 patients with stable cad followed at our institution , 361 met the inclusion criteria .
the main reasons for non - inclusion and exclusion are shown in fig . 1 .
thus , a total of 174 patients completed the 2 sets and had ip assessed .
the study population comprised 86 patients with dm and 88 without this diagnosis ( fig .
the 2,140 initial cad patients screened , 361 met the inclusion criteria and were enrolled , and 174 completed the study protocol .
cad = coronary artery disease ; ckd = chronic kidney disease ; cva = cerebrovascular accident ; ecg = electrocardiogram ; ef = ejection fraction ; et = exercise test ; dm = diabetes mellitus ; ip = ischemic preconditioning ; set = sequential exercise test ( s ) study flow chart . of
the 2,140 initial cad patients screened , 361 met the inclusion criteria and were enrolled , and 174 completed the study protocol .
cad = coronary artery disease ; ckd = chronic kidney disease ; cva = cerebrovascular accident ; ecg = electrocardiogram ; ef = ejection fraction ; et = exercise test ; dm = diabetes mellitus ; ip = ischemic preconditioning ; set = sequential exercise test ( s ) the main demographic , clinical and biochemical characteristics of the 2 groups are presented in table 1 . despite the higher prevalence of previous myocardial infarction in the diabetic population and lipid profile ( higher ldl - cholesterol levels in nondiabetic patients ) , both groups had homogeneous characteristics .
the duration of diabetes was 11.5 8.8 years ( mean sd ) , with a median of 10 years ( interquartiles ranges of 6 and 15 years ) .
table 2 shows the medications used in the two groups of patients.table 1main demographic , biochemical , and clinical characteristics of the study populationtotal n = 174diabetics ( n = 86)non - diabetics ( n = 88 )
p valueage64.1 6.864.3 8.60.84male n ( % ) 73 ( 84.9)76 ( 86.3)0.83hypertension64 ( 80)74 ( 84.1)0.54smokers7 ( 8.8)7 ( 8.6)previous smokers38 ( 48.1)33 ( 40.7)0.6non - smokers34 ( 43.0)41 ( 50.6)previous ami36 ( 46.1)17 ( 22.7)0.004cabg28 ( 34.6)23 ( 26.1)0.25pci24 ( 30.4)28 ( 32.2)0.87ef0.61 0.060.62 0.060.2tri - vessel disease38 ( 52)43 ( 51.2)1.0bi - vessel disease35 ( 48)41 ( 48.8)1.0lad disease63 ( 87.5)74 ( 86.0)1.0collateral circulation34 ( 50.7)38 ( 46.9)0.64collateral grade 2/330 ( 88.2)32 ( 84.2)0.74weight ( kg)74.5 11.073.2 12.70.51height ( m)1.66 8.11.66 9.10.75bmi26.5 2.826.4 3.20.54fasting glycemia143.5 47.099.0 9.40.0001a1c7.35 1.615.63 0.300.0001bun41.9 13.440.1 9.40.32creatinine1.04
0.231.07 0.200.37total cholesterol156.5 34.5168.9 36.30.02ldl cholesterol90.5 27.4102.0 32.70.01hdl cholesterol38.0 9.640.3 9.00.12triglycerides147.2 88.3132.6 72.30.24data are expressed as means standard deviation or as absolute and relative risks
ami stands for acute myocardial infarction , cabg coronary artery bypass surgery , pci percutaneous coronary intervention , ef ejection fraction , lad left anterior descending , bmi body mass index , a1c glycosylated hemoglobin , bun blood urea nitrogen , ldl low - density lipoprotein , hdl high - density lipoproteintable 2classes of medications used by the two groups of diabetic and nondiabetic patientsaspirin / clopidogrelstatinsbeta - blockersacei / arbcalcium blockersdiureticsoadinsulinsdm93.2 % 93.2 % 89.8 % 78.4 % 35.2 % 37.5 % 88.5 % 26.5 % non - dm96.5 % 96.5 % 86.2 % 78.2 % 36.8 % 34.5 % --
dm stands for diabetic patients , non - dm nondiabetic patients , acei angiotensin converting enzyme inhibitors , arb angiotensin receptor blockers , oad oral antidiabetic drugs main demographic , biochemical , and clinical characteristics of the study population data are expressed as means standard deviation or as absolute and relative risks
ami stands for acute myocardial infarction , cabg coronary artery bypass surgery , pci percutaneous coronary intervention , ef ejection fraction , lad left anterior descending , bmi body mass index , a1c glycosylated hemoglobin , bun blood urea nitrogen , ldl low - density lipoprotein , hdl high - density lipoprotein classes of medications used by the two groups of diabetic and nondiabetic patients
dm stands for diabetic patients , non - dm nondiabetic patients , acei angiotensin converting enzyme inhibitors , arb angiotensin receptor blockers , oad oral antidiabetic drugs among the 86 diabetic patients , 62 ( 72 % ) had an improvement in t-1.0 mm consistent with ip . among the 88 nondiabetic patients , 60 ( 68 % ) had an ischemic improvement consistent with ip ( fig . 2 , p = 0.62).fig .
2pie charts showing the number and percentage of diabetic and nondiabetic patients who demonstrated ip .
ip = ischemic preconditioning pie charts showing the number and percentage of diabetic and nondiabetic patients who demonstrated ip .
ip = ischemic preconditioning the t-1.0 mm results demonstrated that diabetic patients who demonstrated ip had an improvement in t-1.0 mm between the 2 sets of 79.4 47.6 s , whereas nondiabetic patients who demonstrated ip had an improvement of 65.5 36.4 s ( table 3 , p = 0.12).table 3t-1.0 mm in exercise test 1 ( et1 ) and exercise test 2 ( et2 ) and the difference between the 2 tests ( et2-et1 ) in diabetic and nondiabetic patients who demonstrated ip ( ip+ ) or who did not demonstrate ip ( ip-)et1et2et2-et1
p value*dm269.2 117.8320.9 132.351.7 63.20.15non - dm275.6 111.9314.6
126.239.0 52.3 dm / ip+274.8 102.8354.3 115.179.4 47.60.12non - dm / ip+296.9 107.7362.4 108.565.5 36.4 dm / ip -254.7 151.6234.7 137.020.0 36.40.80non - dm / ip -230.1 108.7212.4 98.217.7 31.9data are expressed as means standard deviation .
dm stands for diabetes mellitus , ip + ischemic preconditioning present , ip - ischemic preconditioning absent , et , exercise test . *
the p values are from the comparison of et2-et1 among diabetic and nondiabetic patients t-1.0 mm in exercise test 1 ( et1 ) and exercise test 2 ( et2 ) and the difference between the 2 tests ( et2-et1 ) in diabetic and nondiabetic patients who demonstrated ip ( ip+ ) or who did not demonstrate ip ( ip- ) data are expressed as means standard deviation .
dm stands for diabetes mellitus , ip + ischemic preconditioning present , ip - ischemic preconditioning absent , et , exercise test .
* the p values are from the comparison of et2-et1 among diabetic and nondiabetic patients regarding rpp , the group of diabetic patients who demonstrated ip had an improvement of 3,011 2,430 bpm x mmhg , whereas nondiabetic patients had an improvement of 2,081 2,139 bpm x mmhg ( table 4 , p = 0.01).table 4rpp in exercise test 1 ( et1 ) and exercise test 2 ( et2 ) and the difference between the 2 tests ( et2-et1 ) in diabetic and nondiabetic patients who demonstrated ip ( ip+ ) or who did not demonstrate ip ( ip-)et1et2et2-et1
p value*dm / ip+22,841 4,50025,853 4,7423,011 2,4300.01non - dm / ip + 23,094 5,31225,175 5,4852,081 2,139 dm / ip -22,705 4,00022,124 3,686580 2,2500.43non - dm / ip -23,910 5,38022,869 5,3511,050 2,027data are expressed as means standard deviation .
dm stands for diabetes mellitus , ip + ischemic preconditioning present , ip - ischemic preconditioning absent , et exercise test . *
the p values are from the comparison of et2-et1 among diabetic and nondiabetic patients rpp in exercise test 1 ( et1 ) and exercise test 2 ( et2 ) and the difference between the 2 tests ( et2-et1 ) in diabetic and nondiabetic patients who demonstrated ip ( ip+ ) or who did not demonstrate ip ( ip- ) data are expressed as means standard deviation .
dm stands for diabetes mellitus , ip + ischemic preconditioning present , ip - ischemic preconditioning absent , et exercise test . *
the p values are from the comparison of et2-et1 among diabetic and nondiabetic patients table 5 shows the hemodynamic parameters heart rate and blood pressure at baseline and peak exercise in the 4 groups of patients.table 5hemodynamic parameters , heart rate and blood pressure in the 4 groups of patients , diabetic and nondiabetic , according to the demonstration of ipvariableset 1et 2et2-et1 dm / ip + hr ( baseline)79.0 15.186.3 17.07.3 11.0hr ( peak)139.0
18.2149.7 19.36.8 12.6bp ( peak)192.0 25.0191.8 23.90.16 15.4 dm / ip -hr ( baseline)79.0 14.784.4 14.95.4 8.0hr ( peak)141.7 14.3143.0 13.51.3 7.4bp ( baseline)153.3 22.6147.1 19.76.2 14.7bp ( peak)183.7 23.2180.6 22.23.12 14.7non - dm / ip + hr ( baseline)78.0
13.187.1 13.99.1 7.9hr ( peak)139.6 15.4144.3 14.74.7 5.7bp ( baseline)146.7 20.6138.7 18.38.0 11.3bp ( peak)193.5 23.6190.2 24.53.3 12.4non - dm / ip -hr ( baseline)82.5 12.987.1 12.64.6 7.1hr ( peak)139.3 12.4139.6 13.00.35 5.2bp ( baseline)158.6 26.2147.9 26.110.7 14.6bp ( peak)204.6 25.3194.8 28.18.9
dm stands for diabetic patients , non - dm nondiabetic patients , ip + ischemic preconditioning present , ip - ischemic preconditioning absent , et exercise test hemodynamic parameters , heart rate and blood pressure in the 4 groups of patients , diabetic and nondiabetic , according to the demonstration of ip data are expressed as means standard deviation .
dm stands for diabetic patients , non - dm nondiabetic patients , ip + ischemic preconditioning present , ip - ischemic preconditioning absent , et exercise test the improvement in the total exercise time comparing the 2 sets was similar between the groups of diabetic and nondiabetic patients ( 20 39 s versus 17 36 s , respectively , p = 0.60 ) .
the improvement in the frequency and severity of arrhythmias had similar results among diabetic and nondiabetic patients who experienced ip ( 50 % versus 63 % of patients demonstrated improvement in arrhythmias , respectively ; p = 0.41 ) .
the improvement in the st - segment deviation morphology was also similar among diabetic and nondiabetic patients ( 38.5 % versus 48.3 % , respectively , p = 0.41 ) .
when the total group of patients ( n = 174 ) was stratified according to the demonstration of ip , the frequency of diabetic patients as well as the levels of fasting glycemia and glycosylated hemoglobin were similar between the 2 groups , as shown in table 6.table 6frequency of diabetes mellitus , and levels of fasting glycemia and a1c in the study population according to the expression of ipip + ( n = 122)ip - ( n = 52 )
p valuedm n ( % ) 62 ( 50.8)24 ( 46.1)0.57fasting glycemia121.3 42.6118.9 34.10.72a1c6.63 1.66.32 1.20.25data are expressed as means standard deviation and as absolute and relative frequencies
dm stands for diabetes mellitus , a1c glycosylated hemoglobin , ip + ischemic preconditioning present , ip - ischemic preconditioning absent frequency of diabetes mellitus , and levels of fasting glycemia and a1c in the study population according to the expression of ip data are expressed as means standard deviation and as absolute and relative frequencies
dm stands for diabetes mellitus , a1c glycosylated hemoglobin , ip + ischemic preconditioning present , ip - ischemic preconditioning absent in addition , when we stratified patients by quartiles of glycemia and glycosylated hemoglobin , there was no statistical difference in terms of ip demonstration among the different quartiles ( fig .
3graphs showing the percentage of patients who demonstrated ischemic preconditioning ( ip+ in blue ) and who did not demonstrate ischemic preconditioning ( ip - in red ) stratified into quartiles of a1c ( graph a ) and fasting glycemia ( graph b ) .
ip = ischemic preconditioning ; q = quartile ( s ) .
x axis represents the percentage of patients and y axis the quartiles of a1c and fasting glycemia graphs showing the percentage of patients who demonstrated ischemic preconditioning ( ip+ in blue ) and who did not demonstrate ischemic preconditioning ( ip - in red ) stratified into quartiles of a1c ( graph a ) and fasting glycemia ( graph b ) . ip = ischemic preconditioning ; q = quartile ( s ) .
x axis represents the percentage of patients and y axis the quartiles of a1c and fasting glycemia
among the 86 diabetic patients , 62 ( 72 % ) had an improvement in t-1.0 mm consistent with ip . among the 88 nondiabetic patients , 60 ( 68 % ) had an ischemic improvement consistent with ip ( fig . 2 , p = 0.62).fig .
2pie charts showing the number and percentage of diabetic and nondiabetic patients who demonstrated ip .
ip = ischemic preconditioning pie charts showing the number and percentage of diabetic and nondiabetic patients who demonstrated ip .
ip = ischemic preconditioning the t-1.0 mm results demonstrated that diabetic patients who demonstrated ip had an improvement in t-1.0 mm between the 2 sets of 79.4 47.6 s , whereas nondiabetic patients who demonstrated ip had an improvement of 65.5 36.4 s ( table 3 , p = 0.12).table 3t-1.0 mm in exercise test 1 ( et1 ) and exercise test 2 ( et2 ) and the difference between the 2 tests ( et2-et1 ) in diabetic and nondiabetic patients who demonstrated ip ( ip+ ) or who did not demonstrate ip ( ip-)et1et2et2-et1
p value*dm269.2 117.8320.9 132.351.7 63.20.15non - dm275.6 111.9314.6 126.239.0 52.3 dm / ip+274.8 102.8354.3 115.179.4 47.60.12non - dm / ip+296.9 107.7362.4
108.565.5 36.4 dm / ip -254.7 151.6234.7 137.020.0 36.40.80non - dm / ip -230.1 108.7212.4 98.217.7 31.9data are expressed as means standard deviation .
dm stands for diabetes mellitus , ip + ischemic preconditioning present , ip - ischemic preconditioning absent , et , exercise test . *
the p values are from the comparison of et2-et1 among diabetic and nondiabetic patients t-1.0 mm in exercise test 1 ( et1 ) and exercise test 2 ( et2 ) and the difference between the 2 tests ( et2-et1 ) in diabetic and nondiabetic patients who demonstrated ip ( ip+ ) or who did not demonstrate ip ( ip- ) data are expressed as means standard deviation
. dm stands for diabetes mellitus , ip + ischemic preconditioning present , ip - ischemic preconditioning absent , et , exercise test . *
the p values are from the comparison of et2-et1 among diabetic and nondiabetic patients regarding rpp , the group of diabetic patients who demonstrated ip had an improvement of 3,011 2,430 bpm x mmhg , whereas nondiabetic patients had an improvement of 2,081 2,139 bpm x mmhg ( table 4 , p = 0.01).table 4rpp in exercise test 1 ( et1 ) and exercise test 2 ( et2 ) and the difference between the 2 tests ( et2-et1 ) in diabetic and nondiabetic patients who demonstrated ip ( ip+ ) or who did not demonstrate ip ( ip-)et1et2et2-et1
p value*dm / ip+22,841 4,50025,853 4,7423,011 2,4300.01non - dm / ip + 23,094 5,31225,175 5,4852,081 2,139 dm / ip -22,705 4,00022,124 3,686580 2,2500.43non - dm / ip -23,910 5,38022,869 5,3511,050 2,027data are expressed as means standard deviation .
dm stands for diabetes mellitus , ip + ischemic preconditioning present , ip - ischemic preconditioning absent , et exercise test . * the p values are from the comparison of et2-et1 among diabetic and nondiabetic patients rpp in exercise test 1 ( et1 ) and exercise test 2 ( et2 ) and the difference between the 2 tests ( et2-et1 ) in diabetic and nondiabetic patients who demonstrated ip ( ip+ ) or who did not demonstrate ip ( ip- ) data are expressed as means standard deviation
. dm stands for diabetes mellitus , ip + ischemic preconditioning present , ip - ischemic preconditioning absent , et exercise test . *
the p values are from the comparison of et2-et1 among diabetic and nondiabetic patients table 5 shows the hemodynamic parameters heart rate and blood pressure at baseline and peak exercise in the 4 groups of patients.table 5hemodynamic parameters , heart rate and blood pressure in the 4 groups of patients , diabetic and nondiabetic , according to the demonstration of ipvariableset 1et 2et2-et1 dm / ip
+ hr ( baseline)79.0 15.186.3 17.07.3 11.0hr ( peak)139.0
15.1142.1 14.73.2 5.6bp ( baseline)156.5 18.2149.7 19.36.8 12.6bp ( peak)192.0 25.0191.8 23.90.16 15.4 dm / ip -hr ( baseline)79.0 14.784.4 14.95.4 8.0hr ( peak)141.7 14.3143.0 13.51.3 7.4bp ( baseline)153.3 22.6147.1 19.76.2 14.7bp ( peak)183.7 23.2180.6 22.23.12
14.7non - dm / ip + hr ( baseline)78.0 13.187.1 13.99.1 7.9hr ( peak)139.6 15.4144.3 14.74.7 5.7bp ( baseline)146.7 20.6138.7 18.38.0 11.3bp ( peak)193.5 23.6190.2 24.53.3 12.4non - dm / ip -hr ( baseline)82.5 12.987.1 12.64.6 7.1hr ( peak)139.3
12.4139.6 13.00.35 5.2bp ( baseline)158.6 26.2147.9 26.110.7 14.6bp ( peak)204.6 25.3194.8 28.18.9 16.5data are expressed as means standard deviation .
dm stands for diabetic patients , non - dm nondiabetic patients , ip + ischemic preconditioning present , ip - ischemic preconditioning absent , et exercise test hemodynamic parameters , heart rate and blood pressure in the 4 groups of patients , diabetic and nondiabetic , according to the demonstration of ip data are expressed as means standard deviation .
dm stands for diabetic patients , non - dm nondiabetic patients , ip + ischemic preconditioning present , ip - ischemic preconditioning absent , et exercise test
the improvement in the total exercise time comparing the 2 sets was similar between the groups of diabetic and nondiabetic patients ( 20 39 s versus 17 36 s , respectively , p = 0.60 ) .
the improvement in the frequency and severity of arrhythmias had similar results among diabetic and nondiabetic patients who experienced ip ( 50 % versus 63 % of patients demonstrated improvement in arrhythmias , respectively ; p = 0.41 ) .
the improvement in the st - segment deviation morphology was also similar among diabetic and nondiabetic patients ( 38.5 % versus 48.3 % , respectively , p = 0.41 ) .
when the total group of patients ( n = 174 ) was stratified according to the demonstration of ip , the frequency of diabetic patients as well as the levels of fasting glycemia and glycosylated hemoglobin were similar between the 2 groups , as shown in table 6.table 6frequency of diabetes mellitus , and levels of fasting glycemia and a1c in the study population according to the expression of ipip + ( n = 122)ip - ( n = 52 )
p valuedm n ( % ) 62 ( 50.8)24 ( 46.1)0.57fasting glycemia121.3 42.6118.9 34.10.72a1c6.63 1.66.32 1.20.25data are expressed as means standard deviation and as absolute and relative frequencies
dm stands for diabetes mellitus , a1c glycosylated hemoglobin , ip + ischemic preconditioning present , ip - ischemic preconditioning absent frequency of diabetes mellitus , and levels of fasting glycemia and a1c in the study population according to the expression of ip data are expressed as means standard deviation and as absolute and relative frequencies
dm stands for diabetes mellitus , a1c glycosylated hemoglobin , ip + ischemic preconditioning present , ip - ischemic preconditioning absent in addition , when we stratified patients by quartiles of glycemia and glycosylated hemoglobin , there was no statistical difference in terms of ip demonstration among the different quartiles ( fig
3graphs showing the percentage of patients who demonstrated ischemic preconditioning ( ip+ in blue ) and who did not demonstrate ischemic preconditioning ( ip - in red ) stratified into quartiles of a1c ( graph a ) and fasting glycemia ( graph b ) . ip = ischemic preconditioning
x axis represents the percentage of patients and y axis the quartiles of a1c and fasting glycemia graphs showing the percentage of patients who demonstrated ischemic preconditioning ( ip+ in blue ) and who did not demonstrate ischemic preconditioning ( ip - in red ) stratified into quartiles of a1c ( graph a ) and fasting glycemia ( graph b ) .
ip = ischemic preconditioning ; q = quartile ( s ) .
x axis represents the percentage of patients and y axis the quartiles of a1c and fasting glycemia
considering that diabetes is an independent risk factor for the occurrence of major cardiovascular events and mortality [ 5 , 6 ] , it is reasonable to consider that diabetes might damage the protective mechanism of ip in cad patients , leading to worse outcomes .
however , in this study , the presence of type 2 dm did not have any deleterious effects on the myocardial protective mechanism termed ischemic preconditioning . in this context
, our study showed that patients with type 2 dm demonstrated ip in similar frequency and intensity compared with nondiabetic patients and , interestingly , our data indicated an improvement in ischemic parameters associated with diabetes .
the analyses of the data showed a better ischemic response evaluated by rpp in diabetic patients .
thus , this study adds clinical information on some questions that have emerged from contradictory experimental studies .
the improvement in myocardial oxygen consumption , observed by the analysis of rpp , was more pronounced in patients with compared to those without diabetes , indicating better adaptation of the myocardium of diabetic patients after an ischemic insult . moreover , diabetic patients had an improvement in t-1.0 mm greater than that in nondiabetic patients , although it did not reach statistical significance .
analyzes of the total exercise time also confirmed the main results of the study , as times were similar among patients with and without dm .
similarly , the st - segment deviation morphology did not differ among diabetic and nondiabetic patients . because myocardial ischemia is a frequent cause of arrhythmias during treadmill stress tests
, we also assessed the occurrence and complexity of arrhythmias and their improvement during set .
thus , the improvement in the occurrence of arrhythmias also confirmed the main findings of the study , as the occurrence did not differ among diabetic and nondiabetic patients .
interestingly , analyzes of the percentage of patients who demonstrated ip among different quartiles of fasting glycemia and glycosylated hemoglobin showed similar rates of ip demonstration .
this analyzes infer that the differences in the intensity of glycemic control in our population did not prevent the demonstration of this cardioprotective phenomenon .
however , despite our consistent findings , the information from this study is contrary to lee s et al . and ishihara s et al . , who have also studied ip in humans .
studied diabetic and nondiabetic cad patients during percutaneous coronary interventions , and evaluated the action of hypoglycemic agents on ip .
patients underwent consecutive balloon coronary inflations . during the second sequential balloon inflation , patients had less thoracic discomfort , less myocardial lactate production , and lower st - segment deviation .
moreover , the authors observed that diabetic patients treated with glimepiride had higher lactate production compared to nondiabetic patients treated with glimepiride . despite this finding , an important limitation of this study is that there was not a direct comparison of diabetic and nondiabetic patients who had no drug interference in ip evaluation .
studied patients hospitalized due to an acute myocardial infarction and evaluated the effects of preinfarct angina , on the release of cardiac markers of necrosis , ventricular function , and in - hospital death and compared the results among diabetic and nondiabetic patients .
the authors found that in the nondiabetic population , patients with preinfarct angina had a lower release of cardiac markers of necrosis , better recovery of ventricular function , and lower in - hospital mortality , compared to patients with no preinfarct angina . on the other hand , when they analyzed diabetic patients , these variables were not different among patients with and without preinfarct angina .
thus , the authors inferred that diabetes prevented the appearance of ip . despite their findings ,
first , this was a retrospective study , which included a small number of diabetic ( n = 121 ) compared to nondiabetic patients ( n = 490 ) .
many studies have shown that some of these drugs may block ip , and it has been speculated that this interference with ip mechanisms may partially explain the worse prognosis of diabetic patients hospitalized due to an acute myocardial infarction . on the other hand ,
other experimental studies that evaluated in vitro human myocardial tissue [ 41 , 42 ] showed results that match those of the present study .
they evaluated the release of cardiac biomarkers of necrosis and the percentage of tissue viability . among other findings
, the authors found a similar intensity in myocardial protection among diabetic and nondiabetic patients .
additionally , cleveland et al . evaluated the contractile function of isolated right atrial trabeculae from cad patients , resected during coronary artery bypass graft surgery .
they showed that the diabetic group that underwent the preconditioning stimuli had similar improvement in contractile function compared to the nondiabetic group .
moreover , a small study conducted by bilinska and colleagues with diabetic patients treated with glibenclamide , gliclazide , and diet compared the demonstration of ip in these groups with that in nondiabetic patients .
besides the main findings of the study , which were related to the effects of sulfonylureas in the warm - up phenomenon , they showed that the group of diabetic patients on diet ( n = 15 ) had similar improvement in ischemic parameters compared to nondiabetic patients ( n = 17 ) .
of note , this was a secondary result , and the small sample size did not permit to make a definitive conclusion .
this prospective study on ip in humans had a sample size powered enough to compare ip demonstration in 2 populations with matched clinical characteristics and evaluated the possible interference of diabetes on a myocardial protective mechanism .
the group of patients with diabetes was under strict control of hyperglycemia , and this is observed by the controlled levels of fasting glucose and glycosylated hemoglobin in the group of diabetic patients . assuming that hyperglycemia may interfere negatively with ip , it is possible that the result of this study would be different in a population under less strict hyperglycemic control .
, the response of the myocardium to an ischemic insult may probably play an important role in prognosis .
one mechanism that may interfere with such a prognosis may be the presence of a protective myocardial phenomenon , termed ischemic preconditioning . because diabetes is an independent risk factor for the occurrence of major cardiovascular events , it is reasonable that it may compromise this cardioprotective mechanism , leading to worse outcomes .
contrary to the initial expectation , the analysis of our data revealed that diabetic patients showed this protective phenomenon similarly to nondiabetic patients .
this prospective study on ip in humans had a sample size powered enough to compare ip demonstration in 2 populations with matched clinical characteristics and evaluated the possible interference of diabetes on a myocardial protective mechanism .
the group of patients with diabetes was under strict control of hyperglycemia , and this is observed by the controlled levels of fasting glucose and glycosylated hemoglobin in the group of diabetic patients . assuming that hyperglycemia may interfere negatively with ip , it is possible that the result of this study would be different in a population under less strict hyperglycemic control .
, the response of the myocardium to an ischemic insult may probably play an important role in prognosis .
one mechanism that may interfere with such a prognosis may be the presence of a protective myocardial phenomenon , termed ischemic preconditioning . because diabetes is an independent risk factor for the occurrence of major cardiovascular events , it is reasonable that it may compromise this cardioprotective mechanism , leading to worse outcomes .
contrary to the initial expectation , the analysis of our data revealed that diabetic patients showed this protective phenomenon similarly to nondiabetic patients .
in this study , diabetes mellitus did not substantially affect myocardial ip in symptomatic cad patients .
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backgroundthe influence of diabetes mellitus on myocardial ischemic preconditioning is not clearly defined .
experimental studies are conflicting and human studies are scarce and inconclusive.objectivesidentify whether diabetes mellitus intervenes on ischemic preconditioning in symptomatic coronary artery disease patients.methodssymptomatic multivessel coronary artery disease patients with preserved systolic ventricular function and a positive exercise test underwent two sequential exercise tests to demonstrate ischemic preconditioning .
ischemic parameters were compared among patients with and without type 2 diabetes mellitus .
ischemic preconditioning was considered present when the time to 1.0 mm st deviation and rate pressure - product were greater in the second of 2 exercise tests .
sequential exercise tests were analyzed by 2 independent cardiologists.resultsof the 2,140 consecutive coronary artery disease patients screened , 361 met inclusion criteria , and 174 patients ( 64.2 7.6 years ) completed the study protocol . of these , 86 had the diagnosis of type 2 diabetes .
among diabetic patients , 62 ( 72 % ) manifested an improvement in ischemic parameters consistent with ischemic preconditioning , whereas among nondiabetic patients , 60 ( 68 % ) manifested ischemic preconditioning ( p = 0.62 ) .
the analysis of patients who demonstrated ischemic preconditioning showed similar improvement in the time to 1.0 mm st deviation between diabetic and nondiabetic groups ( 79.4 47.6 vs 65.5 36.4 s , respectively , p = 0.12 ) .
regarding rate pressure - product , the improvement was greater in diabetic compared to nondiabetic patients ( 3011 2430 vs 2081 2139 bpm x mmhg , respectively , p = 0.01).conclusionsin this study , diabetes mellitus was not associated with impairment in ischemic preconditioning in symptomatic coronary artery disease patients .
furthermore , diabetic patients experienced an improvement in this significant mechanism of myocardial protection .
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Background
Methods
Study design
Patients
Sequential treadmill exercise tests
Sample size calculation
Statistical analysis
Results
Demonstration of IP
Analyses of secondary ergometric variables
Analysis of glycemic variables
Discussion
Study limitations and strengths
Clinical practice perspectives
Conclusions
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recently
we reported on an analysis of the infrared spectrum of
c2f4 . by observation
of many combination and difference bands , including many from two c - containing isotopologues , and by comparison with mp2 and
density - functional calculations , all 12 fundamentals could be assigned ,
although only five are ir active .
the geometry
was deduced from ro - vibrational analysis of strong ir active bands
and by comparison with theoretical calculations .
however , it is also
instructive to correlate anharmonic constants with some peculiarities
of the reactions of c2f4 .
accordingly , in this
work more anharmonic constants ( altogether 54 ) are derived from the
spectra , and a complete set ( 78 ) is calculated .
the earlier vibrational
assignments of c2f4 were reported at a time
when the unusual properties of this molecule were barely known .
explained this property by positing that the electron attraction
of fluorine leads to a preference of sp- over sp - type carbon : radical addition converts both
c atoms from sp to sp hybridization , which
stabilizes the primary product , lowers the transition state , and thus
accelerates the reaction .
alder reactions , for example ,
which was again explained by stabilization of radical intermediates .
( borden also presented an alternative but related description based
on delocalization of unpaired radicalic electrons to accepting *
orbitals of cf groups . )
the preference
for carbon sp hybridization can also be expected to lower
the force constants for the pyramidalization ( wagging ) vibrations
( 7 , 8 ) and/or increase their anharmonicity .
furthermore , torsion ( 4 ) should be facilitated by
rehybridization and in fact quantum chemistry confirmed this for a
90 twist .
another spectacular property of c2f4 is how
readily it can dissociate thermally to two cf2 molecules .
a c = c bond energy of only 2.95 ev is derived , less than that of a typical c c single
bond ( 4 ev ) .
the low dissociation energy is caused by stabilization
of the resulting two difluorocarbenes by back - bonding from nonbonding
fluorine electrons to the empty carbon p - orbital .
this also explains why cf2 has a singlet
ground state which is more stable than the triplet ( by 2.458 ev , taken
from the band origin of the phosphorescence spectrum ) . whereas in the previous work
, only cc stretching is considered
as the dissociation coordinate , it was argued in ref ( 16 ) that pyramidalization
must also be involved .
this idea is supported in the present work
by the large value found for the anharmonic constant x18 coupling cc stretch with trans pyramidalization .
the
magnitude of x18 as compared to the much
smaller x17 ( coupling cc stretch with
cis pyramidalization ) also supports a valence - bond analysis of weak
double bonds , which was reviewed in ref ( 19 ) .
the samples in natural isotopic abundance were either taken from
commercial c2f4 ( hoechst ) , that contained a
trace of the cyclic dimer , or prepared from c2f4br2 + zn in a high - boiling
alcohol ( diethylene glycol ) ; the comparison allowed for recognizing
absorptions of impurities .
a sample enriched in c to
50% was prepared from chclf2 by co2 laser isotope
separation with enriched cf2 as the primary product .
ir spectra of c2f4 in natural isotopic
abundance
and isotopically enriched were recorded between 370 and 4000 cm with 0.2 cm resolution by a perkinelmer
ftir spectrometer in garching . at the
synchrotron lab ,
the isotopically enriched sample was measured at
moderate 0.1 cm resolution in the far ir region
from 100 to 600 cm ( capturing the band at 210
cm ) , and at high resolution for the ir active
cf stretch bands , see ref ( 1 ) .
the present work focuses on anharmonic constants of c2f4 . only where the constants were
expected to be different for ccf4 and c2f4 ( e.g. , in the case of
fermi resonance )
theoretical values of the anharmonic constants xij were obtained from anharmonic
vibrational
frequency calculations performed using gaussian 03 revision c02 at the mp2 level using
dunning - type correlation - consistent ( cc - pvtz ) orbitals .
the molecular force field at this level of theory
was shown in the previous study to reproduce the fundamental wavenumbers
and the isotopic shifts of c2f4 very satisfactorily
( for details , see ref ( 1 ) ) , and hence was an appropriate choice for computing xij values .
the calculations were performed for each of
the c2f4 , ccf4 , and c2f4 isotopologues .
the spectra were measured previously and the
fundamentals extracted
from them , using a limited number of anharmonic constants ( from combination
and hot bands ) where necessary . for convenience
table 1
lists the fundamentals again . in this
work we derived a large set of experimental anharmonic constants from
combination bands and in particular for the lower - wavenumber
vibrations from hot bands , and report a complete set of theoretically
computed values .
symmetry types refer to a d2h molecule with z axis along the cc bond and the y axis perpendicular
to the plane , as used in ref ( 1 ) for easier comparison with the c2v symmetric ccf4 ; the more conventional designation with x axis along the cc bond and z axis perpendicular
to the plane is given in parentheses .
revised value ( instead of 210 cm ) , based on a more definite
assignment of a hot - band structure and x10,10 , see supporting information .
band origins from high - resolution
spectra ; low - resolution values are 1338.4
and 1187.0 .
upright :
experimental values .
in parentheses : values calculated by mp2/pvtz . where two values or
a range are given for the experimental data
, it may indicate ( a ) a
dependence of the effective xij on the involved levels due to perturbations ( fermi resonances ,
section 4.4 ) or ( b ) an uncertainty due to error
limits ( e.g. , where broad bands are involved ) or an uncertain assignment .
a preferred value is indicated by bold face . for further details on
the derivation of each xij and some isotopic data ,
the anharmonic constants xij were calculated from observed
combination and difference bands
by assuming that anharmonic shifts are additive : for binary combination
bands ( i j ) and for overtones
we used1and2with obvious extensions for more complicated
combinations .
( the xij values are mostly negative , as usual . ) the left - hand side of these
equations should be read as wavenumber of ( name ) . for hot bands ( satellite transitions near i that originate from a thermally populated
lower level j ) we used3again
with obvious extensions .
one can often
observe sequences of equidistant q branches , corresponding to hot
bands starting from higher levels mj , or combination of such sequences ( from mj + nk ) .
equation 3 implies that the distance of a ( first ) hot band from the fundamental
directly indicates the corresponding anharmonic constant .
combination
bands allow for a direct assignment , whereas hot bands must be identified
by their intensity ( controlled by the boltzmann factor ) . in a number
of cases
the supporting information ( si ) compiles the sources for each xij in table s2 , whereas table s1 lists the observed
bands ( with hot bands ) and their isotopic shifts .
two examples of
hot bands , whose shifts are dominated by the large x18 anharmonicity , are shown in figure 1 .
absorbance spectra demonstrating the large x18 derived shifts for hot - bands originating in 8 .
the spectra were recorded
in the garching laboratory , with pressure and path length of 2 bar
and 10 cm ( for the lower trace of each panel ) , and 140 mbar and 4
m ( upper trace ) , respectively . for levels involving more than two quanta of vibration , anharmonic
shifts
this helped to decide between different
assignments of combination bands in a few cases where the harmonic
sums were similar to each other .
however , such additivity need not
apply when the levels involved are subject to fermi resonance , producing
shifts that depend on the separation of nearby interacting levels .
we use eqs 13 to
obtain the experimental values , and hence , table 2 reports phenomenological xij , some of which show a spread in the values suggestive of fermi
resonances ( in particular x18 , x26 , and x77 ) .
fermi
resonances can also be recognized if the apparent xij or the hot - band structure depends
on the isotopologue . for c2f4 ,
fermi resonances
were identified ( see section 4.4 and si ) between:5 and 2+6 ( discussed already in ref ( 1)),1 and 5+6 ( indicated by the hot - band structure
of 1-combinations , which are different in the mixed
isotopologue),2 and 27 ( causing irregularities
in overtone levels of 7 , also contributing to the
large magnitude of x27 and x77).2 and 23 , identified from the
mp2 calculations .
5 and 2+6 ( discussed already in ref ( 1 ) ) , 1 and 5+6 ( indicated by the hot - band structure
of 1-combinations , which are different in the mixed
isotopologue ) , 2 and 27 ( causing irregularities
in overtone levels of 7 , also contributing to the
large magnitude of x27 and x77 ) . 2 and 23 , identified from the
mp2 calculations .
the mp2/cc - pvtz calculations
reproduced the observed wavenumbers
and isotopic shifts very well . supporting
the overall vibrational analysis
exceptions are 15 ( x22 , x28 , x29 , x2,10 , x2,11 , x36 , x3,10 , x45 , x59 , x5,11 , x66 , x68 , x77 , x88 , x10,11 ) out of 54 cases where an observed value is available
for comparison . some of these will be discussed below .
as already
described in the introduction , the peculiar
reactions of c2f4 have to do
with cc stretching and carbon pyramidalization . the discussion will
focus on these coordinates ( sections 4.14.3 ) . for
a planar geometry ,
cc dissociation correlates c2h4 and c2f4 with a pair of carbenes in their lowest triplet states ( figure 2a ) . because
the energy of forming these products is practically the same for the
two molecules
, one could also expect that the potential energy curves
coincide and the cc stretch force constants are equal .
in fact , already
early force - field calculations showed no substantial difference ( 889
n / m for c2f4 and
940
furthermore ,
the short cc distance ( 132 pm , which is 1 pm shorter than in ethylene ) and the high cc stretch wavenumber ( 1873.8 cm , compared to 1625.4 cm for ethylene ) do not point to a weakened bond .
as early as 1965 , the low dissociation energy of c2f4 was attributed to switching over to a channel producing two
singlet carbenes and to the stabilization ( by 2.458 ev , see introduction ) of singlet cf2 by back bonding , so that this channel is energetically favored by 4.9 ev .
this path hence leads over an avoided
crossing of potential surfaces ( figure 2a ) .
if the avoidance is strong , one could expect an influence at least
on the anharmonicity x11 ( 1 is dominated by cc stretching ) , even if not on the force
constant at the bottom of the potential energy well .
table 2 shows that the magnitude of x11 ( calculated 7.8 cm ) is not unusually
large for such a high - frequency vibration , though noticeably larger
than for ethylene ( x22 = 2.3 cm ) . that the increase
in magnitude is only moderate
can be taken as a sign that the avoidance
along this coordinate is not very strong .
hence , the back reaction
( recombination ) will have a high barrier in planar geometry . whereas
the measured recombination barrier is not , in fact , zero , it is only
minor ( 9 kj / mol = 0.09 ev ) .
so ,
a lower - energy path must lead around the barrier , out of the drawing
plane of figure 2a , that is , with distortion
different from cc stretch .
potentials for ground - state cc dissociation
of c2f4 , leading over an avoided crossing to
two singlet carbenes .
in ( a ) it is assumed that the one - sided pyramidalization ( q7 + q8 ) begins only after overcoming the barrier , whereas
( b ) shows the preferred path , where this distortion is already fully
developed on the barrier and is preceded by a trans pyramidalization
( q8 ) , while the cc distance is steadily increasing .
it was argued in ref ( 16 ) that recombination of
two cf2 molecules should
be easiest if one of them points with its filled n orbital
to the empty p orbital of the other .
hence , the dissociation
path will change from pure cc stretch to include also pyramidalization
( figure 2b ) , and the potential should be lowered
in energy in the direction of q7 ( the coordinate of 7 ) and/or q8 . in turn
in fact x18 has an unusually large magnitude , although x17 is in the normal range ( table 2 ) .
( the magnitude of x18 is not
caused by secondary effects such as fermi resonance , as we shall see
in section 4.4.3 . )
we can conclude that on
stretching the cc bond the molecule first bends toward trans pyramidalization
( q8 ) ( figure 2b ) .
the one - sided
nonplanar bending ( figure 2 ) is a superposition
of q7 and q8 .
the reason why q8 is
active earlier than q7 can be understood by a valence - bond
model for weak double bonds . in this model
several symmetric arrangements
of carbenes ( a c
of scheme 2 ) were considered : whereas with
two triplet carbenes all arrangements ( a d ) lead to bonding
interaction , with two singlet carbenes bonding occurs only in the
trans - bent case ( c ) ; this corresponds to a double donor
one can add that the cis - bent case with singlet carbenes would
lead only to antibonding ( scheme 2d ) .
malrieu
and trinquier derived a criterion for trans - bending ( deriving even
the angle ) by comparing the standard double - bond energy with the singlet
their findings were confirmed by many examples , in particular with
heavier - element homologues .
we can apply
this rule not only to the equilibrium geometry of c2f4 but also to its dissociation path , if we consider in the
comparison the double - bond energy that is reduced on extension of
this bond .
the rule thus predicts that on cc extension c2f4 is distorted from planar geometry to a trans - bent structure ,
and this rationalizes the large magnitude of x18 . in the triplet case ,
the interaction
can be bonding in all arrangements . in the singlet case , ( a )
is antibonding ,
( b ) nonbonding , ( c ) bonding , ( d ) antibonding .
more recently , borisov et al . found evidence
for trans pyramidalization
on extension of the cc bond also from molecular orbital calculation
( at high level : mller plesset perturbation theory of
second , third and fourth order ) : the
authors found that the onset is relatively sudden on cc extension
to 142 pm and that the distortion lowers the energy appreciably .
( the
large x18 anharmonicity is an indication
that this pyramidalization already begins not far from the s0 minimum .
in fact , a cc stretch by 10 pm from 132 to 142 pm
is
just about the vibrational amplitude in the 1 =
1 level . )
although borisov et al . did not find an energy minimum with
such a distortion , they suggested that it plays a crucial role in
the low - energy real intermediate found by buravtsev , kolbanovskii
et al . in thermal reactions of c2f4 and recombination
of cf2 ( see ref ( 31 ) and literature quoted therein and section 4.3 ) .
one could suppose that in order to lower the activation
energy
for dissociation the only necessary deviation from simple cc stretching
is distortion toward the 8 coordinate ( q8 ) .
however , there is experimental
evidence that the asymmetric pyramidalization ( corresponding to superposition
of q7 and q8 as in figure 2b ) is already crucial in the transition state : the pre - exponential
factor found in the rate of thermal c2f4 dissociation
( 2.8 10 s ) is clearly larger than would be typical for
a simple bond splitting ( 10 s ) .
this
corresponds to a raised entropy of activation , such as that which
happens if an additional degree of freedom is activated in the transition
state .
the most plausible candidate is free internal rotation , as
it can be expected in the asymmetric structure with a single dative
bond but not in symmetric geometry with double donor acceptor
bond .
this picture is also consistent with a stepwise cleavage of
the double donator acceptor bond on c2f4 dissociation and its stepwise formation on cf2 recombination .
it is worth noting that the molecule with one - sided pyramidalization
has an electron distribution ( figure 2b ) that
is zwitterionic and correlates in planar geometry with the 2ag state with two electrons excited to * , as
already pointed out in ref ( 16 ) .
a fully analogous dissociation path can also be
expected for the
other systems considered by malrieu and trinquier .
in fact , according
to ref ( 32 ) ground - state
( ir induced ) dissociation of diazomethane ( h2cnn ) follows
a path with a pyramidalized methylene group to produce n2 + singlet ch2 ; the out - of - plane deformation was experimentally
confirmed . in the backward reaction ,
n2 points with its
n electron pair to the empty p orbital of the singlet
methylene ( see refs ( 32 and 33 ) for a calculated potential in two dimensions , which would look similar
for c2f4 ) .
hence ,
the out - of - plane deformation on dissociation of c2f4 is not an isolated phenomenon . applying
their energetic criterion to the equilibrium geometry of c2f4 , trinquier and malrieu found that this molecule is
not far from the limit , where it would become nonplanar , and suggested
that the 8 force constant may be significantly lower
than that of ethylene .
this is in fact
confirmed in the following way : analytical expressions for a valence
force field model for planar x2y4 were given
by herzberg . besides the masses mx and my , bond lengths l1 ( for xx ) and l2 ( xy ) and the angle ( half of yxy ) , both 7 and 8 only depend on the force constant for pyramidalization
( k/l2 ) . with equilibrium distances and angles from ref ( 1 ) and i = ( 2ci )
one derives this force constant from
7 and 8 ( with identical results
for c2f4 and c2f4):4both values should be identical
in this valence - force field model .
in fact for ethylene , the value
is 22.9 n / m , whether derived from 7 or 8 .
the pyramidalization force constant
derived from 7 of c2f4 is
slightly higher than in ethylene ( by 17% ) , but the same quantity derived
from 8 is only 49% of the ethylene value .
( of course ,
these values are not quantitatively meaningful , as the discrepancy
highlights that a pure valence - force field model is not sufficient
for wagging of c2f4 . )
so in fact the restoring
force for the 8 vibration is significantly smaller
than in ethylene and than that expected on the basis of the same pyramidalization
force constant as for 7 .
this is consistent with
the prediction of the valence - bond model favoring the trans - bent geometry .
the slightly raised force constant for 7 could
be due to repulsive nonbonding interaction in cis pyramidalization .
such a geometry would be involved in the hypothetical transition state
of the diels
alder addition of c2f4 to
butadiene , a reaction that has not been observed .
it was argued that
an increased energy associated with this distortion might contribute
to inhibiting the reaction .
however , an
increase in the force constant of only 17% is hardly sufficient to
support this explanation , and the other effects discussed in ref ( 7 ) , in particular the competing
fast radical reactions , are probably more important . applying
again the force - field formulas of herzberg ,
the force constant for torsion , k/l2 , is found to be 30.3
n / m for both ethylene and tetrafluoroethylene .
that is , near the potential
minimum the c = c bond in c2f4 behaves
just like a normal double bond .
consequently , the torsional barrier
at a 90 twist ( which is a measure of the bond strength )
would be identical for both molecules ( 2.8 ev ) if the two c cf2 groups remained planar ; this was confirmed by a high - level
calculation .
however , pyramidalization
lowers this barrier by 0.57 ev ( at mp2 level , consistent with ref ( 35 ) ) .
if this stabilization is activated early along q4 ,
it could give rise to substantially negative values for x44 and especially for x47 and x48 .
however , x44 and x47 are actually slightly positive
( 0.8 and 0.3 cm ) and x48 has no unusual magnitude ( 0.7 to 1.05 cm ) .
evidently , the stabilizing effect of pyramidalization becomes
effective only at high torsional angles .
the corresponding values
for ethylene are 2.4 , 8.9 , and 7.2 cm , respectively .
the nondiagonal anharmonicities xi4 and x4j are
generally smaller in magnitude than in ethylene . but this may simply
have to do with the relative vibrational amplitudes , which are much
larger for torsion of ch2 than of cf2 due to
the relative masses . in the twisted - pyramidalized structure
of c2f4 ,
the triplet ( t1 ) is only
0.013 ev lower than the singlet
( s0 ) .
it
can therefore act as a real intermediate and undergo radical - type
reactions , further assisted by its low energy that is calculated at
2.1 ev .
this has been a widespread interpretation
of some peculiarities of c2f4 reactions ( see ,
for example ref ( 7 ) ) .
however , buravtsev , kolbanovskii et al . found in shock - tube experiments
( with c2f4 diluted by ar and adiabatically compressed )
an intermediate at much lower energy ( 0.8 to 1 ev ) with uv and visible
absorption ( 235 and 500 nm ) ( see refs ( 31,36 , and 37 ) and the
literature quoted there ) .
they supposed it to be a singlet diradicaloid
of c2f4 with a geometry corresponding not to
an energy minimum but to a point on a slope of the potential ( see ,
for example ref ( 31 ) ) .
however , as these points are not stationary there are too many
( nearly ) isoenergetic locations with shifted electronic transitions ,
so that the electronic absorption would be smeared out .
it was therefore
suggested that the observed intermediate
is another low - energy isomer , tetrafluoro - ethylidene cf3cf ( electronic absorption in an ar matrix : ) .
however , its energy ( 1.9 ev ) seems to be nearly as high as that of the twisted - pyramidalized
triplet .
( in one study the twisted - pyramidalized c2f4 triplet is calculated at 0.90 ev , but computational details are not given , and previous theoretical
results are not discussed . )
some more anharmonicities of table 1 have unusually large magnitudes or show a range
of values and/or deviate from prediction . in many of them
fermi resonances
play a role , as will be explained here . in section4.4.3 , however , it is shown that x18 is hardly influenced by such effects and its large magnitude mainly
results from the overall shape of the potential energy surface .
fermi resonances mix two nearby levels of the
same symmetry , which differ by three units in the quantum numbers .
the fermi - induced
repulsion of the levels depends in a nonlinear way on both the magnitude
of the fermi interaction parameter and on the original ( i.e. , unperturbed )
energetic distance of the levels .
this separation will , in general ,
change ( a ) on isotopic substitution and ( b ) on adding a quantum of
another vibration ( i.e. , in analogous combination bands ; similarly
also in higher overtones , in which case a quantum number dependence
must also be taken into account ) .
another method of characterizing fermi resonance interactions relies
on intensity borrowing caused by the mixing of levels . according
to the mp2 calculations , the great majority of xij change by 1 cm on isotopic substitution
the few exceptions are
listed in table 3 and are compared to experimental
values . identified ( fr13 ) and
the interpretation in the line influenced by
is based on the assumption that an xij is affected by such a resonance if at least one
of the levels i and j is involved in the fermi interaction .
the table shows that
the calculated isotopic shift is sometimes
much larger than in the experiment , for instance x26 , x27 .
also x56 for c2f4 is probably
too large , as the two observed bands 5+6+11 and 5+6+12 are close to their harmonic positions
( table s1 in si ) .
it seems that at the
present level of theory , the calculation does not reliably reproduce
the fermi resonance induced shifts , at least the stronger ones .
this
is not surprising given the highly sensitive and nonlinear dependence
of fermi shift on the unperturbed level separations .
the same deficiency will also affect those xij connected with at least one level ( or a
combination thereof ) involved in a resonance listed above ( examples
in figure 3a ) .
in fact , 10 of the 15 deviations
listed at the end of section 3 can be attributed
in this way to the resonances fr1 and fr2 with possible contributions
of fr3 .
the exceptions are x36 , x3,10 , x66 , x68 , and x88 . in
all 42 other cases
it is worthwhile to consider
the two strongest fermi resonances ,
fr1 ( 2/27 ) and fr2 ( 5/2+6 ) , in c2f4 in some detail .
the effect of these resonances
is summarized in figure 3 . some levels involved
in fermi resonances and observed transitions .
solid arrows : ir , broken arrows : raman ; double arrows ( with interaction matrix element ) indicate repulsion
between level pairs .
the anharmonic shifts involving x77 are only nominal , because the fermi perturbation is
strong : 6x77 = 18.1 is
less than 3 times
(= 8.2 ) and 4x77 is only
9.9 . ( but 2x77 + 4x77 = 6x77 , as is obvious from the level scheme , and the raman
anharmonic shifts practically coincide with those from the ir spectra . )
( b ) three - level fermi resonance 5/2+6/27+6 and
their combinations with 9 for c2f4 and c2f4 .
the indicated level energies are from the spectra except those in
parentheses , which are calculated .
the broken horizontal lines are
the estimated unperturbed positions ( see the si ) , and the signed numbers are the calculated shifts .
the 16.4 cm magnitude of the interaction term ( with its expected dependence
on quantum numbers ) has been derived
from band shifts and intensities for a series of levels built on 2/27 for all three isotopologues .
bands for
27+x ( x = 9 , 11 ) borrow their entire intensity from 2+x . the detailed analysis
is provided in si .
it
is also evident that x77 , x27 and x22 are affected by
the fermi resonance ( and probably other x2i , if the level separations differ ) and that x77 is level dependent because of the varying
separation .
the fermi term is almost entirely responsible for the
+ 8.2 cm shift of 27 for c2f4 and those of the heavier isotopologues
( table 3 under fr1 ) . in the latter
the fermi - induced
shift is increased , because the levels 2 and 27 ( before perturbation ) are closer together than in c2f4 and are practically degenerate in c2f4 .
the success of these calculations
shows that the additional mixing of 2 with 23 ( fr3 ) is apparently a weak perturbation at most .
the
fermi resonance fr2 between 5 and 2+6 was qualitatively discussed in ref ( 1 ) .
a more complete understanding
of the resonance shifts and pattern of intensity borrowing requires
the three - level scheme shown in figure 3b ;
this figure also shows combinations with 9 , as they
are all ir active and were directly observed .
analysis based on this
model has enabled the magnitude of the fermi term to be determined
as 11 cm ; the fr1 term of 16.4 cm was used without change .
significantly , the model predicts varying
fr shifts that even differ in sign ( originating from the different
repulsions in figure 3b ) and can thus account
for the unusual isotopic shifts observed between c2f4 and c2f4 .
it explains a substantial deviation of the observed isotopic shift
5 = 51.9 from the value of 5 = 43.1
expected from the teller redlich product rule , which is valid
for harmonic oscillators .
additionally
the relative intensities of combination bands and their isotopic dependence
are well predicted .
it is also remarkable that according to the model
the largest part of the anharmonicity x26 is due to fermi resonance .
as described in ref ( 1 ) , the lower symmetry in ccf4 leads to strong mixing of 5 with 9 ( which are nearly degenerate in c2f4 ) .
the two levels repel each other
( even in the harmonic approximation ) and give rise to two infrared - active
transitions .
it is sometimes difficult to disentangle them and their
combination bands from the spectra .
if the large magnitude of some
anharmonicities is caused by fermi resonance , a crucial question is
whether this might also be the case for x18 .
if so , the conspicuous value of x18 could be largely a matter of the character of the resonance and
less a consequence of global features of the potential for dissociation ,
which we considered in section 4.1 .
in fact ,
weak perturbations of the 1 level and its combinations
by the 1/5+6 fermi resonance are indicated by the slight x18 dependence on the level and on isotopic substitution and
the change of the hot - band structure of 1 combinations
in ccf4 , as explained in the si .
a quantitative estimate of the strength
of the 1/5+6 interaction can be deduced from the intensities of 5+6 combinations relative to those of the corresponding
1 combinations , assuming that the former borrow
their full strength from the latter .
the 5+6+12 band ( 2444.3 cm )
has 12% the strength of the 1+12 band ( 2428.7 cm ) , see figure 1a .
similarly , the 5+6+11 shoulder ( 3072 cm ) is < 1% as intense as 1+11 ( 3056.2 cm ) and clearly visible only with longer
path length than shown in figure 1b .
from both
observations , one can estimate a fermi term ( w ) of
no more than 2.5 cm , causing a shift ( and
consequent change in x18 ) of < 0.5 cm .
this is actually an upper bound , because such weak
bands can also be expected without intensity borrowing .
hence , fermi
resonance affects the magnitude of x18 only by a little ( by < 3% ) , and we can maintain the conclusions
of section 4.1 on the shape of the potential .
a large set of anharmonic
constants of c2f4 was derived from spectroscopic
data , and a complete set was calculated .
most of the calculated and experimental values agree very well , and
nearly all deviations can be attributed to fermi resonances . the values
were used to interpret peculiarities of the potential and reactions
of this molecule .
the facile thermal cc dissociation of c2f4 is due to curve crossing of the ground state
( 1ag ) with
the two - electron excited zwitterionic 2ag state .
the crossing
barely alters the properties of the potential curve ( cut purely as
a function of cc stretch ) near the equilibrium geometry ; the force
constant for cc stretching ( 1 in c2f4 , 2 in c2h4 ) is nearly
identical for the two molecules .
only a slightly larger magnitude
of the anharmonicity x11 compared to the
corresponding x22 of ethylene provides
a hint of a weakly avoided crossing in planar geometry of c2f4
. however , the large magnitude of x18 leads us to conclude that the dissociation path bends
early toward
q8 , i.e. to trans pyramidalization of the two carbon atoms
( figure 2b ) .
this is rationalized by a valence - bond
model , which describes the stretched cc bond as a double donor acceptor
bond ( scheme 2c ) .
it is also consistent with
the tendency of fluorine to induce sp hybridization at
carbon , although this preference alone would also allow cis pyramidalization ,
q7 , which is not involved early in dissociation .
on further
cc stretching , one of the two donor acceptor bonds breaks ,
and the pyramidalization becomes fully one - sided already in the transition
state ( figure 2b ) . in this way , free internal
rotation of the cf2 groups becomes possible , providing
an additional degree of freedom that rationalizes the high pre - exponential
factor in the rate constant of dissociation .
this structure with perpendicular
arrangement of the two cf2 groups appears very plausible
for the back reaction .
thus , one cf2 group approaches with
its filled n orbital the empty p orbital of the other
cf2 group ( figure 2b ) .
the easy
trans pyramidalization on minor cc stretching is probably also the
reason that the corresponding 8 force constant is
lowered , although all other force constants are similar to those in
ethylene .
the preference of fluorine for sp hybridization
at
carbon has been invoked to explain not only the easy radical addition
and other reactions , but also the low - lying , twisted - pyramidalized
triplet , which is nearly isoenergetic with the singlet that has a
saddle point there .
this is indicated by x44 , x47 , and x48 , which have no unusual values .
very recently ,
high - level theoretical studies investigated
electronic ground and excited states of c2f4 and their dependence on some coordinates , mainly
to discover the fate of this molecule after electronic excitation
and make comparisons with a corresponding time - resolved experiment
( ref ( 16 ) ) . for the
ground state , the stabilizing effect of pyramidalization in the twisted
molecule was confirmed .
interestingly , the excited molecule enters
the s1/s0 conical intersection from the 2ag state , where the molecule has a large one - sided pyramidalization ,
a 90 cc twist and a cc bond length ( 1.51 ) similar to
that of a single bond . in the present work ,
the transition state for
s0 dissociation was said to result from avoided crossing
of the 2ag and 1ag ( i.e. , s0 ) states ,
also with large one - sided pyramidalization but at longer cc distances
and perhaps without twist
. it would be interesting to check whether
these geometrical differences are large enough to generate an excited - state
barrier , because evidence was found in ref ( 16 ) that cc dissociation does not begin in the excited
state .
|
compared to ethylene and its nonfluorinated
derivatives , c2f4 is peculiar in many reactions .
it very easily
adds to radicals and prefers formation of four - membered rings over
diels alder reactions .
this has been rationalized by the preference
of fluorine for carbon sp3 hybridization , which is possible
on opening of the double bond .
another property , the thermal dissociation
of the c = c bond , has been explained by the stabilization of
the product ( cf2 ) by back - bonding .
here , it is attempted
to correlate such properties with vibrational constants , in particular
for c = c stretching and twisting and for carbon pyramidalization .
the only force constant found to be lowered compared to ethylene is
that for trans pyramidalization ( 8 ) , and cc bond
softening on 8 distortion is indicated by the conspicuously
large magnitude of anharmonic constant , x18 .
both observations can be rationalized by a valence - bond model that
predicts a trans bent structure on weakening the cc bond .
conclusions
are drawn about the dissociation path and peculiarities of the potential .
other anharmonicities , both experimental and calculated and some in 12c13cf4 and 13c2f4 , are also discussed . in particular
some strong fermi
resonances are identified and their effects accounted for .
|
Introduction
Methods
Results
Discussion
Conclusion
|
environmental factors play a major role in the etiology of cancers , cardiovascular disease , and other chronic diseases .
these factors are diverse in nature and include diet , drugs , cosmetics , household chemicals , pollutants , or infectious agents .
exposures to these factors vary widely between populations , and between individuals within the same population . therefore , their measurement is essential to : ( i ) study associations in epidemiological studies with disease outcomes and assess their contribution to disease risk , ( ii ) monitor exposures to disease risk factors in population studies and ( iii ) assess subject compliance in clinical trials or large intervention studies ( 13 ) .
exposure measurements have traditionally relied on the use of questionnaires and self - reporting . however , these methods are known to be error - prone and biased .
molecular biomarkers , on the other hand , are more direct and objective indicators of exposure .
indeed , biomarkers of exposure have been increasingly used since the early 1980s , thanks to rapid progress in analytical techniques and the establishment of large cohorts with extensive biospecimen collections .
biomarkers of exposure can be compounds present in the environment and absorbed in the gut after ingestion , inhaled in lungs , or absorbed through the skin .
they can also be metabolic end - products derived from environmental compounds that were metabolized by the liver and other tissues , and the microbiota .
they may also be macromolecular indicators of environmental effects ( e.g. enzymes , proteins or rna transcripts related to the status of a nutrient or toxic agent ) . over the past 30 years
several hundred biomarkers of exposure have been measured and reported in blood , urine and other biospecimens in various populations . however , this information is scattered over hundreds of publications under many diverse titles and subject headings .
this makes the identification of these biomarkers along with their comparative performance , their field of application and their concentration ranges in different populations difficult .
historically , most biomarkers of exposure have been measured individually using compound - specific assays . however , with the emergence of various omics technologies there is an increasing tendency to characterize exposures more comprehensively .
indeed , modern mass spectrometry techniques now allow the measurement of thousands of compounds in blood , urine or other biospecimens in a single analytical run .
these developments are leading , increasingly , to the reporting of data from multiple markers of exposure .
modern omics technologies should also allow the translation into practice of the concept of the exposome ( the totality of exposures of a particular individual over lifetime ( 4,5 ) ) and the development of exposome - wide association studies ( ewas ) ( 2,68 ) .
these newly emerging trends in exposure science , combined with the growing volume of comprehensive exposure data being published , make the establishment of a centralized , online database on biomarkers of exposure particularly critical . to date
, relatively little effort has been directed to collecting and organizing data on biomarkers of exposure .
the expocastdb database contains information on exposure to environmental chemicals such as pahs , pcbs , nonylphenols , or pesticides ( http://actor.epa.gov/actor/faces/expocastdb/home.jsp ) .
expocastdb contains information on compound concentrations in various environmental matrices but very limited data in biospecimens .
the comparative toxicogenomics database ( ctd ) is the only online database containing a large number of concentration values in blood , urine and other biospecimens extracted from the scientific literature ( 9 ) .
the ctd contains about 35 000 concentration values in various biospecimens for 250 organic and inorganic compounds . while expocastdb and ctd are very useful and important databases
it contains comprehensive information on almost 500 biomarkers of exposure with concentrations , correlations with exposure estimates and temporal reproducibility , as well as other details on study population and analytical methods .
all data in exposome - explorer was acquired from a careful review and analysis of nearly 500 peer - reviewed publications , with a particular focus on dietary and pollution exposures .
all data in exposome - explorer was compiled through extensive literature analysis along with manual curation and computer - assisted validation .
literature searches were conducted using the web of science ( wos ) . only peer - reviewed publications describing original work with biomarker measurements in observational studies conducted in human populations
publications on intervention studies , analytical method development , associations of biomarker with biological status or disease , biomarkers of biological status ( e.g. inflammation , oxidative stress , disease ) , or animal studies were not considered for this initial stage of development .
articles not available online were omitted . for pollutants , general rather than occupational exposures were prioritized .
exposure , biological monitoring ) and biospecimens ( blood , serum , plasma ,
adduct ) . for dietary biomarkers , data on the correlations between food or food compound intake and biomarker concentrations
citations were searched for according to several intake synonyms ( intake , consumption , diet , recall ,
citations were searched for by common pollutant chemical group , such as polycyclic aromatic hydrocarbons ( pah ) , polychlorinated biphenyls ( pcb ) , polybrominated diphenyl ethers ( pbde ) , polybrominated biphenyls ( pbb ) , polychlorinated dibenzodioxins / furans ( pcdd / f ) , heterocyclic amines ( hca ) , phthalates and disinfection byproducts ( dbp ) . to search for biomarker reproducibility values ,
reliability , reproducibility , repeatability , intra - subject , inter - subject , within - subject , between - subject. full - record citations from the wos were downloaded in the bibtex format ( http://www.bibtex.org/ ) and handled with bibdesk , an open - source bibliography manager for bibtex libraries ( http://bibdesk.sourceforge.net/ ) .
those rated as relevant were then manually described using a series of attributes ( tags ) related to exposures , study design , type of numerical data , populations , biospecimens , biomarkers and confounding variables to facilitate the selection of references for annotators .
the bibdesk smart groups functionality and a combination of criteria based on the tags were used to dynamically generate a priority list of publications for further annotation .
full - texts from these articles were then retrieved and submitted to annotators for detailed analysis and upload of the data to exposome - explorer using the annotation interface .
a password protected annotation interface was used for the manual uploading of all data from scientific publications to exposome - explorer .
, the annotator is guided through successive steps to ensure comprehensive capture of the following information :
publication with its bibliographic details ( title , authors , year , journal , pubmed id).subject groups studied in the publication , and the populations to which they belong .
each population is named with a short informative summary sentence ( e.g. cases and controls in a case - control study on breast cancer ) .
all ) or several ( e.g. soy consumers and soy non - consumers ) subject groups according to different criteria ( e.g. by gender , by ethnicity , by smoking status ) .
a reference to a cohort ( e.g. epic ) can also be indicated in this data field .
age , height , weight , bmi , group size , gender , health condition , exclusion of supplement users , smoker proportion , country of origin and ethnicity are also specified when available.samples defined for each subject group .
these describe number and time of collections ( e.g. baseline , 1 year ) of different biospecimens .
biospecimens include urine , whole blood , serum , plasma , hair , nails , adipose tissue , breast milk , or fractions such as plasma phospholipids or red blood cell membranes.biomarkers .
chemical information ( e.g. structure , chemical formula , average mass , monoisotopic mass ) is automatically generated by the structure server .
identifiers and links to external databases such as cas , pubchem ( 10 ) , chebi ( 11 ) , hmdb ( 12 ) and foodb ( www.foodb.ca ) are provided.biomarker measurements . for each sample or biospecimen type ,
also included is the analytical method , the original measurement units as described in the publication , and specimen - specific concentration value adjustments ( e.g. normalizing to
lipid for blood , or a list of regression variables ) . arithmetic or geometric mean , as well as median
concentration values are compiled along with the minimum and maximum values , standard deviation , percentiles , confidence intervals , measurement size ( number of subjects from which the concentration value was calculated ) , as well as the proportion of subjects for which the biomarker was detected .
information on the inclusion or exclusion of zero values in the calculated concentration is also given .
this was mostly done for pollutant biomonitoring studies where a chemical is often detected only in a small proportion of the studied population .
some authors used a threshold ( e.g. detected in at least 30% samples ) .
below this threshold , the geometric mean was not calculated and the authors considered the compound to be not detected in the studied population . in certain cases ,
the compound used to express the concentration is given if it is different from the compound or compound class being measured ( e.g. polyphenols in urine expressed as gallic acid).correlations . to compile data on the correlations between specific biomarker measurements and food or dietary compound intake ,
information was collected on the intake of different foods , food groups or dietary compounds together with the method used for dietary assessment ( record or questionnaire , food composition database , food coverage and time period ) .
food items in exposome - explorer are linked to the foodb database ( www.foodb.ca ) . for food groups ,
the list of individual foods considered to calculate the intake of that particular food group is indicated if described in the publication .
for dietary compounds , the inclusion of dietary supplements in the calculation of their intake is indicated when mentioned in the publication .
correlation coefficients ( pearson 's product moment or spearman 's rank - order ) are collated together with p - value , confidence intervals , statistical significance ( yes/no ) , and correlation size ( number of measurements from which the correlation coefficient was calculated ) .
adjustment of intake or biomarker measurements prior to calculation of the correlation coefficient ( e.g. energy intake by residual method or
creatinine ) as well as a list of regression variables included in the calculation ( e.g. age , smoking status , bmi , gender ) , and the use of measurement error de - attenuation are also indicated.temporal reproducibility of biomarker measurements in an individual is an important characteristic of the biomarker .
a high reproducibility is required when only one sample per subject is available for biomarker measurement , as in most large cohort studies .
reproducibility is usually measured on repeated samples collected in a small group of individuals over a given time interval ( generally weeks or months ) .
reproducibility is principally described by the intraclass correlation coefficient ( icc ) , defined as the ratio of between - subject variance to the sum of within- and between - subject variance .
the higher the icc value , the higher the reliability of the measurement . in some cases , within- and between - subject coefficient of variation ( cv ) , as well as within- and between - subject
reproducibility size ( number of measurements from which the reproducibility value was calculated ) and the confidence interval are also indicated in the database .
publication with its bibliographic details ( title , authors , year , journal , pubmed i d ) .
each population is named with a short informative summary sentence ( e.g. cases and controls in a case - control study on breast cancer ) .
all ) or several ( e.g. soy consumers and soy non - consumers ) subject groups according to different criteria ( e.g. by gender , by ethnicity , by smoking status ) . a reference to a cohort ( e.g. epic )
age , height , weight , bmi , group size , gender , health condition , exclusion of supplement users , smoker proportion , country of origin and ethnicity are also specified when available .
these describe number and time of collections ( e.g. baseline , 1 year ) of different biospecimens .
biospecimens include urine , whole blood , serum , plasma , hair , nails , adipose tissue , breast milk , or fractions such as plasma phospholipids or red blood cell membranes . biomarkers .
chemical information ( e.g. structure , chemical formula , average mass , monoisotopic mass ) is automatically generated by the structure server .
identifiers and links to external databases such as cas , pubchem ( 10 ) , chebi ( 11 ) , hmdb ( 12 ) and foodb ( www.foodb.ca ) are provided .
also included is the analytical method , the original measurement units as described in the publication , and specimen - specific concentration value adjustments ( e.g. normalizing to
lipid for blood , or a list of regression variables ) . arithmetic or geometric mean , as well as median
concentration values are compiled along with the minimum and maximum values , standard deviation , percentiles , confidence intervals , measurement size ( number of subjects from which the concentration value was calculated ) , as well as the proportion of subjects for which the biomarker was detected .
information on the inclusion or exclusion of zero values in the calculated concentration is also given .
this was mostly done for pollutant biomonitoring studies where a chemical is often detected only in a small proportion of the studied population .
some authors used a threshold ( e.g. detected in at least 30% samples ) .
below this threshold , the geometric mean was not calculated and the authors considered the compound to be not detected in the studied population . in certain cases ,
the compound used to express the concentration is given if it is different from the compound or compound class being measured ( e.g. polyphenols in urine expressed as gallic acid ) .
correlations . to compile data on the correlations between specific biomarker measurements and food or dietary compound intake ,
information was collected on the intake of different foods , food groups or dietary compounds together with the method used for dietary assessment ( record or questionnaire , food composition database , food coverage and time period ) .
food items in exposome - explorer are linked to the foodb database ( www.foodb.ca ) . for food groups ,
the list of individual foods considered to calculate the intake of that particular food group is indicated if described in the publication . for dietary compounds ,
the inclusion of dietary supplements in the calculation of their intake is indicated when mentioned in the publication .
correlation coefficients ( pearson 's product moment or spearman 's rank - order ) are collated together with p - value , confidence intervals , statistical significance ( yes/no ) , and correlation size ( number of measurements from which the correlation coefficient was calculated ) .
adjustment of intake or biomarker measurements prior to calculation of the correlation coefficient ( e.g. energy intake by residual method or
creatinine ) as well as a list of regression variables included in the calculation ( e.g. age , smoking status , bmi , gender ) , and the use of measurement error de - attenuation are also indicated .
temporal reproducibility of biomarker measurements in an individual is an important characteristic of the biomarker .
a high reproducibility is required when only one sample per subject is available for biomarker measurement , as in most large cohort studies .
reproducibility is usually measured on repeated samples collected in a small group of individuals over a given time interval ( generally weeks or months ) .
reproducibility is principally described by the intraclass correlation coefficient ( icc ) , defined as the ratio of between - subject variance to the sum of within- and between - subject variance .
the higher the icc value , the higher the reliability of the measurement . in some cases , within- and between - subject coefficient of variation ( cv ) , as well as within- and between - subject variance ( var )
reproducibility size ( number of measurements from which the reproducibility value was calculated ) and the confidence interval are also indicated in the database .
exposome - explorer 's annotation interface uses a number of features to help complex , heterogeneous , literature - derived data to be easily and systematically translated into organized electronic data .
controlled vocabularies for compounds , foods , experimental methods , specimens , cohorts and units can be created and are fully documented in both the annotation interface and the database .
this helps to avoid the inclusion of duplicate vocabulary items , such as different spellings or synonyms for a same item .
hierarchical associations of populations , samples and measurements can be represented through the creation of parent - children relations .
for instance , populations stratified into different sub - groups can be easily described , or the association of several samples taken at different collection times .
all data uploaded to the database via the exposome - explorer annotation interface is automatically validated , thereby preventing the uploading of erroneous records .
error checks include the usual database integrity verifications such as the presence of mandatory fields , checks on input text size , checks on allowable values or the uniqueness of specific values .
the error checking utilities also include application - specific consistency verifications such as correlations can not be created between measurements of different subject groups or measurement size can not exceed subject group size. these checks ensure the highest consistency for data collected by different annotators .
newly uploaded data is also manually and systematically checked by the database 's chief curator .
repetitive insertion of similar data is facilitated via the support of record duplication and the ability to edit several records at once .
error messages are displayed to guide annotators toward making correct data entries or fixing erroneous entries .
exposome - explorer 's public user interface allows intuitive browsing and searching of almost any data type in the database ( figure 1 ) .
data can be retrieved through the quick search functionality or through specific searches offered on the website 's different menu pages .
distinct areas are shown : structure and chemical information ( a ) , concentrations ( b ) , correlations ( c ) and reproducibility ( d ) collated from several publications .
biomarkers menu lists the biomarkers , biospecimens , analytical methods and cohorts documented in the database .
each item has its own summary page with both general details ( name and classification ) and all data related to this item in the database .
correlations menu lists the correlation values between different food or dietary compound intakes and the corresponding biomarker measurements .
these correlations can be searched according to the food type , dietary compound , or biomarker .
the data can be filtered according to a variety of parameters including the type of biospecimen or the method used to assess the dietary exposure .
reproducibility menu lists the biomarker reproducibility values , which can also be filtered according to biomarker classification ( as well as other parameters ) and ranked in decreasing or increasing order .
the data search menu lists all biomarker concentration values and allows searches over all data fields .
searches by chemical structure are also possible , as well as browsing biomarker classes and subclasses . the
the list can be filtered by title , authors , year or pubmed i d .
full - text for these publications can be accessed via pubmed links or direct publisher urls . for every annotated publication , the totality of collected data including the bibliographic information , detailed subject descriptions , and biomarker data ( concentration values , dietary intake values , correlation values and reproducibility values ) is displayed in a single page .
tables on exposome - explorer 's different webpages can be easily adjusted to suit user - specific needs .
several hidden columns are available and can be shown in order to provide more details for the default display .
every original value is listed with its corresponding bibliographic citation , making it possible to link each value back to its metadata .
default conversions from highly diverse to standardized units are possible while original values are preserved .
tables can be exported in different formats ( e.g. csv , tab ) and reused in other programs ( e.g. excel , r ) in order to conduct more specific analyses .
ruby on rails is a web framework which employs the model - view - controller ( mvc ) design pattern .
the exposome - explorer data is stored in a mysql relational database ( http://www.mysql.com/ ) .
hierarchical associations of records ( trees ) are implemented as a nested set model inside a single database column of the tables using the rails gem ancestry ( https://github.com/stefankroes/ancestry ) .
highly diverse units and their conversions are transparently handled with the phys - units library ( https://github.com/masa16/phys-units/ ) , which is a ruby implementation of the gnu units software ( http://www.gnu.org/software/units/ ) .
website global search is implemented using the wishart 's unearth gem , which uses elasticsearch indexing ( https://www.elastic.co/ ) .
the web interface is built with the bootstrap front - end framework ( http://getbootstrap.com/ ) .
to date , data from a total of 480 publications have been entered into this first release of exposome - explorer .
this includes 10 510 concentration values for 692 biomarker entries , among which 8,861 concentrations correspond to 488 single dietary and pollutant biomarkers .
approximately one third of these biomarkers are dietary biomarkers ( table 1 ) . for dietary biomarkers ,
almost half of the concentrations are related to fatty acids , followed by carotenoids and polyphenols . with regard to pollutant biomarkers , about two thirds of the concentration data in exposome - explorer
are related to pcbs and pbdes , followed by pahs , pcdd / fs and phthalates .
some concentration data are also available for other biospecimens such as hair , nails , adipose tissue or breast milk .
exposome - explorer currently contains the most complete and comprehensive information on exposure biomarkers ever compiled from peer - reviewed literature .
it is also the first publicly available , web - enabled database specifically dedicated to exposure biomarkers .
we believe it provides a good starting point for selecting markers of interest or for defining panels of biomarkers that can be used in exposome - wide association studies . through exposome - explorer ,
biomarker concentrations can be compared in different cohorts or population groups at different levels of exposure ( e.g. consumers and non - consumers of a particular food ) , or between different geographical areas .
all of the database 's manually curated information is fully linked with other online databases and with the original publications .
the high granularity of the data in the database should allow users to conduct very diverse and advanced analyses or comparisons across publications .
looking both at the range of studied compounds and the number of corresponding studies , exposome - explorer also allows users to quickly identify poorly studied exposures or biomarkers that may require further work for validation .
plans for future enhancements to exposome - explorer include its extension to other categories of exposures ( nutritional status , pesticides , occupational exposures ) , the addition of other types of biomarkers ( dna and protein adducts ) , and the inclusion of more information characterizing biomarkers such as their half - life and other pharmacokinetic parameters .
plans are also being made to add putative or non - validated biomarkers identified in ( pre)clinical studies , but never measured in populations .
overall , we believe exposome - explorer will help in the generation of hypotheses for discovery of new biomarkers to be tested in the laboratory . it should also help in evaluating the performance of existing biomarkers and integrating exposure data based on biomarkers with data collected with other technologies .
exposome - explorer should contribute to the translation of the exposome into practice in epidemiological research .
european commission : exposomics fp7-kbbe-2012 ; nutritech fp7-kbbe-2011 - 5 ; joint programming initiative foodball 201417 .
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exposome - explorer ( http://exposome-explorer.iarc.fr ) is the first database dedicated to biomarkers of exposure to environmental risk factors .
it contains detailed information on the nature of biomarkers , their concentrations in various human biospecimens , the study population where measured and the analytical techniques used for measurement .
it also contains correlations with external exposure measurements and data on biological reproducibility over time .
the data in exposome - explorer was manually collected from peer - reviewed publications and organized to make it easily accessible through a web interface for in - depth analyses .
the database and the web interface were developed using the ruby on rails framework .
a total of 480 publications were analyzed and 10 510 concentration values in blood , urine and other biospecimens for 692 dietary and pollutant biomarkers were collected . over 8000 correlation values between dietary biomarker levels and food intake as well as 536 values of biological reproducibility over time were also compiled .
exposome - explorer makes it easy to compare the performance between biomarkers and their fields of application
. it should be particularly useful for epidemiologists and clinicians wishing to select panels of biomarkers that can be used in biomonitoring studies or in exposome - wide association studies , thereby allowing them to better understand the etiology of chronic diseases .
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INTRODUCTION
DATA COLLECTION
DATA COMPILATION
QUERYING THE DATABASE
DATABASE IMPLEMENTATION
DATA STATISTICS
CONCLUSIONS AND FUTURE ENHANCEMENTS
FUNDING
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the aspergilli are filamentous fungi , which are multicellular eukaryotes with a relatively simple life cycle .
many of them have long been used in food production , industrial fermentation , and agriculture . on the other hand , a few , such as a. fumigatus , a. flavus , a. niger , a. nidulans , and a. terreus , are opportunistic fungal pathogens , causing life - threatening invasive aspergillosis ( ia ) in immunosuppressed patients [ 1 , 2 ] , in which a. fumigatus is the predominant pathogen [ 35 ] .
the crude mortality for ia is 6090% and remains around 2942% even when treatment is given .
the main reasons for patient death are late diagnosis and the low efficiency of the drug therapies available to treat ia .
it maintains cell shape and provides osmotic protection [ 7 , 8 ] and has therefore been recognized for a long time as an ideal drug target .
indeed , several cell - wall - targeted drugs , such as echinocandins , caspofungin , micafungin , and anidulafungin , have been introduced as therapies .
for example , echinocandins , which inhibit synthesis of -1,3-glucan , a crucial component of the cell wall are effective in the treatment of invasive fungal infections including ia .
unfortunately , the echinocandins also trigger an increase of chitin [ 9 , 10 ] , which partially compensates for the loss of -1,3-glucan and reduces the efficiency of treatment due to the complicated mechanism of cell wall biogenesis in a. fumigatus .
therefore , a more profound understanding of the mechanisms of cell wall biosynthesis in a. fumigatus would help to improve the efficiency of drug therapies , especially for drugs which target the cell wall .
the cell wall of a. fumigatus is composed of a unique -1,3/1,4-glucan skeleton with chitin and galactomannan covalently linked to the nonreducing ends of -1,3-glucan . the cell wall is mainly coated with gpi proteins , which contain n- and o - glycans [ 11 , 12 ] . while there is no doubt that glycosylation is involved in cell wall organization , the functional importance of protein glycosylation in cell wall organization has , until recently , remained poorly understood .
however , during the past few years , it has become increasingly evident that glycosylation is vital for cell wall synthesis and thus vital for growth and morphology of a. fumigatus .
basically , all eukaryotes possess three types of protein glycosylation , n - glycosylation of asparagine residues , o - glycosylation of threonine and serine residues , and glycosylphosphatidylinositol - anchoring ( gpi - anchoring ) of the c - terminus of some proteins .
humans lacking individual glycosyltransferases suffer from severe congenital diseases , known as carbohydrate - deficient glycoprotein syndromes ( cdgs ) [ 1214 ] .
clearly , the sugar components of proteins play a major role in embryonic and postembryonic development of humans as well as of all higher eukaryotes .
however , the molecular details leading to cdgs are only vaguely understood . during the past 20 years
it is now known that carbohydrates play increasingly important roles in regulating the development of higher organisms . however , the mechanism by which carbohydrates play a role in development and disease is still unclear .
our knowledge of protein glycosylation comes mainly from investigation of the model yeast s. cerevisiae and of mammalian cells .
although investigation of the model yeast has been very useful in elucidating the biochemical features of protein glycosylation at the cellular level , they can not reveal the complicated functions of glycosylation in the development of multicellular eukaryotes .
therefore , investigation of glycosylation in the multicellular fungus a. fumigatus not only helps understand the mechanism of cell wall synthesis in this species but also provides insights into the role of glycosylation in the development of multicellular eukaryotes .
this paper concentrates on protein glycosylation in a. fumigatus , which will be discussed with respect to the enzymatic pathways involved and their functional importance .
furthermore , the utility of a. fumigatus as a model for glycosylation during development of multicellular eukaryotes will be outlined .
the cell wall of a. fumigatus is mainly composed of -1,3-glucans that are highly branched with -1,6 linkages . together
they constitute a three - dimensional network with a large number of nonreducing ends , to which chitin , galactomannan , and -1,3/1,4-glucan are covalently anchored . the cell wall is mainly coated with gpi proteins , which contain n- and o - glycans derived primarily from the process of glycosylation [ 11 , 17 ] .
deletion of gel2 leads to slower growth , abnormal conidiogenesis , an altered cell wall composition , and reduced virulence [ 18 , 19 ] .
it has been proposed that gel2p is responsible for the elongation of -1,3-glucan side chains of -1,3/1,6 branched glucan to provide new nonreducing ends .
ecm33p is also involved in maintaining proper cell wall architecture though its function is unknown .
disruption of ecm33 results in morphogenetic aberrations such as defects in conidial separation , increase of chitin in conidial cell walls , rapid conidial germination , and increased virulence [ 20 , 21 ] .
it is clear that glycoproteins are directly , as structural components of the cell wall , and indirectly , as enzymes required for cell wall synthesis , involved in maintaining proper cell wall architecture in a. fumigatus .
increased chitin synthesis has been known as an important compensatory response to cell wall stress both in s. cerevisiae and filamentous fungi [ 2226 ] . in s. cerevisiae , the cell wall is required to maintain cell shape , which is essential for the formation of a bud and hence cell division .
the yeast cell remodels its rigid structure to accommodate cell expansion during vegetative proliferation , mating pheromone - induced morphogenesis , and nutrient - driven filamentation through the cell wall integrity ( cwi ) signaling pathway .
cell wall defects or damage induces the cells to activate the cwi pathway to survive , and the compensatory mechanism characterized by an increased chitin content is triggered .
the cwi signaling pathway in s. cerevisiae is activated in response to low osmolarity , thermal stress , or mating pheromone and polarized growth .
it is comprised of a family of cell surface sensors coupled to the small g - protein rho1p , which activates the cwi mapk cascade via protein kinase c ( pkc1p ) .
this signaling cascade activates the expression of genes encoding for cell wall proteins that stabilize the cell wall .
meanwhile , activated rho1p also activates a set of additional effectors such as bni1p and bnr1p formin proteins , skn7p transcription factor , and the sec3p exocyst component , which regulate a diverse set of processes including -glucan synthesis at the site of cell wall remodeling , gene expression related to cell wall biogenesis , organization of the actin cytoskeleton , and secretory vesicle targeting to the growth site .
a family of cell surface sensors is responsible for detecting and transmitting the status of the cell wall to rho1p .
these sensor molecules include wsc1p ( hcs77p / slg1p ) [ 2931 ] , wsc2p and wsc3p , and mid2p and mtl1p [ 32 , 33 ] . among these cell wall stress sensors , wsc1p and mid2p
appear to be the most important and serve a partially overlapping role in cwi signaling .
reduced o - mannosylation leads to incorrect proteolytic processing of these proteins , which in turn results in impaired activation of the pkc1 pathway and finally causes cell death in the absence of osmotic stabilization .
more recently , n - glycan is found to be directly involved in mid2p sensing .
it has been shown that both the extent of the n - linked glycan and its distance from the plasma membrane affect mid2p function .
these observations demonstrate that n- and o - glycosylation are important for cwi sensing and thus important for cell wall biogenesis and polarized growth in yeast .
the presence of a. fumigatus genes encoding for proteins homologous to the yeast rho1p , rho3p , and cdc42p suggests a similar mechanism for the cwi pathway .
indeed , it has been recently shown that afcdc42/cdc42 , afrho1/rho1 , and afrho3/rho3 are highly expressed in the mutant devoid of cwh41p ( glucosidase i ) , which suggests an activation of these genes induced by cell wall damage . also ,
increased expression and activation of the a. fumigatus mpkap , an ortholog of the s. cerevisiae mpk1p , is also induced by cell wall damage [ 38 , 39 ] .
it is becoming clear that , as in yeast , defects in cell wall integrity also trigger the cwi mapk cascade in a. fumigatus . on the other hand ,
in contrast to yeast , little is known about the cell wall stress sensors in a. fumigatus . in the last release of the a. fumigatus genomic database ( http://www.aspergillus.org.uk/indexhome.htm?secure/sequence_info/index.php~main ) , only one protein ( afua_5g09020 )
is annotated as a homologue of the wsc4p , which does not appear to contribute to cwi signaling in yeast .
therefore , the a. fumigatus cell wall stress sensor molecule remains to be identified and investigated .
the precursor of all mannose residues found in galactomannan , glycoprotein , and gpi anchor in a. fumigatus is gdp - mannose .
the activation of mannose initiates from formation of mannose 6-phosphate ( man-6-p ) , which occurs by one of two routes : direct phosphorylation of mannose by hexokinase or interconversion from fructose 6-phosphate ( fru-6-p ) via phosphomannose isomerase ( pmi ) , and the latter pathway requires three enzymes : pmi , phosphomannomutase ( pmm ) , and gdp - mannose pyrophosphorylase ( gmpp ) .
fru-6-p is converted to man-6-p by pmi , and then man-6-p is converted to mannose 1-phosphate ( man-1-p ) by pmm .
subsequently , man-1-p is ligated with the guanosine 5-triphosphate molecule ( gtp ) to form gdp - mannose by man-1-p guanylyltransferase [ 4063 ] .
the interconversion of man-6-p and fru-6-p catalysed by pmi is the first committed step in the synthesis of man - containing sugar chains and provides a link between glucose metabolism and mannosylation .
pmi deficiency is the cause of carbohydrate - deficient glycoprotein syndrome type ib ( cdg - ib , omim 602579 ) in humans , but the clinical symptoms and aberrant glycosylation can be corrected with dietary mannose supplements .
genes encoding for pmis have been investigated in several fungal species , such as s. cerevisiae , candida albicans , a. nidulans , and cryptococcus neoformans [ 4851 ] .
the pmi mutant shows a significantly reduced growth rate at high concentrations of mannose .
biochemical and genome - wide analysis reveals that excess mannose leads to an accumulation of man-6-p , which mainly inhibits the activity of phosphoglucose isomerase ( pgi ) and thus represses glycolysis , protein biosynthesis , and cell wall biogenesis .
the a. nidulans mana1 mutant exhibits abnormal ballooning of hyphal tips and eventually ceases to grow . disrupted man1 mutant of c. neoformans leads to poor capsule formation , reduced polysaccharide secretion , morphological abnormalities , and attenuated virulence .
in a. fumigatus , pmi activity is essential for viability and plays a central regulatory role in both glycosylation and energy production .
deletion of the a. fumigatus pmi1 gene leads to uncoupling of the link between energy production and glycosylation and accumulation of man-6-p , which then results in defects in cell wall integrity , conidiation , and morphology . although extracellular mannose can rescue the growth of pmi deficient mutants in a. fumigatus , both lower and higher concentrations of mannose lead to a reduction in the levels of -glucan in the cell wall and an accumulation of man-6-p .
the phenotypes associated with the mutant under mannose starvation are mainly due to an insufficient supply of gdp - man required for cell wall synthesis .
the abnormal morphology associated with the pmi1 mutant under mannose - replete conditions is mainly ascribed to an accumulation of man-6-p , which can not efficiently enter glycolysis , instead becoming trapped in a cycle of dephosphorylation and rephosphorylation resulting in depletion of intracellular atp .
it should be pointed out that the pmi in a. fumigatus mainly catalyzes the conversion of fru-6-p to man-6-p , and its binding affinity for man-6-p is similar to that of yeasts but different from the ones from bacteria or animals ( table 1 ) .
this suggests that it may be possible to design a specific inhibitor for fungal pmis .
several gmpps have been identified and characterized in different species [ 5559 ] . in s. cerevisiae and c. albicans , gmpp is essential [ 60 , 61 ] , while in leishmania mexicana gmpp is not required for viability .
repression of gmpp in yeast leads to pleiotropic phenotypes including cell lysis , failure of cell separation , impaired budding and hyphal switching , clumping and flocculation , and cell wall defects .
repression of expression of a. fumigatus gmpp srb1 , a homologue of yeast srb1/psa1/vig9 , leads to hyphal lysis , a defective cell wall , impaired polarity maintenance , and branching site selection , as well as rapid germination and reduced conidiation .
in contrast to yeast , inducible repression of srb1 expression in a. fumigatus does affect the ability to direct polarity establishment and septation .
these reports imply that mannose activation is specifically crucial for the synthesis and organization of the cell wall and thus essential for survival of fungal species .
this further suggests that glycosylation is essential for the viability of pathogenic fungal species such as a. fumigates , and inhibitors that specifically block mannose activation in fungi may be potential drugs to treat fungal infections .
n - glycosylated proteins contain oligosaccharides that are n - glycosidically linked to the -amido group of asparagine .
this type of glycoprotein has been intensively studied in many model systems from yeast to human cells with respect to their structure , biosynthesis , and function .
it has been shown that the formation of the highly variable n - linked oligosaccharides is initiated by the assembly of a lipid - linked oligosaccharide glc3man9glcnac2-pp - dol by a series of glycosyltransferases located on the cytoplasmic and luminal faces of the er membrane .
the most complete understanding of biosynthesis of the lipid bound precursor has been obtained from s. cerevisiae and from mammals . as far as it is known , the corresponding reactions proceed almost identically in other eukaryotes . subsequently , the dol - pp - linked glc3man9glcnac2 is transferred as a whole to an asparagine residue within an n - x - t / s consensus sequence of a nascent peptide , which is catalyzed by the oligosaccharyltransferase ( ost ) , and then the n - glycosylated proteins are modified in a species - specific manner and transferred through the secretory pathway to the cell surface where they either get exported or anchored to the plasma membrane , to the extracellular matrix , or to the cell wall ( figure 1 ) .
the ost complex consists of at least eight different subunits , including ost1p , ost2p , wbp1 , stt3p , swp1p , ost4p , ost5p , and ost3p / ost6p [ 6467 ] .
although the function of each subunit is still unclear , stt3p is believed to be the catalytic subunit [ 6870 ] , and its homologues are found in almost all eukaryotes .
it appears that the a. fumigatus stt3 is also essential as no viable knockout mutant has been recovered .
repression of the stt3 gene in a. fumigatus leads to a severe retardation of growth and a slight defect in cell wall integrity .
further analysis shows that repression of stt3 upregulates expression of the genes responsible for glucan and chitin synthesis , especially gel1 , gel2 , fska , chse , and chsg .
indeed , an increase of cell wall mannoprotein and chitin was observed following repression of the stt3 gene .
however , this upregulation of chitin is not accompanied by an activation of the mpka kinase .
indeed , only the unfolded protein response ( upr ) is induced . as the upr has been shown to be involved in cwi signaling in a. fumigatus ,
it is likely that upr , instead of the mpka - dependent cwi signaling pathway , is the major compensatory mechanism induced by repression of the n - glycosylation in a. fumigatus .
once glc3man9glcnac2 is transferred to proteins , the n - glycan is processed sequentially in the er and golgi .
n - glycan processing is initiated by the removal of the glucose residues catalyzed by er glucosidase i and glucosidase ii . in mammalian cells ,
n - linked glycan plays a decisive role as a quality control ( qc ) of the folding of secretory proteins , which is composed of calnexin , calreticulin , udp - glucose : glycoprotein glucosyltransferase ( gt ) and glucosidase ii ( gii ) and is essential for cellular survival [ 7577 ] .
n - glycans initially serve to increase the hydrophilicity of the as - yet - unstructured nascent polypeptides .
subsequently , the two outermost glucose residues of the n - glycan are removed by the sequential action of glucosidase i ( gi ) and gii to the monoglucosylated form , which is recognized and bound by calnexin , a type i er membrane lectin , and calreticulin , its soluble relative . for many glycoproteins ,
the interaction with calnexin or calreticulin slows down the rate of folding but increases efficiency .
gii - catalyzed removal of the third glucose residue follows the dissociation of folding substrates from calnexin and is required for release of native polypeptides from the er and transport to their final destination .
the folding sensor gt adds back a terminal glucose to promote reassociation of nonnative polypeptides released from calnexin , thus prolonging their retention in the er folding environment .
cycles of de-/reglucosylation might be protracted until the polypeptide released from calnexin fulfills quality control requirements .
when correct folding is not achieved , an er - specific n - glycan - dependent pathway of degradation removes the misfolded proteins .
when n - glycosylation is inhibited , the most commonly observed effect is the generation of misfolded , aggregated proteins that fail to reach a functional state [ 75 , 76 ] . before entering the qc system ,
the outermost glucose residue of the n - glycan is trimmed by er glucosidase i .
a human inherited glucosidase i deficiency has been reported to result in neonatal birth with severe generalized hypotonia and dysmorphic features . unlike mammalian cells ,
s. cerevisiae lacks a calnexin cycle and gt and only has an effective mannosidase i - dependent erad system [ 80 , 81 ] . the yeast glucosidase i ( cwh1p ) is encoded by the cwh41 gene .
mutational defects in the cwh41 gene cause severe and selective instability of glycoprotein kre6p , a putative golgi glucan synthase required for -l,6-glucan synthesis [ 23 , 83 , 84 ] .
some filamentous fungi have been proposed to possess n - glycan - dependent qc of glycoprotein folding based on fungal genome sequence data . recently ,
evidence that filamentous fungi possess an n - glycan - dependent qc system has been reported in a. fumigatus [ 37 , 86 ] .
indeed , calnexin ( aas68033 ) , glucosidase ii , and gt have been annotated in the last release of the a. fumigatus genomic database ( figure 2 ) .
zhang et al . reported that deletion of the cwh41 gene in a. fumigatus results in defective n - glycan processing of the proteins secreted by a. fumigatus .
although afcwh41 is not essential for hyphal growth and virulence , a severe reduction in conidial formation , abnormalities of polar growth and septation , and a temperature - sensitive deficiency of cell wall integrity were documented .
also , the genes encoding rho - type gtpases ( rho - type gtpase / cdc42 ) were upregulated , which suggests that the cwi pathway was activated in the mutant .
after processing by two er -glucosidases , the n - glycan is further processed by the action of various 1,2-mannosidases , which can remove one or more of the four 1,2-linked mannose residues . in mammalian cells , man9glcnac2 is converted to man5glcnac2 by the action of er and golgi -mannosidases , which is the precursor for complex , hybrid , and high - mannose n - glycans . in s. cerevisiae , a specific er 1,2-mannosidase converts man9glcnac2 into man8glcnac2 , which is elongated in the golgi to form an outer chain containing up to 200 residues of mannose [ 89 , 90 ] .
a. saitoi and trichoderma reesei have been found to produce n - glycan structures containing five mannose units ( man5glcnac2 ) , suggesting further processing of the man9glcnac2 precursor [ 91 , 92 ] .
the n - glycans on mature secreted glycoprotein produced by a. fumigatus are man6glcnac2 , man7glcnac2 , and man8glcnac2 , in which man6glcnac2 is the major glycoform .
these observations demonstrate that n - glycan synthesis in filamentous fungi seems to differ from that in yeast and is similar to that in higher eukaryotes ( figure 3 ) .
although small n - glycans are commonly found on glycoproteins of a. fumigatus , hex5 - 13hexnac2 glycans on the galactomannoproteins , and man5 - 9glcnac2 as well as galf1man5 - 7glcnac2 structures on other secreted glycoproteins have been identified in a. fumigatus [ 93 , 94 ] . the enzyme ( udp - galp mutase ) required to synthesize the requisite udp - galf donor has been shown to be an important factor in biosynthesis of the cell wall in a. fumigatus , while the gene / enzyme responsible for the transfer of galf has not been identified .
recently , the a. fumigatus och1 , a key mannosyltransferase for synthesis of elaborated protein n - glycans in yeast , has been identified .
deletion of the och1 gene results in a reduction of sporulation in the presence of high calcium concentrations .
this evidence suggests that polymannosylated n - glycans exist in a. fumigatus and certain proteins engaged in sporulation require n - glycan outer chains to be fully functional .
the -mannosidases have been classified into two groups : class i and class ii [ 102 , 120 , 121 ] .
class i -mannosidases include er man9-mannosidase , endomannosidase , and golgi mannosidase i. class ii -mannosidases include the lysosomal mannosidases , golgi mannosidase ii , yeast vascular mannosidase [ 98 , 99 ] , and er -mannosidase ii [ 122 , 123 ] .
several golgi -mannosidases have been cloned and characterized from penicillium citrinum [ 124 , 125 ] , a. saitoi , a. oryzae , t. reesei , and a. nidulans [ 102 , 129 ] .
these enzymes are all monomeric with a molecular weight of 5060 kda and show the maximal activity in the semiacidic condition ( ph 46 ) .
class i -mannosidase is known to play an important role in the processing of mannose - containing glycans . in drosophila melanogaster , deletion of the golgi mannosidase i ( mas-1 ) results in viable progeny , and the null organisms synthesize the same range of oligosaccharides as the wild - type ones , albeit at different ratios . in s. cerevisiae , disruption of the er -mannosidase gene does not prevent outer chain synthesis . in the last release of the tigr database ( http://www.aspergillus.org.uk/indexhome.htm?secure/sequence_info/index.php~main ) , nine a. fumigatus genes are annotated to encode -mannosidases , including xp_749038.1 , xp_754794.1 , xp_751252.1 , xp_751819.1 , xp_752444.1 , xp_752825.1 , xp_753592.1 , xp_751114.1 , and xp_750572.1 . among them , msdsp ( xp_752825.1 ) has been identified to encode a class i 1 , 2-mannosidase and acts on man8glcnac2 to produce man6glcnac2 .
deletion of the msds gene leads to a defect in n - glycan processing , as well as a reduction of cell wall components ( including -glucan , -glucan , mannoprotein , and chitin ) and reduced conidiation .
. in mammalian cells , free oligosaccharides ( fos ) are generated by ost - mediated hydrolysis of glc3man9glcnac2-pp - dolichol in the lumen of the er or peptide n - glycanase ( pngase)-mediated de - n - glycosylation of newly synthesized glycoproteins either in the er or the cytosol .
fos that are liberated in the lumen of the er can be transported into the cytosol , where they are trimmed by an endo--d - n - acetylglucosamine h ( endo h)-like enzyme and the -mannosidase man2c1p in order to yield an oligosaccharide , man5glcnac , that can be imported directly into lysosomes to be degraded ( figure 4 ) [ 122 , 123 , 131135 ] . in humans and cattle [ 137139 ] , a deficiency in -mannosidase results in the lethal disease mannosidosis , a rare lysosomal storage disease with a collection of clinical symptoms including progressive mental retardation , impaired hearing , dysostosis multiplex , immune defects , elevation of serum and urinary oligosaccharide levels , and an enlargement of lysosomes in most cell types resulting from the accumulation of undegraded oligosaccharides .
the rat man2c1p is involved in oligosaccharide catabolism of misfolded glycoproteins in the lumen of the er which have been retrotranslocated into the cytoplasm for proteolytic disposal [ 131133 ] .
a proteolytically cleaved version of the rat man2c1p has been found in the lumen of the er where it is believed to be involved in the early stages of glycoprotein maturation ( also called er -mannosidase ii ) ( figure 4 ) [ 122 , 123 ] .
the yeast cytosolic -mannosidase ams1p , a counterpart of man2c1p , is also involved in the processing of fos .
since the yeast png1p is mainly localized to the cytosol , it is proposed that the png1p - generated fos may both be generated and processed in the cytosol .
the role of the yeast ams1p is to aid in recycling macromolecular components of the cell under nutrient deprivation . interestingly , after its synthesis in the cytosol , the ams1p is translocated into the vacuole by the cytosol - to - vacuole targeting pathway , which suggests a common feature shared by the s. cerevisiae ams1p and its mammalian counterparts .
however , the yeast ams1p only participates in recycling or utilizing of oligosaccharide but not in processing of n - glycan ( figure 4 ) .
it should also be noted that no structural studies have been performed on the products that can be generated from man8glcnac2 by ams1p , and the ultimate fate of such products remains obscure . on the other hand , two png1p - independent fos pools , man3glcnac2 and man8glcnac2 , are also seen in s. cerevisiae .
the pool comprising small fos ( man3glcnac2 ) appears to be disposed of by unknown enzymes in the vacuole .
the pool containing mainly man8glcnac2 may be generated and disposed of along the secretory pathway .
similarly , a. nidulans -mannosidase iic is also proposed to be involved in oligosaccharide catabolism .
both a. nidulans-mannosidase iic and s. cerevisiae ams1p are not essential for normal cellular function since disruption of these genes has no visible effect on growth or morphology [ 98 , 99 , 102 ] .
in contrast to its counterpart in yeast or a. nidulans , the a. fumigatus ams1p is required for normal cellular function .
deletion of the a. fumigatus ams1 leads to a severe defect in conidial formation , especially at a higher temperature .
these results show that the afams1 gene is required for morphogenesis and cellular function in a. fumigatus .
the involvement of the afams1 gene in polarized growth demonstrates that the processes involved in fos regulation are important for a. fumigatus .
it is likely that the ams1p is involved in cell wall synthesis and thus polarity through the cwi pathway .
therefore , probably the afams1 mutant could serve as a simple model to investigate the mechanism of -mannosidosis . functional analyses of the genes required for n - glycosylation reveal that protein
n - glycosylation is important for cell wall synthesis , morphogenesis , and polarized growth in a. fumigatus .
2-d gel analysis reveals that deletion of the cwh41 gene encoding glucosidase i in a. fumigatus leads to er stress , which induces overexpression of hsp70 and calnexin chaperone and activates the erad .
meanwhile , the proteins required for actin rearrangement are found to be underexpressed or missing , which is consistent with the observation of random localization of actin fibers in the mutant .
these observations , for the first time , clearly suggest that n - glycosylation contributes to proper folding and trafficking in a. fumigatus .
it appears that proteins involved in cell wall biosynthesis in a. fumigatus are more dependent on the n - glycan - dependent folding system . as in yeast
, cell wall defects also trigger the cwi signaling pathway in a. fumigatus , which activates downstream effectors that regulate cell wall biogenesis and polarized growth .
zhang et al . [ 37 , 86 ] have proposed that the proteins required for cell wall synthesis or cell wall stress sensing are substrates of a. fumigatus cwh41p and require glucose trimming for their proper localization and function .
misfolding of these proteins would cause cell wall defects , which then leads to activation of the erad and rho - type gtpases - mediated cwi pathway .
moreover , activation of cdc42 in the cwi pathway also activates sepa , an upstream organizer of actin ring formation at septation sites , and thus causes abnormal polarized growth associated with the afcwh41 mutant ( figure 5 ) .
although the phenotypes associated with different n - glycosylation mutants vary , the finding that all of these mutants exhibit phenotypes associated with cell wall defects , abnormal polarization , and morphological changes can all be explained by this proposed model . obviously , more investigations are needed to identify and characterize all of the proteins affected by n - glycosylation in a. fumigatus .
this information would be key to understanding the complex compensatory mechanisms participating in cell wall biosynthesis in a. fumigatus , which would serve as a basis to develop new antifungal therapies , as well as help to elucidate the molecular mechanism of human diseases associated with defects in glycosylation .
o - mannose glycosylated proteins were first discovered in yeast and filamentous fungi , and recently this type of glycoproteins has also been described in mammals .
the o - mannosylation most likely occurs in all animals , with the exception of nematodes ( e.g. , caenorhabditis elegans ) ; it is also not detected in plants ( arabidopsis thaliana , oryza sativa ) .
however , it has also been discovered in one bacterial species ( mycobacterium tuberculosis ) . in mammalian cells ,
the inner o - linked mannose is elongated with the first addition of a n - acetylglucosamine and then various sugars . in the case of yeast ,
the o - mannose type carbohydrate chain starts with a serine / threonine - linked mannose , which is extended to an oligomannose chain . in a. fumigatus ,
the o - linked glycans on cell wall mannoproteins are found to be glc1 , 6man , galf1 , 6man1 , 6man , galf1 , 5galf1 , 6man1 , 6man and galf1 , 5[galf1,5]3 galf1 , 6man , while only a single mannose residue was detected on secreted proteins .
a further type of protein o - glycosylation , in which a single -o - linked glcnac residue is linked to serine and threonine occurs in animals , plants , and filamentous fungi , but not in s. cerevisiae . for this type of protein modification ,
protein o - mannosylation is initiated by a family of protein o - mannosyltransferases ( pmts ) that are evolutionarily conserved from yeast to human [ 145 , 146 ] . in s. cerevisiae
a total of seven pmt family members ( scpmt17p ) are present [ 104 , 147 ] , which fall into three major groups of homology : ( i ) pmt1/5/7 , ( ii ) pmt2/3/6 , and ( iii ) pmt4 .
genes with significant homology to pmts have been cloned in humans , mice , and drosophila [ 148150 ] .
specific protein substrates that are o - mannosylated by scpmt1p , scpmt2p , or scpmt4p have been described in s. cerevisiae [ 151153 ] . in comparison with s. cerevisiae , the pmt family is less redundant in higher eukaryotes . in drosophila
only two pmt family members are present ( rotated abdomen and twisted ) [ 149 , 150 ] . the same is true for mice and humans ( pomt1 and pomt2 ) [ 148 , 150 ] .
mutations in human pomt1 , a homologue of the yeast pmt4 , cause walker - warburg syndrome ( wws ) , which is characterized by severe congenital muscular dystrophy , neuronal migration defects , and structural abnormalities of the eye . targeted deletion of pomt1 in mice results in embryonic lethality due to defects in the formation of the reichert 's membrane , the first basement membrane to form in the embryo .
mutations of the drosophila pmt homologues alter muscle structures and the alignment of adult cuticle . the pmt family is crucial for viability , cell wall integrity , and morphogenesis in several fungal species , such as s. cerevisiae , s. pombe , c. albicans and c. neoformans , a. nidulans , and a. fumigatus [ 108 , 109 , 111115 , 145 , 156 ] . in s. cerevisiae , single pmt1 mutants fail to grow in anaerobic conditions on some media .
the pmt1,2,3-triple mutants grow only in osmotically stabilized medium , whereas the pmt1,2,4- and pmt2,3,4-triple mutants are not viable in any conditions , indicating that pmt protein activity is essential in s. cerevisiae , although individual genes are dispensable .
c. albicans contains five pmt genes .
the pmt1 mutants are viable , but they are defective in undergoing cellular differentiation from yeast to a true hyphal growth form under some conditions .
the virulence of the pmt1 null mutant is significantly attenuated , which is likely due to reduced o - glycosylation of the c. albicans adhesin als1p .
the pmt phenotypes are closely linked to alterations in cell wall components , including cell wall mannoproteins and polysaccharides . in s. pombe only one member of each pmt subfamily
is present , namely , oma1 , oma2 , and oma4 . deletion of oma2 , as well as simultaneous deletion of oma1 and oma4 is lethal .
characterization of the viable s. pombe oma1d and oma4d single mutants shows that reduced o - mannosylation results in abnormal cell wall and septum formation , therefore severely affecting cell morphology and cell - cell separation . in c. neoformans ,
recently , willger et al . showed that pmt2 is an essential gene , and the double pmt1pmt4 deletion is lethal .
filamentous fungi , such as a. fumigatus , a. nidulans , neurospora crassa , and fusarium gramineum , contain only three pmt genes that belong to the pmt1 , pmt2 , and pmt4 subfamilies , respectively [ 107 , 108 , 113 ] .
all single pmt mutants in a. nidulans are viable but showed reduced growth at elevated temperatures and defects in morphogenesis [ 111 , 112 ] .
the double deletion pmta / pmtc ( orthologues of the pmt2 and pmt4 ) and pmtb / pmtc ( orthologues of pmt1 and pmt4 ) are synthetically lethal .
have shown that a single deletion of a. fumigatus pmt1 results in temperature - sensitive phenotypes .
when the a. fumigatus pmt1 mutant was grown on solid complete medium at 37c , no difference was found between the mutant and the wild type .
a strongly retarded growth , however , was observed when this mutant was grown at 42c and 50c .
this temperature - sensitive phenotype could be complemented by the addition of 1 m sucrose in the media .
further analysis shows that the mannoprotein , -glucan , and chitin in the cell wall of the mutant grown at 37c are increased , while -glucan is reduced . when the a. fumigatus pmt1 mutant was cultured at 42c ,
the -glucan was increased , while the -glucan was decreased , and the mannoprotein and chitin content remained unchanged .
moreover , deficient conidiation and reduced germination have been documented at 42c . as compared with the s. cerevisiae pmt1 mutants , the a. fumigatus pmt1 mutant , as well as the c. albicans and s. pombe pmt1 mutants , shows more severe defects in cell wall integrity .
this significant phenotype could be explained by the fewer members of pmt family presented in a. fumigatus , c. albicans , and s. pombe .
however , in a recent study by mouyna et al . , the a. fumigatus pmt1 mutant does not show any visible phenotype . in the report by zhuo et al .
, the pmt1 deletion mutant was constructed by replacement of the entire coding region of the pmt1 in a. fumigatus strain cea17 ( pyrg ) with a pyrg cassette .
therefore , the genetic background of the pmt1 null mutant is pyrg pmt1 , while in the report by mouyna et al .
, the pmt1 mutant was constructed by transformation of a. fumigatus strain ku80 with a deletion cassette containing the e. coli phleomycin phosphotransferase gene ( phle ) .
therefore , the major differences may be due to the different genetic background of the strains used in these two reports .
the single pmt2 or double pmt1pmt4 deletion(s ) are lethal [ 114 , 115 ] .
fang et al . reported that reduced expression of pmt2 leads to retarded growth , cell wall defects , abnormal polarity , and reduced conidiation ; however , no temperature - sensitive growth was found .
interestingly , this is the first time that pmt2p is revealed to be involved in polarized growth .
these observations suggest that a. fumigatus pmt2p is required for cell wall synthesis and morphogenesis and its function is distinct from that of a. fumigatus pmt1p .
disruption of a. fumigatus pmt4 leads to abnormal mycelial growth , poor conidiation , and abnormal polarity .
although an increased sensitivity to echinocandin , a 1,3-glucan synthase inhibitor , was observed in the a. fumigatus pmt4 null mutant , glucan synthase activity and 1,3-glucan content were not affected .
in contrast to its counterpart in c. albicans , a. fumigatus pmt4 is not required for full virulence .
the different functions associated with different a. fumigatus pmts are likely due to their different substrate specificities .
further investigation of the pmt mutants will be helpful for understanding their molecular mechanism , which will not only increase our understanding of the function of o - mannosylation in a. fumigatus , but also may deepen our understanding of the molecular basis of the human walker - warburg syndrome ( wws ) which features mutations in pomt1 , a homologue of a. fumigatus pmt4p , and results in a failure of polarized growth during neuronal migration .
gpi anchoring is a conserved glycosylation process in eukaryotes , which enables many cell surface proteins such as cell surface enzymes , receptors , and adhesion molecules to be covalently anchored to the cell membrane .
the core structure of the gpi anchor consists of a lipid group , myoinositol , glucosamine , several mannose residues , and a phosphoethanolamine group , which ultimately connects the gpi anchor to the protein via an amide bond .
although the number of mannose groups and the position of side - chains on the gpi anchors vary widely between species , a common core structure of etnman3glcn - pi is conserved in all gpi - anchored proteins found in protozoa , yeast , plants , and mammals ( figure 6 ) .
gpi anchoring is not essential in mammals at a cellular level as several gpi - deficient cell lines have been established .
however , an acquired gpi - anchoring deficiency in haematopoietic stem cells causes paroxysmal nocturnal haemoglobinuria , a rare but serious human disease .
also an overexpression of pig - p , a protein of unknown function required for gpi anchor synthesis , has been noted in fetal down syndrome brain .
in contrast to mammals , gpi anchor synthesis is essential in s. cerevisiae . in s. cerevisiae , many gpi - anchored proteins are known to be involved in morphogenesis and cell wall organization .
one type is the gpi - mannoproteins covalently linked to cell wall -1,6-glucan which play important biological functions in filamentation , mating , flocculation , or adhesion to the external matrix [ 161167 ] .
the second type are the gpi proteins associated with the plasma membrane which possess enzymatic activities able to modify cell wall polymers and are involved in altering cell morphology , such as -glucanase and -glucosyltransferase [ 168170 ] .
recent studies in a. fumigatus suggest that at least nine gpi - anchored proteins are common to filamentous fungi and yeast .
five of them are homologues of putative gpi - anchored yeast proteins that have been shown to play a role in cell wall morphogenesis .
the gpi anchor is assembled at the er in multiple steps catalyzed by the concerted actions of approximately 20 proteins .
the first step of gpi anchor synthesis is initiated by the transfer of n - acetylglucosamine ( glcnac ) from udp - glcnac to phosphatidylinositol ( pi ) , which is catalyzed by the glycosylphosphatidylinositol - n - acetyl - glucosaminyltransferase ( gpi - gnt ) complex .
the mammalian gpi - gnt complex consists of seven proteins , including pig - a , pig - h , pig - c , pig - p , gpi1 , pig - y , and dpm2 .
all except dpm2 have structural and functional counterparts in s. cerevisiae , where they are known as gpi3p , gpi15p , gpi2p , gpi19p , gpi1p , and eri1p , respectively .
pig - a / gpi3p is believed to possess the catalytic domain because gpi3p binds a photoactivatable udp - glcnac analog and is a member of glycosyltransferase family 4 of retaining glycosyltransferases .
the roles of the other subunits in the gpi - gnt complex are as yet unclear , but they may mediate regulatory interactions . in yeast ,
gpi anchoring is essential for viability and plays an important role in the biosynthesis and organization of the cell wall .
a gpi3 temperature - sensitive mutant is not viable at 37c [ 116 , 117 ] .
, it is postulated that the introduction of mutations in gpi3/gpig-1 genes allows for minimal level of product function and survival when growing the mutant cells below the restrictive temperatures .
however , the mechanism , by which the defect in gpi anchoring leads to a lethal phenotype in these two species , is poorly understood .
it has been shown that a. fumigatus gpi anchors possess five mannose residues with a phosphoethanolamine linked on the first three residues [ 174 , 175 ] .
the a. fumigatus pig - a gene , the homologue of the gpi3/pig - a gene in yeast , has been investigated .
also , an increased content of -glucan and mannoprotein was observed in the mycelial cell wall of the afpig - a mutant .
unlike the temperature - sensitive or conditional lethal phenotype seen in the yeast gpi3 mutant , afpig - a can survive at temperatures from 30c to 50c . completely blocking gpi anchor synthesis in a. fumigatus afpig - a leads to cell wall defects , abnormal hyphal growth , rapid conidial germination , and aberrant conidiation . in vivo assays
therefore , the gpi anchor seems not essential for viability , but required for cell wall integrity , morphogenesis and virulence in a. fumigatus .
indeed , this is the first report that a deficiency in gpi - anchor synthesis does not lead to a temperature - sensitive or conditional lethal phenotype in microbes , which provides an opportunity to identify the basic function of gpi anchoring in fungi .
during the past 50 years , proteins and nucleic acids have dominated the field of biology .
the enormous advances in the analysis of complex carbohydrates have enabled us to investigate the structure and function of carbohydrates , and the field has developed enormously .
it is now known that carbohydrates play very important roles , especially in the regulation of development of higher organisms .
however , the mechanisms by which carbohydrates play a role in development and diseases are still poorly understood .
our knowledge of protein glycosylation comes mainly from the investigation of s. cerevisiae and mammalian cells .
although investigations of the model yeast and mammalian cells have been very useful in elucidating the biochemical features of protein glycosylation , these investigations at the cellular level can not reflect the complicated functions of glycosylation in the development of multicellular eukaryotes .
therefore , more model systems have been introduced , such as caenorhabditis elegans , drosophila melanogaster , and mice . however , these model systems are still too complex since deletion of individual glycosyltransferase genes in these systems sometimes leads to fetal death or nonvisible phenotypes .
as compared with s. cerevisiae , c. elegans , d. melanogaster , or mice , a. fumigatus seems to be an ideal model for investigation of the function of glycosylation since a. fumigatus is a multicellular eukaryote with a relative simple life cycle , in which it undergoes a series of developmental events that require polarized growth .
recent progress shows that a. fumigatus has evolved an intact n - glycan - dependent qc system , which is present in mammalian cells but not in yeast . disruption of either processing or degradation of n - glycan in a. fumigatus leads to phenotypes such as cell wall defects and abnormal polarity .
based on investigations of s. cerevisiae and filamentous fungi , it is likely that glycosylation first evolved to ensure synthesis of the fungal cell wall and only later did the n - glycan - dependent qc system evolve to ensure precisely controlled cell wall synthesis and polarized growth which are important for multicellular development . however , this hypothesis is controversial .
. showed that the n - glycan - dependent qc system is functional in entamoeba , trichomonas , cryptococcus , and s. pombe , but is not functional in some fungi such as giardia and plasmodium , theileria , encephalitozoon , toxoplasma , cryptosporidium , and tetrahymena .
they proposed that the n - glycan - dependent qc system was likely present in the common ancestor of extant eukaryotes and was secondarily lost from some eukaryotes .
for example , the s. cerevisiae kre5 is believed to be the gt ortholog that no longer glucosylates misfolded glycoproteins but is instead thought to be involved in -1,6-glucan synthesis .
of course , the possibility that the s. cerevisiae kre5 is the ancestor of gt can not be excluded .
it remains unclear where is the evolutionary origin of glycosylation , what is the basic function of glycosylation at the early stages of evolution , and how glycosylation is regulated .
definitely , the answers to these questions will enable us to understand the basic function and regulation of glycosylation in the development of multicellular eukaryotes and help to understand more complex functions in higher eukaryotes .
on the other hand , the investigation of a. fumigatus is also a key to understanding complex compensatory mechanisms of cell wall biosynthesis and may provide a new strategy for drug development . during the past few years
, the framework of the biosynthetic pathways of glycosylation in a. fumigatus has been delineated .
functional analyses of some of the genes in this pathway have shown that glycosylation is required for cell wall synthesis , polarity , morphogenesis , and cellular function in a. fumigatus ( figure 7 and table 2 ) .
however , a detailed understanding of this pathway remains unknown , such as details regarding the synthesis of the n - glycan precursor , the precise molecular mechanism of n - glycan processing , qc of protein folding , and modification of the gpi anchor .
moreover , the molecular mechanisms by which glycosylation plays a role in morphogenesis and development of a. fumigatus are vaguely understood .
therefore , the future direction would be looking for those key proteins that are affected by glycosylation and identifying the signal transduction pathways that link glycosylation and development , through genetic , biochemical , cell biological , and proteomic studies .
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glycosylation is a conserved posttranslational modification that is found in all eukaryotes , which helps generate proteins with multiple functions .
our knowledge of glycosylation mainly comes from the investigation of the yeast saccharomyces cerevisiae and mammalian cells .
however , during the last decade , glycosylation in the human pathogenic mold aspergillus fumigatus has drawn significant attention .
it has been revealed that glycosylation in a. fumigatus is crucial for its growth , cell wall synthesis , and development and that the process is more complicated than that found in the budding yeast s. cerevisiae .
the present paper implies that the investigation of glycosylation in a. fumigatus is not only vital for elucidating the mechanism of fungal cell wall synthesis , which will benefit the design of new antifungal therapies , but also helps to understand the role of protein glycosylation in the development of multicellular eukaryotes .
this paper describes the advances in functional analysis of protein glycosylation in a. fumigatus .
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1. Introduction
2. Cell Wall Organization and Its Compensatory Mechanism in
3. Importance of Glycosylation in
4. Biosynthesis and Function of N-Glycosylation in
5. Biosynthesis and Function of O-Glycosylation
6. Biosynthesis and Function of GPI Anchoring in
7. Outlook
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coronary artery disease ( cad ) remains the principal cause of mortality and morbidity around the world [ 1 , 2 ] .
although the mechanisms are still not entirely resolved , previous studies have demonstrated that an imbalance between t - helper 1- ( th1- ) mediated and th2-mediated immune functions contributes to the development of atherosclerosis .
recently , th17 , which are a newly defined subset different from th1 and th2 , reactive to autoantigens are involved in the pathogenesis of several autoimmune diseases [ 4 , 5 ] .
in addition to th1 , th2 , and th17 lineage , regulatory t cells ( tregs ) , a special subset of cd4 t cells , inhibit atherosclerosis development by attenuating activated t cell responses [ 6 , 7 ] .
inspiringly , previous clinical and experimental studies from our laboratory have found that th17/tregs functional imbalance exists during atherogenesis , indicating that the imbalance of th17/tregs plays a critical role in cad [ 8 , 9 ] .
hence , these findings have showed that cd4 t cell subpopulations play an important role in the initiation and progression of atherosclerosis .
it is well known that cd4 t cells are divided into different subsets depending on the cytokines they produce .
interferon- ( ifn- ) , the principal cytokine of th1 cells , is proinflammatory and exacerbates atherosclerotic disease , while the th2 cytokine such as interleukin- ( il- ) 4 is considered to be mainly atheroprotective and can neutralize th1 cytokine activity [ 1012 ] .
moreover , hashmi and zeng have found that il-17 and il-17 induced cytokines ( il-6 and il-8 ) were significantly increased in acs patients .
interestingly , tregs exert antiatherosclerosis partly by secretion of anti - inflammatory cytokine transforming growth factor ( tgf-1 ) , which can in turn induce the expression of forkhead box transcription factor p3 ( foxp3 ) and promote differentiation of tregs derived from cd4cd25 t cells [ 15 , 16 ] . therefore , these findings suggest that cytokines as those mentioned above are also essential for the formation and progression of the atherosclerotic plaque
. the galectin family is characterized by conserved carbohydrate recognition domains ( crd ) that can bind glycosylated proteins .
galectins are involved in a wide range of biologic processes such as cell adhesion and migration , proliferation and apoptosis , tumor biology , and regulation of the immune system .
galectin- ( gal- ) 9 is a member of the galectin family of carbohydrate - binding proteins comprising two carbohydrate recognition domains connected by a linker peptide .
gal-9 is mainly expressed by eosinophils , endothelial cells , macrophages , dcs , kupffer cells , vascular endothelial cells , intestinal epithelial cells , and in particular t lymphocytes .
furthermore , gal-9 is believed to function predominantly as a multifaceted player in adaptive and innate immunity , especially in t cell development and homeostasis [ 2023 ] .
accumulating evidence shows that gal-9 induces apoptosis in subsets of differentiated t cells , particularly in th1 and th17 cells , and stimulates the activity of tregs [ 19 , 2428 ] .
indeed , treatment with recombinant gal-9 moderated the progression of experimental autoimmune encephalomyelitis ( eae ) , arthritis , and diabetes in animal models [ 19 , 2931 ] , by reducing the number of th1 and th17 cells and downregulating circulating ifn- levels .
t cell immunoglobulin mucin- ( tim- ) 3 has been identified as a receptor for gal-9 .
although gal-9 can function in a tim-3-independent fashion , the immune - regulatory effects of gal-9 are largely attributed to gal-9-tim-3 pathway [ 3235 ] .
more recently , foks et al . demonstrated that anti - tim-3-ab administration promoted atherosclerotic plaque formation , which indicated that tim-3-gal-9 pathway may be concerned with the development of atherosclerosis .
thus , it is reasonable to postulate that gal-9 may be involved in atherosclerosis based cad .
in addition , several findings strongly support the original experimental observations that gal-3 plays an important role in the underlying heart failure ( hf ) processes and that elevation of gal-3 is associated with disease progression and poor outcome in hf [ 3739 ] .
herein , we investigate serum gal-9 levels in chinese patients with cad , and the severity of coronary arteries stenosis was evaluated by gensini score .
furthermore , ifn- , il-4 , il-17 , tgf-1 , high - sensitivity c - reactive protein ( hs - crp ) , and the classical atherosclerosis risk factors were evaluated .
additionally , th1 , th2 , th17 , and cd4cd25foxp3 tregs differentiation were detected in pbmc cultures exposed to gal-9 .
232 patients presenting at the department of cardiology of huazhong university of science and technology affiliated union hospital between jan .
2013 with suspected or established cad were recruited , including 149 males and 83 females .
they were divided into four groups : ( 1 ) the control group , which consisted of 50 subjects with nca ( 28 men and 22 women , mean age 56.7 11.7 ) ; ( 2 ) sap group ( 25 men and 15 women , mean age 62.4 10.0 , inclusion criteria : typical chest discomfort that was associated with horizontal or downsloping st - segment depression > 1 mm in an exercise test ) ; ( 3 ) nsteacs group ( 60 men and 30 women , mean age 60.6 9.9 , which included unstable angina pectoris ( uap ) and non - st - segment elevation myocardial infarction ( nstemi ) , inclusion criteria : chest pain at rest with definite ischemic electrocardiographic changes , t - wave inversions and/or st - segment changes , or typical ischemic chest pain which persists for more than 20 min , elevated serum myocardial necrosis markers concentration and dynamic evolution , but not the typical st - elevation electrocardiogram ) ; ( 4 ) stemi group ( 36 men and 16 women , mean age 54.1 12.3 , inclusion criteria : myocardial infarction that was confirmed by a significant increase of serum creatine kinase mb , troponin i levels , and elevation of st - segment )
. furthermore , all participants underwent coronary angiography after admission and completed a standardized questionnaire about previous and present illness , medication history , and smoking status .
cardiovascular - interrelated factors , such as age , body mass index ( bmi ) , gender , and ejection fraction ( ef ) were estimated via physical examination , ultrasonic cardiography ( ucg ) , and electrocardiogram ( ecg ) , respectively .
patients with the following characteristics were excluded from study enrollment : valvular heart disease , thromboembolism , autoimmune diseases ( systemic lupus erythematosus , rheumatoid arthritis , etc . ) , collagen disease , disseminated intravascular coagulation , severe liver and kidney disease , symptomatic heart failure , trauma or surgery , or malignant disease .
the study was approved by the ethics committee of tongji medical college of huazhong university of science and technology , and all participants were provided written informed consent prior to study entry .
two experienced cardiologists , who were not aware of the patients ' clinical and biochemical results , visually examined angiographic images to assess the extent and severity of cad .
cad diagnosis was made according to the presence of 50% stenosis in 1 main coronary artery .
the stenosis severity of cad was assessed by gensini score as previously described . in the ami group ,
the blood was drawn with a 21-gauge needle for clean venipuncture of an antecubital vein and the samples were collected into sodium heparin vacutainers ( becton - dickinson ) .
after centrifugation , the serum obtained was stored at 80c until further use to measure the concentration of gal-9 .
the level of gal-9 was measured by elisa according to the manufacturer 's instructions ( r&d systems ) .
in addition , the levels of ifn- , il-17 ( bolingkewei biotechnology , china ) , il-4 , hs - crp ( huijia biotechnology , china ) , and tgf-1 ( fumeng gene biotechnology , china ) were measured by elisa , following the manufacturer 's instructions .
the minimal detectable concentrations were 28 pg / ml for gal-9 , 1.8 pg / ml for ifn- , 1.8 pg / ml for il-17 , 0.4 pg / ml for il-4 , 22 pg / ml for hs - crp , and 8 pg / ml for tgf-1 .
the intra - assay and interassay coefficients of variation for all elisa were < 5% and < 10% , respectively .
all the other biochemical measurements , including serum total cholesterol ( tc ) , triglyceride ( tg ) , low - density lipoprotein cholesterol ( ldl - c ) , high - density lipoprotein cholesterol ( hdl - c ) , apolipoprotein a - i ( apoa - i ) , apolipoprotein b ( apob ) , lipoprotein(a ) , creatinine , fasting plasma glucose ( fpg ) , uric acid , and cardiac troponin i ( ctni ) , were carried out by the biochemical laboratory of our cardiovascular institute using standard methods . peripheral blood mononuclear cells ( pbmcs ) from nca ( excluded from hypertension , diabetes , and dyslipidemia ) were isolated by ficoll - hypaque density gradient centrifugation ( pharmacia lkb technology , uppsala , sweden ) .
cells from the interphase were collected and washed twice with dulbecco 's phosphate buffered saline ( d - pbs ) .
remaining erythrocytes were removed using lysis buffer ( 4.14 g nh4cl , 0.5 g khco3 , and 18.6 mg na2edta in 500 ml water , ph = 7.4 and sterilized ) for 5 min on ice .
pbmcs were resuspended in rpmi1640 ( lonza , verviers , belgium ) supplemented with 2.5% fcs , penicillin ( 100 u / ml)/streptomycin ( 100 u / ml ) , and sodium pyruvate ( 1 mm , sigma ) .
pbmcs were stimulated with anti - human cd3 and anti - human cd28 antibodies ( both 2 g / ml , sanquin ) in the presence of recombinant gal-9 ( m ) ( 0.010.1 m , prospec - tany technogene ltd .
, israel ) for 24 h. cells were allocated into tubes and washed once in phosphate buffered saline ( pbs ) . for tregs analysis
, the cells were incubated with anti - cd4-fitc - hab and anti - cd25-apc - hab ( bd pharmingen ) . after the surface staining
, the cells were stained with anti - foxp3-pe - hab ( bd pharmingen ) after fixation and permeabilization according to the manufacturer 's instructions . for analysis of th1 , th2 , and th17 cells ,
the cells were stimulated with phorbol myristate acetate ( pma , 20 ng / ml , alexis biochemicals , san diego , ca ) and ionomycin ( 1 g / ml , alexis biochemicals ) for 4 h in the presence of 2 mol / ml monensin ( alexis biochemicals ) . the incubator was set at 37c under a 5% co2 environment .
after culture for 4 hours , the cells were collected for staining according to the instructions .
fixation and permeabilization were necessary before staining with anti - ifn--pe- , anti - il-4-pe- , or anti - il-17-pe- hab ( bd pharmingen ) .
continuous variables are summarized as mean standard deviation ( sd ) , and categorical data are presented as percentages . the shapiro - wilk test was used to assess the normality of distribution of continuous variables . in order to compare two groups ,
continuous variables were tested using the independent samples t - test for normally distributed data and the mann - whitney u test for nonnormally distributed data ; the chi - square test was used for categorical variables . when three or more groups were compared , one - way anova was used .
if significance was found , newman - keuls test was performed for post hoc analysis to detect the difference among groups .
multiple stepwise regression analysis was used to evaluate the influence of different variables on gal-9 and to adjust for covariates .
independent factors were sex , age , ctni , and the metabolic - related variables including bmi , fpg , lipid profiles , and hs - crp . to determine the independent predictors for the presence and severity of cad , all the conventional risk factors associated with cad were tested in multiple stepwise regression analysis .
the prevalence of smoking and the levels of tg , lipoprotein(a ) , fpg , creatinine , hs - crp , and ctni were significantly higher in patients with cad compared to patients with nca group ( all p < 0.05 ) .
however , other biochemical results , including tc , hdl - c , ldl - c , and uric acid , were similar between nca and cad patients .
compared with stemi group , patients in sap and nsteacs groups showed markedly higher hdl - c levels and age and lower levels of lipoprotein(a ) , fpg , hs - crp , and ctni ( all p < 0.01 ) . compared to patients with sap ,
the use of aspirin , -blockers , and statins was rare in patients with acs ( all p < 0.05 ) , whereas the levels of lipoprotein(a ) and hs - crp were markedly higher in patients with acs ( all p < 0.01 ) .
a significant increase of creatinine levels was observed in patients with stemi compared with nsteacs group ( p < 0.05 ) and an obvious decrease of uric acid levels was found in patients with stemi compared to sap group ( p < 0.01 ) .
unexpectedly , the distribution of hypertension , diabetes mellitus , dyslipidemia , and family history was similar among patients with acs and sap . among the total 232 study participants , serum gal-9 levels ranged from 1733.86 to 5259.39 pg / ml .
compared with the nca group , patients with cad had significantly lower levels of gal-9 ( 3283.55 587.59 versus 3565.97 544.37 pg / ml , p < 0.05 ; figure 1(a ) ) .
in addition , we found that serum gal-9 levels were significantly lower in the stemi ( 3126.36 637.7
pg / ml ) than those in the sap group ( 3607.91 541.35 pg / ml ) or the nca group ( stemi versus sap and nsteacs versus sap , all p < 0.01 ; stemi versus nca and nsteacs versus nca , all p < 0.01 ; figure 1(b ) ) .
interestingly , serum gal-9 levels did not differ significantly between patients with nsteacs and stemi ( p > 0.05 ) , nor was there a difference between the sap and nca groups ( p > 0.05 ; figure 1(b ) ) .
as shown in figure 2 , serum il-17 , il-4 , ifn- , and tgf-1 were detected in each group .
the il-17 and ifn- concentrations in patients with stemi ( il-17 , 57.16 16.14 pg / ml ; ifn- , 13.43 5.29
pg / ml ) and nsteacs ( il-17 , 47.68 14.46 pg / ml ; ifn- , 11.28 4.23
pg / ml ) were significantly higher compared with those in patients with sap ( il-17 , 26.94 9.09 pg / ml ; ifn- , 6.05 2.41 pg / ml ) and nca groups ( il-17 , 24.00 10.14 pg / ml ; ifn- , 5.83 2.12 pg / ml ) ( all p < 0.01 ; figures 2(a ) and 2(c ) ) .
il-4 concentrations showed no difference in any of the groups ( stemi , 7.25 3.82 pg / ml ; nsteacs , 7.22 3.87 pg / ml ; sap , 8.12 4.48 pg / ml ; nca , 6.83 3.25
tgf-1 concentrations in patients with stemi ( 4.08 2.13 ng / ml ) and nsteacs ( 4.73 2.71 ng / ml ) were significantly lower than those in patients with sap ( 8.03 3.88 ng / ml ) and nca ( 9.02 4.86 ng / ml ) ( all p < 0.01 ; figure 2(d ) ) . importantly , gal-9 levels were negatively correlated with il-17 ( r = 0.45 , p < 0.001 ; figure 3(a ) ) and ifn- ( r = 0.53 , p < 0.001 ; figure 3(c ) ) but positively associated with tgf-1 ( r = 0.58 , p < 0.001 ; figure 3(d ) ) . however , gal-9 levels showed no correlation with il-4 concentrations ( r = 0.04 , p = 0.528 ; figure 3(b ) ) . to detect the involvement of gal-9 in the induction of differentiation of human cd4 t cells
gal-9 expanded the number of cd4cd25foxp3 tregs ( control , 6.99 1.61% ; gal-9 ( 0.01 m ) , 9.24 2.04% ; gal-9 ( 0.03 m ) , 10.27 3.04% ; gal-9 ( 0.1 m ) , 11.24 3.23% ) and decreased the development of th17 ( control , 0.628 0.239% ; gal-9 ( 0.01 m ) , 0.418 0.171% ; gal-9 ( 0.03 m ) , 0.214 0.064% ; gal-9 ( 0.1 m ) , 0.059 0.015% ) dose dependently ( figures
4(c ) and 4(d ) ) , with resulting increase in secretion of tgf-1 ( control , 165.3 48.6 pg / ml ; gal-9 ( 0.01 m ) , 227.8 72.4 pg / ml ; gal-9 ( 0.03 m ) , 312.9 81.7 pg / ml ; gal-9 ( 0.1 m ) , 528.1 97.4
pg / ml ) and suppressed il-17 production ( control , 132.4 23.6 pg / ml ; gal-9 ( 0.01 m ) , 110.3 25.7
pg / ml ; gal-9 ( 0.03 m ) , 81.9 18.0 pg / ml ; gal-9 ( 0.1 m ) , 56.6 13.8
however , the development of th1 and th2 cells and secretion of ifn- and il-4 in the supernatant were similar in the gal-9 untreated and treated pbmcs ( figures 4(a ) , 4(b ) , and 4(e ) ) .
as we expected , gal-9 levels were found to be negatively correlated with hs - crp ( r = 0.37 , p < 0.001 ; figure 5(a ) ) , the gensini score ( r = 0.23 , p = 0.001 ; figure 5(b ) ) , fpg ( r = 0.15 , p = 0.027 ; figure 5(c ) ) , and lipoprotein(a ) ( r = 0.16 , p = 0.012 ; figure 5(d ) ) but positively associated with creatinine ( r = 0.21 , p = 0.002 ; figure 5(e ) ) and age ( r = 0.14 , p = 0.028 ; figure 5(f ) ) . to determine
which variables were independently associated with gal-9 , multiple stepwise regression analysis using gal-9 as the dependent variable identified sex , age , bmi , fpg , tc , tg , hdl - c , ldl - c , hs - crp , lipoprotein(a ) , creatinine , uric acid , ctni , and therapeutic use of statins as independent variables . after adjustments , only hs - crp ( b = 72.158 , p < 0.001 ) , lipoprotein(a ) ( b = 8.333 , p = 0.045 ) , creatinine ( b = 5.228 , p < 0.001 ) , and use of therapeutic statins ( b = 362.29 , p < 0.001 ) retained significance for independently predicting gal-9 ( table 3 ) . in order to determine which variables
gensini score was the dependent variable , and sex , age , bmi , fpg , lipid profiles , gal-9 , hs - crp , uric acid , traditional risk factors , and statins were independent variables .
after adjustments , only gal-9 ( b = 0.145 , p = 0.011 ) , age ( b = 0.276 , p < 0.001 ) , and lipoprotein(a ) ( b = 0.280 , p <
in the present study , we found that participants with cad had lower serum gal-9 levels compared with nca . furthermore , serum gal-9 levels in the acs group were significantly lower than those in the sap or nca group .
moreover , gal-9 was found to be independently associated with hs - crp , lipoprotein(a ) , and creatinine .
notably , gal-9 suppressed t - helper 17 ( th17 ) and expanded regulatory t cells ( tregs ) as analyzed within the activated cd4 t cell population , resulting in decreased il-17 production and increased secretion of tgf-1 .
to the best of our knowledge , this is the first study to investigate the relation of serum gal-9 levels with the presence and the severity of coronary arteries stenosis .
mor et al . have reported that cd4cd25 tregs deficiency enhanced atherosclerotic lesion development in ldlr mice , and adoptive transfer of cd4cd25 tregs attenuated the initiation and progression of atherosclerosis in apoe mice [ 42 , 43 ] . moreover
, our laboratory has detected that th17/treg functional imbalance exists in patients with acs , suggesting a potential role for th17/treg imbalance in plaque destabilization and the onset of acs [ 8 , 9 ] .
of note , gal-9 is a multifunctional modulator of t cell immunity with apoptotic effects on th17 cells and stimulatory activity on tregs in autoimmune diseases . in line with this publication
, we found that gal-9 was able to induce cd4cd25foxp3 tregs development and inhibit th17 differentiation in activated pbmcs of nca .
therefore , we speculated that gal-9 plays an immune - regulatory role in atherosclerosis via its effects on tregs and th17 cells .
acs patients have significantly higher il-17 and il-17 induced cytokines ( il-6 and il-8 ) , while the pleiotropic cytokine ifn- is a proinflammatory regulator that is expressed at high levels in atherosclerotic lesions .
in addition , it has been demonstrated that increased expression of tgf-1 is a stabilizing factor in human atherosclerotic plaques . in this study
, we also found that il-17 and ifn- concentrations in patients with stemi and nsteacs were significantly higher compared with those in patients with sap and nca , while tgf-1 concentrations in patients with stemi and nsteacs were significantly lower than those in patients with sap and nca .
these results again confirmed that inflammatory cytokines mentioned above have differential effects in atherosclerosis based cad .
interestingly , gal-9 levels were showed to be negatively correlated with il-17 and ifn- but positively associated with tgf-1 .
furthermore , we found that gal-9 was able to increase secretion of tgf-1 and decrease il-17 secretion in activated pbmcs .
consequently , our data in vitro imply a protective role for gal-9 in atherosclerosis via expanding tgf-1 and suppressing il-17 secretion .
however , the precise effect and mechanism of gal-9 in atherosclerosis are still to be elucidated in atherosclerosis animal models .
surprisingly , we found that the development of th1 cells and secretion of ifn- in the supernatant were similar in gal-9 untreated and treated pbmcs , since zhu et al . reported that gal-9 induces apoptosis in th1 cells .
this discrepancy may be ascribed to the diverse concentrations of gal-9 and different incubation periods . additionally , il-4 concentrations in the supernatant showed no difference in gal-9 untreated and treated pbmcs .
this result further confirms that the effect of il-4 is still controversial [ 45 , 46 ] , reflecting its dual pro- and anti - inflammatory character .
moreover , there are numerous publications showing that gal-9 has proinflammatory effects in addition to its immune - suppressive effects , especially through its actions on cells of the innate immune system like dc and nk cells [ 47 , 48 ] .
thus , these results indicate that gal-9 has bidirectional effects like many immune - regulatory molecules .
our data showed that cad patients had lower serum gal-9 levels than those without cad , and gal-9 was independently associated with the gensini score in patients with cad .
these results suggest that low serum gal-9 levels are associated with the presence and the severity of coronary arteries stenosis .
previous studies have elucidated that serum biomarkers are recognized as important tools for prediction , diagnosis , risk stratification , and therapeutic decision - marking for patients with cad .
accumulating studies demonstrated that atherosclerotic lesion instability and rupture induced by inflammation are the major mechanisms of acs [ 50 , 51 ] .
recently , a large number of clinical experiments have reported that hs - crp is not only a biomarker of inflammations and atherosclerosis , but also a marker of atheromatous plaque vulnerability [ 5254 ] . in our study , patients with acs had higher serum hs - crp levels compared to patients with sap . of note , gal-9 was found to be independently associated with hs - crp .
hence , low serum concentrations of gal-9 may take part in the onset of acs and may act as a predictor of atheromatous plaque vulnerability .
however , whether a causal relationship exists between the two processes is still to be studied .
several lines of evidence indicate that lipoprotein(a ) is an independent risk factor for patients with cad [ 55 , 56 ] .
all these findings together suggest that gal-9 might be a potential independent biomarker of cad .
interestingly , our data showed that serum gal-9 levels were higher in patients of cad complicated with t2 dm than those of cad without t2 dm ( data not shown ) .
's study reporting that serum gal-9 levels are elevated in the patients with type 2 diabetes ( t2 dm ) and chronic kidney disease ( ckd ) .
however , serum gal-9 levels were decreased in cad and elevated in the patients with t2 dm .
this paradox could be attributed to the fact that atherosclerosis is highly multifactorial , tregs and gal-9 being only some factors among many others .
the exact mechanism is still to be elucidated but different diseases may have divergent immune states .
worth noting is the fact that the serum gal-9 level seems relatively higher in our study than in the previous studies [ 22 , 57 ] .
the differences among these studies might be explained , at least partly , by different experimental designs , different number and characteristics of participants recruited , ethnical differences , medical treatments , lack of standardization of methods for the determination of serum gal-9 levels , and different ways in which samples were handled . nevertheless , several limitations of the present study should be considered .
firstly , the number of study participants ( 232 ) was relatively small . secondly , given the cross - sectional design of the study , the causal relationship between gal-9 level and the severity of coronary artery stenosis can not be determined .
thirdly , different methods of assessment of the coronary stenosis and the criterion for diagnosis of the diseased artery may lead to another result . finally , although we accounted for confounding of traditional cardiovascular risk factors , a potential for uncontrolled or residual confounding that could influence the relationship between gal-9 and cad would be plausible . in conclusion
, we have shown that low serum gal-9 levels are associated with the presence and the severity of coronary arteries stenosis .
importantly , gal-9 can expand cd4cd25foxp3 tregs and inhibit th17 development in activated pbmcs , leading to increased secretion of tgf-1 and decreased il-17 secretion .
our observations provide evidence of the role of gal-9 in cad and that gal-9 is an independent negative predictor of acs , which may also at least partially explain the protection of gal-9 against coronary atherosclerotic plaque vulnerability .
large - scale population - based clinical studies and further experimental studies are needed to elucidate the exact effect and potential biological mechanisms of gal-9 in the pathogenesis of cad and to evaluate whether gal-9 is a potential novel biomarker of cad .
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background .
recently , several studies suggest that galectin-9 ( gal-9 ) might play a pivotal role in the pathogenesis of autoimmune diseases .
however , the exact role of gal-9 in atherosclerosis remains to be elucidated .
methods .
serum gal-9 , high - sensitivity c - reactive protein ( hs - crp ) , interferon- ( ifn- ) , interleukin- ( il- ) 4 , il-17 , and transforming growth factor- ( tgf- ) 1 were measured .
the effect of gal-9 on peripheral blood mononuclear cells ( pbmc ) was investigated in patients with normal coronary artery ( nca ) .
results . the lowest level of gal-9 was found in the st - segment elevation myocardial infarction ( stemi ) group , followed by the non - st - segment elevation acs ( nsteacs ) , the nca , and the stable angina pectoris ( sap ) groups , respectively .
additionally , gal-9 was found to be independently associated with hs - crp , lipoprotein(a ) , and creatinine .
notably , gal-9 was also noted to be an independent predictor of the gensini score .
moreover , gal-9 suppressed t - helper 17 ( th17 ) and expanded regulatory t cells ( tregs ) , resulting in decreased il-17 production and increased secretion of tgf-1 .
conclusions .
serum gal-9 is associated with not only coronary artery disease ( cad ) , but also the severity of coronary arteries stenosis .
gal-9 can expand tregs and suppress th17 development in activated pbmc , implying that gal-9 has the potential to dampen the development of atherosclerosis and may be a new therapy for cad .
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1. Introduction
2. Materials and Methods
3. Results
4. Discussion
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clozapine remains an ultimate option for patients with treatment resistant schizophrenia ( kane 2012 ) .
efficacy of clozapine against positive symptoms of schizophrenia was confirmed in numerous studies and meta - analyses ( chakos et al .
2001 ) , including analysis published by the cochrane library ( asenjo lobos et al .
however , treatment with clozapine is associated with very severe metabolic side - effects ( newcomer 2005 ) .
clozapine - induced weight gain is very common ( wetterling 2001 ) , and so is impaired fasting plasma glucose levels . in patients with schizophrenia weight gain
is associated with impaired physical functioning and negative body appraisal ( bachmann et al . 2012 ) , both of which affect quality of life . while treatment with clozapine may reduce mortality by reducing suicide rate , mortality due to clozapine - associated weight gain will diminish reduction in the suicide rate almost entirely over 10 years by the increased mortality associated with weight gain of 10 kg ( fontaine et al .
also , obesity is linked with numerous secondary health problems ( pressure overload on lungs , joints and bones ) and is an important risk factor for life - threatening diseases , such as cardiovascular disease , type 2 diabetes and certain cancers .
several mechanisms are considered as taking role in clozapine - induced weight gain , including antagonism at histamine h1 receptors ( kroeze et al .
various receptors , including aforementioned 5-ht1b and 5-ht1c , regulate activity of the hypothalamic nuclei ( particularly the arcuate nucleus ( arc ) , which plays the key role in appetite regulation ) .
activity of arc is also regulated by several hormones of anorexigenic properties : leptin , pancreatic polypeptide ( pp ) , cholecystokinin ( cck ) , glucagon - like peptide-1 ( glp-1 ) , oxyntomodulin ( oxm ) , peptide yy ( pyy ) and the first discovered orexigenic substance - ghrelin ( druce et al . 2004 ) .
agouti - related peptide ( agrp ) is one of the hypothalamic hormones that works by increasing appetite and decreasing metabolism and energy expenditure , thus leading to weight gain .
it was identified in 1997 based on its sequence similarity with agouti signaling peptide ( asip ) , a protein synthesized in the skin that controls coat color .
compared with asip , agrp is physiologically expressed in the hypothalamus ( shutter et al .
agrp is also expressed in the adrenal gland , testes , kidneys , and lungs .
agrp induces obesity by antagonism of the melanocortin receptors ( mc3-r and mc4-r , subtypes implicated in weight regulation , via metabolism and appetite control ) ( ollmann et al .
these neurons make peptides that potently stimulate food intake not only by increasing neuropeptide y ( npy ) signaling , but also by reducing melanocortin signaling via the release of agrp ( morton and schwartz 2001 ) .
activity of the agrp / npy neurons is modulated by leptin , released from the adipose tissue , and insulin .
the adiposity signals ( insulin and leptin ) are secreted in proportion to body fat content and act in the hypothalamus to inhibit anabolic and stimulate catabolic , effector pathways ( schwartz et al .
the increase of food intake following a single central administration of agrp is sustained for up to a week ( hagan et al . 2000 ) , while the response to npy is sustained over hours , rather than days .
this study was undertaken with the purpose to determine if patients with schizophrenia on clozapine monotherapy have higher fasting levels of agrp compared with healthy control . in order to provide more accurate measurements ,
biochemical and anthropometric measurements were combined with body composition determined using bioelectric impedance analysis ( bia ) , which provides accurate measurements of body fat , lean mass and body water ( bosy - westphal et al .
this is the first study to investigate such combination of these parameters in subjects with schizophrenia treated with clozapine .
data for 24 european caucasian adult hospitalized patients with paranoid schizophrenia ( 295.30 according to dsm - iv , f20.0 according to icd-10 ) was included into the study .
these subjects were on clozapine monotherapy for at least two months prior the assessments with a minimum dose of 100 mg / day .
most patients was in stable phase of the disease ( i.e. no acute psychosis ) .
control group was 24 healthy subjects and was gender- and age - matched with patients in the clozapine group .
health status of the control subjects was determined on the basis of basic physical examination , including vital signs and an interview .
all patients and volunteers included in the study expressed their written informed consent for participation in this study .
the blood samples for the chemistry panel were collected between 7 am and 8 am , after ensuring at least 8 h of overnight fasting .
glucose , lipids , calcium and uric acid levels were measured using a dirui cs-400 analyzer ( dirui , china ) .
homocysteine chemiluminescence assessments were performed using an immulite 2,000 analyzer ( siemens , germany ) , insulin immunochemistry assessments were performed using a cobas e411 analyzer ( roche diagnostics , switzerland ) and albumin levels were assessed using a cobas integra 800 analyzer ( roche diagnostics , switzerland ) .
levels of agrp were measured in blood serum using elisa ( enzyme - linked immunosorbent assay ) method . prior to assays ,
serum samples were stored at 80 c for up to 6 months .
elisa assays were performed using commercial kits ( intra - assay : cv < 10 % , inter - assay : cv < 12 % ) manufactured by raybiotech ( usa ) , according to protocol provided by its manufacturer ( all samples were 2-fold diluted ) . metabolic syndrome and abdominal obesity were defined according to international diabetes foundation ( idf ) criteria ( alberti et al .
kg / m , 2530 kg / m and 30 kg / m were defined as normal weight , overweight and obesity , respectively . raised triglycerides ( tga ) level 150 mg / dl and/or total cholesterol ( tc ) 200 mg / dl and/or reduced hdl cholesterol level < 40 mg / dl for men and < 50 mg / dl for women and/or raised ldl cholesterol level 135 mg / dl were interpreted as dyslipidemia .
corrected calcium was calculated using the formula : corrected calcium [ mg / dl ] = measured total calcium [ mg / dl ] + 0.8 ( 4.0 serum albumin [ g / dl ] ) .
insulin resistance was estimated from fasting glucose and insulin results by homeostasis model assessment and quicki index , using the formula : homa1-ir = ( fasting plasma glucose [ mg / dl ] insulin [ mu / l])/405 . homa2-ir index was calculated using a calculator downloaded from http://www.dtu.ox.ac.uk .
quicki index ( lower numbers reflect greater insulin resistance ) was calculated using the formula : 1/(log ( fasting insulin [ mu / l ] ) + log ( fasting plasma glucose [ mg / dl ] ) ) .
height was measured with a wall - mounted height measure to the nearest 0.5 cm .
weight was measured with a spring balance that was kept on a firm horizontal surface .
subjects wore light clothing , stood upright without shoes and weight was recorded to the nearest 0.5 kg .
body mass index ( bmi ) was calculated as body weight in kilogram divided by the height in meter squared ( kg / m ) .
waist , abdominal and hip circumference was measured using a non - stretchable fiber measuring tape .
waist - to - hip ratio ( whr ) was calculated as waist circumference divided by hip circumference .
whr cut - off points were defined according to who recommendations ( 0.85 for women and 0.9 for men ) .
fat mass index ( fmi ) was calculated as total body fat in kilogram divided by the height in meter squared ( kg / m ) .
excess body fat according to fmi classification ranges were defined as fmi > 6 for men and fmi > 9 for women
body composition was measured using a maltron bf-906 body fat analyzer ( maltron , uk ) , single frequency bioelectrical impedance analyzer to determine resistance and reactance at 50 hz .
standard operating conditions were observed by a trained operator including preparation of the participant , electrode placement and operation .
the measurement using bia was taken immediately prior to anthropometry measurements with participants lying supine , in a rested state .
simple descriptive statistics ( means , standard deviations , 95 % confidence interval [ ci ] ) were generated for all continuous variables . for discrete variables number of patients and percentages are given .
skewed variables were transformed to follow normal distribution using log , square root , inverse square root or square transformation .
means , standard deviations , and confidence intervals are reported for non - transformed variables , results of tests are reported for transformed or non - transformed variables . if transformation resulted in normal distribution , two - tailed t - test
was used to assess inter - group differences , otherwise variables were analyzed using mann
associations were tested by pearson s ( for variables with normal distribution ) or spearman 's ( for other variables ) correlation coefficients . the significant level was set at p < 0.05 .
for group of patients treated with clozapine the mean age was 38.812.6 and 39.912.3 for the control group ( p = 0.62 ) . in both groups
there were 12 men and 12 women . in the clozapine group 12 subjects smoked cigarettes and 8 in the control group ( p = 0.38 ) .
the mean duration of monotherapy with clozapine was 60.579.4 [ 95 % ci : 27.094.1 ] months and mean clozapine dose was 341.1148.6 [ 95 % ci : 278.4403.8 ] mg / day .
detailed results for anthropometric measurements and laboratory tests are shown in table 1 .
table 1results of anthropometric measurements and laboratory testsclozapine
n = 24control
n = 24weight [ kg]78.114.2(72.184.1)72.615.1(66.278.9 )
p = 0.08
t = 1.38bmi [ kg / m]27.13.6(25.528.6)24.83.5(23.326.2 )
p = 0.01
t = 2.25fmi [ kg / m]8.93.1(7.610.2)7.73.0(6.49.0 )
p = 0.09
t = 1.38abdominal circumference [ cm]96.59.4(92.5100.4)85.511.6(80.690.3 )
p <
0.001
z = 3.58waist circumference [ cm]91.112.1(86.096.2)82.410.6(77.886.8 )
p = 0.005
z = 2.66hip circumference [ cm]99.08.4(95.5102.5)95.97.1(92.998.9 )
p = 0.08
t = 1.39whr0.920.08(0.880.95)0.860.08(0.820.89 )
p = 0.005
z = 2.66sbp [ mm hg]121.713.6(115.9127.4)136.717.9(129.1144.2 )
p = 0.001
z = 3.26dbp [ mm hg]81.28.5(77.684.8)82.812.1(77.687.9 )
p = 0.30
t = 0.50uric acid [ mg / dl]4.51.4(3.95.1)4.31.3(3.74.8 )
t = 0.57homocysteine [ mol / l]14.5 4.4(12.616.4)13.6 5.0(11.515.7 )
p = 0.25
t = 0.67tc [ mg / dl]194.2 53.2(171.7216.6)216.6 65.3(189.0244.1 )
p = 0.06
t = 1.54hdl [ mg / dl]43.5 12.6(38.248.8)55.1 14.3(49.161.1 )
p
41.9(104.8140.3)128.3 39.7(111.5145.0 )
p = 0.29
t = 0.54tga [ mg / dl]140.3 120.4(89.4191.1)104.3 81.4(69.9138.6 )
p = 0.07
t = 1.50fpg [ mg / dl]103.5 31.7(90.1116.8)87.8 11.7(82.892.7 )
p = 0.03
t = 1.88insulin [ u / ml]11.8 8.2(8.3 - 15.3)7.7 3.2(6.3 - 9.1 )
p = 0.02
t = 2.08homa1-ir3.3 3.4(1.84.7)1.7 0.8(1.32.0 )
p = 0.009
t = 2.46homa2-ir1.6 1.1(1.12.1)1.0 0.4(0.81.2 )
p = 0.01
t = 2.26quicki0.15 0.01(0.140.15)0.16
0.01(0.150.16 )
p = 0.007
t = 2.52corrected calcium [ mg / dl]8.6 0.9(8.29.0)8.7
p = 0.37
t = 0.33data given as : mean standard deviation ( 95 % ci)bmi = body mass index ; fmi = fat mass index ; whr = waist - to - hip ratio ; sbp = systolic blood pressure ; dbp = diastolic blood pressure ; tc = total cholesterol ; hdl = high density lipoproteins ; ldl = low density lipoproteins ; tga = triglycerides ; fpg = fasting plasma glucose ; homa1-ir = homoeostasis model assessment of insulin resistance 1 ; homa2-ir = homoeostasis model assessment of insulin resistance 2 ; quicki = quantitative insulin sensitivity check index results of anthropometric measurements and laboratory tests data given as : mean standard deviation ( 95 % ci ) bmi = body mass index ; fmi = fat mass index ; whr = waist - to - hip ratio ; sbp = systolic blood pressure ; dbp = diastolic blood pressure ; tc = total cholesterol ; hdl = high density lipoproteins ; ldl = low density lipoproteins ; tga = triglycerides ; fpg = fasting plasma glucose ; homa1-ir = homoeostasis model assessment of insulin resistance 1 ; homa2-ir = homoeostasis model assessment of insulin resistance 2 ; quicki = quantitative insulin sensitivity check index we have found no inter - group differences for body composition analysis . detailed results for bia analysis are shown in table 2 . lean body mass was higher in men in the whole study sample ( 60.16.4 [ 95 % ci : 57.4 - 62.8 ] vs. 43.85.4 [ 95 % ci : 41.546.1 ] kg , t = 9.56 , p < 0.001 ) and in the clozapine group ( 59.65.7 [ 95 % ci : 56.063.3 ] vs. 45.37.0 [ 95 % ci : 40.9 - 49.8 ] kg , t = 5.48 , p < 0.001 ) .
similarly , basal metabolic rate was higher in men in the whole study sample ( 1,707.7182.3 [ 95 % ci : 1,630.71,784.6 ] vs. 1,337.3138.4 [ 95 % ci : 1,278.8 - 1,395.7 ] kcal / day , t = 7.92 , p < 0.001 ) and in the clozapine group ( 1,701.2138.2 [ 95 % ci : 1,613.41,789.0 ] vs. 1,362.7173.0 [ 95 % ci : 1,252.71,472.5 ] kcal / day , t = 5.29 , p < 0.001 ) .
n = 24control
n = 24total body fat [ kg]25.6 8.8(21.829.2)22.4 8.7(18.726.1 )
p = 0.10
t = 1.25total body fat [ % ] 32.6 8.4(29.136.2)28.9 7.1(25.831.8 )
p = 0.06
t = 1.67lean body mass [ kg]52.5 9.6(48.456.5)51.4 10.8(46.855.9 )
p = 0.36
t = 0.36lean body weight [ % ] 67.6 8.1(64.271.0)71.1 7.1(68.174.1 )
p = 0.07
t = 1.61total body water [ l]38.4 7.0(35.441.3)37.7 7.9(34.340.9)p = 0.36 t = 0.35total body water [ % ] 49.9 5.4(47.652.1)52.1 5.2(49.854.2 )
p = 0.09
t = 1.42basal metabolic rate [ kcal / day]1,532.0
p = 0.39
t = 0.26data given as : mean standard deviation ( 95 % ci ) results of body composition analysis data given as : mean standard deviation ( 95 % ci ) there were no significant difference for fasting serum levels of agrp between the clozapine group and the control group ( 15.008.65 [ 95 % ci : 11.3418.65 ] vs. 15.336.82 [ 95 % ci : 12.4518.22 ] pg / ml , t = 0.32 , p = 0.37 ) , see fig . 1 .
women had significantly lower levels of agrp in the whole study group ( 12.535.65 [ 95 % ci : 10.1414.92 ] vs. 17.808.66 [ 95 % ci : 14.1421.45 ] pg / ml , t = 2.47 , p = 0.009 ) and in the control group ( 12.905.77 [ 95 % ci : 9.2216.57 ] vs. 17.777.14 [ 95 % ci : 13.2322.31 ] pg / ml , t = 1.82 , p = 0.04 ) , but the difference was not significant for the clozapine group ( 12.175.75 [ 95 % ci : 8.5115.83 ] vs. 17.8210.28 [ 95 % ci : 11.28 - 24.36 ] pg / ml , t = 1.64 , p = 0.06).fig .
1mean fasting agrp serum levels [ pg / ml ] in subjects on clozapine and in the control group ( p = 0.37 ; horizontal bars indicate means ) .
mean fasting agrp serum levels [ pg / ml ] in subjects on clozapine and in the control group ( p = 0.37 ; horizontal bars indicate means ) .
for the whole study group significant correlations of agrp levels were found for total body fat [ % ] ( r = 0.34 , p = 0.02 ) , lean body mass [ kg ] ( r = 0.38 , p = 0.006 ) , lean body mass [ % ] ( r = 0.34 , p = 0.02 ) , body water [ l ] ( r = 0.38 , p = 0.006 ) , body water [ % ] ( r = 0.34 , p = 0.02 ) and homocysteine levels ( r = 0.29 , p = 0.04 ) .
for the clozapine group significant correlations of agrp levels were found for total body fat [ % ] ( r = 0.48 , p = 0.02 ) , basal metabolic rate ( r = 0.42 , p = 0.04 ) , lean body mass [ % ] ( r = 0.49 , p = 0.01 ) , body water [ % ] ( r = 0.49 , p = 0.01 ) . for the control group
significant correlations of agrp levels were found only for basal metabolic rate ( r = 0.42 , p = 0.04 ) . in the clozapine group
there were no significant differences for agrp levels between subjects with or without excess body fat ( based on fmi value ) ( p = 0.59 ) , idf - defined metabolic syndrome ( p = 0.33 ) , smokers and non - smokers ( p = 0.15 ) , subjects with bmi < 25 kg / m and with bmi 25 kg / m ( p = 0.09 ) , subjects with and without impaired fasting glucose ( p = 0.16 ) , subjects with and without abdominal obesity ( p = 0.11 ) , subjects with and without dyslipidemia ( p = 0.09 ) , and subjects with and without homa-1ir defined insulin resistance ( p = 0.07 ) . in the control group there were no significant differences for agrp levels between subjects with or without excess body fat ( based on fmi value ) ( p = 0.20 ) , idf - defined metabolic syndrome ( p = 0.44 ) , smokers and non - smokers ( p = 0.35 ) , subjects with bmi < 25 kg / m and with bmi 25 kg / m ( p = 0.12 ) , subjects with and without impaired fasting glucose ( p = 0.36 ) , subjects with and without abdominal obesity ( p = 0.10 ) , subjects with and without dyslipidemia ( p = 0.37 ) , and subjects with and without homa-1ir defined insulin resistance ( p = 0.13 ) . in the whole study group there was no significant differences for agrp levels between subjects with or without excess body fat ( based on fmi value ) ( p = 0.30 ) , idf - defined metabolic syndrome ( p = 0.43 ) , smokers and non - smokers ( p = 0.29 ) , subjects with bmi < 25 kg / m and with bmi 25 kg / m ( p = 0.39 ) , subjects with and without impaired fasting glucose ( p = 0.29 ) , subjects with and without dyslipidemia ( p = 0.11 ) , and subjects with and without homa-1ir defined insulin resistance ( p = 0.33 ) . there was a significant difference for agrp levels between subjects with or without abdominal obesity ( 13.466.50 [ 95 % ci : 9.0917.83 ] vs. 16.926.93 [ 95 % ci : 12.7321.11 ] pg / ml , t = 1.80 , p = 0.04 ) .
the main objective of the present study was to find out whether there is a significant difference in fasting serum levels of agrp peptide between patients with schizophrenia on clozapine monotherapy and age- and sex - matched healthy controls .
we have found the difference was not significant ( clozapine : 15.008.65 , control : 15.336.82 pg / ml , p = 0.37 ) .
we do not know pre - treatment values , so we can not determine whether and how this parameter changed during therapy with clozapine . to our knowledge ,
no data are available on the effect of clozapine on blood agrp concentrations in humans .
limited data are available for olanzapine , which has clinical and pharmacological properties similar to clozapine .
results of these studies show that treatment with olanzapine does not affect agrp levels ( basoglu et al .
however , in animal study it was demonstrated that administration of olanzapine or clozapine upregulates npy and agrp and downregulates proopiomelanocortin in the arcuate nucleus of the hypothalamus ( ferno et al .
we do not know studies demonstrating correlation between blood levels of agrp and levels in the hypothalamus , but since it was showed recently that agrp neurons are unique among hypothalamic neurons by being the predominant neuronal subtype situated outside the blood
brain barrier ( olofsson et al . 2013 ) , we may assume that changes in hypothalamic expression are reflected in changes in blood levels .
the finding that women had significantly lower levels of agrp in the whole study group ( 12.535.65 vs. 17.808.66 pg / ml , p = 0.009 ) and in the control group ( 12.905.77 vs. 17.777.14 pg / ml , p = 0.04 ) , but not in the clozapine group ( 12.175.75 vs. 17.8210.28 pg / ml , p = 0.057 ) , might indicate that the testes are important source of agrp , as shown previously ( shutter et al .
however , several more recent studies showed no difference between men and ( gavrila et al .
2001 ) , therefore it may indicate that plasma agrp levels reflect central agrp concentrations .
it was previously reported that total level of agrp is higher in obese subjects ( katsuki et al .
2001 ) , although in another study it has been demonstrated that agrp levels were lower in normal weight group compared with women with anorexia , probably due to increased leptin levels ( moriya et al .
we did not measure levels of leptin or acylated ghrelin , but we found in the same group of patients that there were no differences for desacyl ghrelin levels between patients taking clozapine and control group ( wysokiski et al .
while there was a significant difference in bmi values between the clozapine group and the control group , we found no differences between both groups for fmi values . compared with bmi ,
fmi is a better indicator of central obesity since it is much more sensitive to body fat content ( kelly et al .
moreover , there were no differences for any of the bia results between both groups .
these indicate that there was a substantial similarity between both groups in terms of body fat content , which is important considering that agrp levels may affect or result more from the amount of adipose tissue than from body weight per se .
on the other hand , we have found negative correlations between agrp levels and total body fat ( r = 0.34 and 0.48 in the whole study group and clozapine group , respectively ) .
these may indicate the effect of leptin on the expression of agrp , but require further studies .
additionally , there were positive correlations with lean body mass ( r = 0.38 and 0.49 in the whole study group and clozapine group , respectively ) , body water ( which amount is negatively correlated with the amount of body fat : r = 0.96 , p < 0.001 ) ( r = 0.34 and 0.49 in the whole study group and clozapine group , respectively ) and basal metabolic rate ( r = 0.42 both in the clozapine group and control group ) , also being in line with the above - mentioned mechanism . in animal studies
it has been found that high - fat diet leads to decreased expression of agrp in the hypothalamus , probably in the mechanism moderated by leptin ( staszkiewicz et al .
we have no detailed data on diet patterns of our study subjects , but reports are consistent that patients with schizophrenia consume higher amounts of high - fat foods compared with healthy population ( dipasquale et al .
. this might be one of the reasons why we have found no differences between our study groups . also ,
level of physical activity ( usually lower in hospitalized patients ) might affect mechanisms regulating agrp levels .
finally , another important factor is the effect of treatment mediated by other anabolic or catabolic neuropeptides . here , two most important peptides are leptin ( which decreases agrp levels ) and ghrelin ( which has opposite effect ) and have also been studied most extensively ( sentissi et al .
2008 ) . since both leptin and ghrelin ( andrews 2011 ) may regulate activity of agrp / npy neurons in the arcuate nucleus , effects of antipsychotics on these neuropeptides must be taken into consideration . therefore
, a study focused on interactions within this regulatory system would be very important for better understanding of this problem . based on our results
however , the low number of study subjects limited the probability of finding inter - group differences due to lack of statistical power . due to the cross - sectional study design causal relationships
can not be established and the effect of previous antipsychotic treatment can not be excluded .
dual - energy x - ray absorptiometry ( dxa ) should be used to measure body composition and adipose tissue mass more accurately .
due to complex structure of interactions between anabolic and catabolic neuropeptides , a longitudinal study comparing these interactions are crucial for understanding mechanisms of treatment - induced weight gain .
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aim : agouti - related peptide ( agrp ) is one of the hypothalamic hormones that works by increasing appetite and decreasing metabolism , thus leading to weight gain .
the aim of the study was to find out if agrp level in subjects with schizophrenia on clozapine monotherapy is higher compared with healthy controls .
methodology : we determined fasting serum agrp levels in 24 subjects with schizophrenia on clozapine monotherapy and 24 healthy , age- and sex - matched controls .
biochemical and anthropometric measurements were combined with body composition analysis .
results : there was no difference for agrp levels between patients taking clozapine and control group ( 15.008.65 vs. 15.336.82 pg / ml , p = 0.37 ) .
we found negative correlations between agrp levels and total body fat ( r = 0.34 and 0.48 in the whole study group and clozapine group , respectively ) and positive correlations with lean body mass ( r = 0.38 and 0.49 in the whole study group and clozapine group , respectively ) , body water ( r = 0.34 and 0.49 in the whole study group and clozapine group , respectively ) and basal metabolic rate ( r = 0.42 both in the clozapine and control groups ) .
there were no correlations with age , height , weight , body mass index , fat mass index , abdominal , waist or hip circumferences , waist - hip ratio , blood pressure , total cholesterol , hdl , ldl , triglycerides , uric acid , glucose , insulin , clozapine dose or treatment duration , duration of treatment with antipsychotics and markers for insulin resistance .
conclusion : we can not conclude that treatment with clozapine is associated with increased level of agrp .
we did not find previously described differences in agrp levels between obese and non - obese subjects or associations between agrp and various metabolic parameters .
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Introduction
Material and methods
Results
Discussion
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echocardiographic measurements were performed in 19992001 ( baseline ) and 20072009 ( follow - up ) examinations of the hoorn study .
the hoorn study is a population - based cohort study on glucose metabolism and cardiovascular diseases , previously described in detail ( 10 ) . at baseline , 290 individuals with normal glucose metabolism ( ngm ) , 187 with impaired glucose metabolism ( igm ) , and 345 with type 2 diabetes participated . at follow - up ,
167 ( 20% ) individuals were excluded a priori because of incomplete baseline data ( n = 26 ) , mental incompetence to participate ( n = 12 ) , or death ( n = 129 ) . of the remaining 655 individuals , 441 ( 67% ) participated .
thirteen ( 3% ) of those were excluded in the present analyses because of missing echocardiography data at follow - up . to restrict the study population to individuals at risk of developing lv
systolic and/or diastolic dysfunction , we also excluded 34 individuals ( 8% ) with an lv ejection fraction < 50% or an la volume index > 40 ml / m at baseline .
the local ethics committee approved the study , and written informed consent was obtained from all participants .
echocardiography was performed at baseline and follow - up with the use of an hp sonos 5500 echocardiography system ( 24 mhz transducer ) according to a standardized protocol consisting of two - dimensional , m - mode , and pulsed wave doppler assessments as previously described ( 13 ) . at follow - up , this protocol was expanded with tissue doppler assessments of mitral annular velocities .
a set of three markers of lv diastolic dysfunction was determined : la volume index , lv mass indexed to height to the power of 2.7 ( 14 ) , and the ratio of early ( e ) mitral valve flow to early ( e ) diastolic lengthening velocity ( e / e ) using tissue and pulsed wave doppler assessments .
incident heart failure was considered present if signs and symptoms were accompanied by lv systolic ( lv ejection fraction < 35% ) and/or diastolic dysfunction ( 15 ) .
presence of incident lv diastolic dysfunction ( grade 2 or 3 ) was assessed according to international recommendations ( 16 ) . in the present analyses ,
we primarily focused on previously described ultrasound - derived distensibility coefficients of carotid , brachial , and femoral arteries to assess stiffness of the arterial wall ( 10 ) .
additionally , systemic arterial compliance was determined according to the time - decay method and by calculating the ratio of stroke volume to aortic pulse pressure ( 17 ) .
augmentation index was determined using applanation tonometry . in a subset of the study population ,
carotid - femoral pulse wave velocity was approached by measuring carotid - femoral transit time , adjusted for height in the statistical analyses .
reproducibility of arterial ultrasound measurements at baseline was assessed by calculating intraobserver intersession coefficients of variation as previously described ( 10 ) . at follow - up ,
intraobserver intersession coefficients of variation in 10 individuals ( aged 70.3 3.6 years ) were comparable : 4.2 , 13.1 , 4.1 , and 6.1% for lv ejection fraction , la volume , lv mass , and e / e , respectively .
for assessment of glucose status , all participants except those with previously diagnosed diabetes underwent a standard 75-g oral glucose tolerance test and were classified into ngm , igm ( impaired fasting glucose and/or impaired glucose tolerance ) , and type 2 diabetes groups according to the 1999 world health organization criteria ( 18 ) .
health status , medical history , medication use , and smoking habits were assessed by questionnaires ( 10 ) .
waist circumference , blood pressures ( systolic , mean arterial , and pulse pressures ) , and levels of cholesterol , insulin , hba1c , and c - reactive protein were determined as previously described ( 10 ) .
descriptive statistics are presented as means sd or , in the case of a skewed distribution , as median ( interquartile range ) .
the f test for anova in the case of continuous variables or tests in the case of proportions were performed to test for differences between groups of glucose status . in the case of skewed distributions ,
the above tests were done on log - transformed data . for every variable that had >
10% missing data , the number missing is presented in the legends of tables 13 .
linear regression analyses , adjusted for age and sex , were performed to assess associations of glucose status and arterial stiffness with markers of lv systolic and diastolic dysfunction at follow - up .
regression models with arterial stiffness as an independent variable were subsequently adjusted for mean arterial pressure to investigate to what extent these associations were explained by elevated blood pressure .
for investigation of changes in markers , lv systolic and diastolic dysfunction according to glucose status or arterial stiffness , we adjusted all models for baseline values of these markers ( models assessing e / e adjusted for baseline la volume index ) .
lv systolic and diastolic dysfunction in type 2 diabetes and arterial stiffness might partially be due to underlying factors , like overweight , hypertension , insulin resistance , or previous cardiovascular events ( 19 ) .
therefore , we additionally analyzed which potentially underlying or mediating factors contribute to these associations and to what extent by adding these factors one by one or simultaneously to the models ( supplementary data ) .
a secondary purpose of these analyses was to investigate potential residual confounding by these factors .
product terms were entered to test for effect modification by sex or medication use at follow - up and for interactions of glucose status with arterial stiffness or prior cardiovascular disease .
associations of arterial distensibility coefficients , systemic arterial compliance , and arterial compliance coefficients were shown per lower unit to reflect associations with greater arterial stiffness .
additional analyses , adjusted for age and sex , were performed to investigate whether diabetes duration had an effect on markers of lv systolic and diastolic function .
p values < 0.05 , or < 0.10 in case of interaction analyses , were considered statistically significant .
statistical analyses were performed with spss for windows ( version 15.0 ; spss , chicago , il ) .
echocardiography was performed at baseline and follow - up with the use of an hp sonos 5500 echocardiography system ( 24 mhz transducer ) according to a standardized protocol consisting of two - dimensional , m - mode , and pulsed wave doppler assessments as previously described ( 13 ) . at follow - up , this protocol was expanded with tissue doppler assessments of mitral annular velocities .
a set of three markers of lv diastolic dysfunction was determined : la volume index , lv mass indexed to height to the power of 2.7 ( 14 ) , and the ratio of early ( e ) mitral valve flow to early ( e ) diastolic lengthening velocity ( e / e ) using tissue and pulsed wave doppler assessments .
incident heart failure was considered present if signs and symptoms were accompanied by lv systolic ( lv ejection fraction < 35% ) and/or diastolic dysfunction ( 15 ) .
presence of incident lv diastolic dysfunction ( grade 2 or 3 ) was assessed according to international recommendations ( 16 ) .
in the present analyses , we primarily focused on previously described ultrasound - derived distensibility coefficients of carotid , brachial , and femoral arteries to assess stiffness of the arterial wall ( 10 ) .
additionally , systemic arterial compliance was determined according to the time - decay method and by calculating the ratio of stroke volume to aortic pulse pressure ( 17 ) .
augmentation index was determined using applanation tonometry . in a subset of the study population ,
carotid - femoral pulse wave velocity was approached by measuring carotid - femoral transit time , adjusted for height in the statistical analyses .
reproducibility of arterial ultrasound measurements at baseline was assessed by calculating intraobserver intersession coefficients of variation as previously described ( 10 ) . at follow - up ,
intraobserver intersession coefficients of variation in 10 individuals ( aged 70.3 3.6 years ) were comparable : 4.2 , 13.1 , 4.1 , and 6.1% for lv ejection fraction , la volume , lv mass , and e / e , respectively .
for assessment of glucose status , all participants except those with previously diagnosed diabetes underwent a standard 75-g oral glucose tolerance test and were classified into ngm , igm ( impaired fasting glucose and/or impaired glucose tolerance ) , and type 2 diabetes groups according to the 1999 world health organization criteria ( 18 ) .
health status , medical history , medication use , and smoking habits were assessed by questionnaires ( 10 ) .
waist circumference , blood pressures ( systolic , mean arterial , and pulse pressures ) , and levels of cholesterol , insulin , hba1c , and c - reactive protein were determined as previously described ( 10 ) .
descriptive statistics are presented as means sd or , in the case of a skewed distribution , as median ( interquartile range ) . the f test for anova in the case of continuous variables or tests in the case of proportions
were performed to test for differences between groups of glucose status . in the case of skewed distributions ,
the above tests were done on log - transformed data . for every variable that had >
10% missing data , the number missing is presented in the legends of tables 13 .
linear regression analyses , adjusted for age and sex , were performed to assess associations of glucose status and arterial stiffness with markers of lv systolic and diastolic dysfunction at follow - up .
regression models with arterial stiffness as an independent variable were subsequently adjusted for mean arterial pressure to investigate to what extent these associations were explained by elevated blood pressure . for investigation of changes in markers , lv systolic and diastolic dysfunction according to glucose status or arterial stiffness , we adjusted all models for baseline values of these markers ( models assessing e / e adjusted for baseline la volume index ) .
lv systolic and diastolic dysfunction in type 2 diabetes and arterial stiffness might partially be due to underlying factors , like overweight , hypertension , insulin resistance , or previous cardiovascular events ( 19 ) .
therefore , we additionally analyzed which potentially underlying or mediating factors contribute to these associations and to what extent by adding these factors one by one or simultaneously to the models ( supplementary data ) .
a secondary purpose of these analyses was to investigate potential residual confounding by these factors .
product terms were entered to test for effect modification by sex or medication use at follow - up and for interactions of glucose status with arterial stiffness or prior cardiovascular disease .
associations of arterial distensibility coefficients , systemic arterial compliance , and arterial compliance coefficients were shown per lower unit to reflect associations with greater arterial stiffness .
additional analyses , adjusted for age and sex , were performed to investigate whether diabetes duration had an effect on markers of lv systolic and diastolic function .
p values < 0.05 , or < 0.10 in case of interaction analyses , were considered statistically significant .
statistical analyses were performed with spss for windows ( version 15.0 ; spss , chicago , il ) .
a total of 394 individuals in the age range of 5087 years were included in the present analyses , 87 ( 22% ) of whom had igm and 128 ( 32% ) of whom had type 2 diabetes . compared with individuals who were not included ,
these individuals were younger and less likely to have type 2 diabetes and had more favorable levels of arterial stiffness and lv systolic and diastolic dysfunction at baseline ( data not shown ) .
individuals with type 2 diabetes were younger , had higher bmi and blood pressure , and had greater arterial stiffness and more severe lv systolic and diastolic dysfunction at baseline compared with individuals without type 2 diabetes ( table 1 ) .
heart failure incidence was not significantly different : 23 ( 21% ) in type 2 diabetes versus 15 ( 19% ) in igm and 24 ( 15% ) in ngm .
incidence of lv diastolic dysfunction grade 2 or 3 was higher with deteriorating glucose status : 88 ( 72% ) in type 2 diabetes versus 52 ( 61% ) in igm and 55 ( 31% ) in ngm .
characteristics according to glucose status the presence of type 2 diabetes was associated with more severe levels of all markers of lv systolic and diastolic dysfunction : lv ejection fraction , la volume index , lv mass index , and e / e ( table 2 ) .
after adjustment for age and sex , lv ejection fraction at follow - up was 2.98% lower in individuals with type 2 diabetes at baseline compared with that in subjects with ngm .
individuals with type 2 diabetes had a 3.71 ml / m higher la volume index , 5.86 g / m higher lv mass index , and 1.64 higher e / e. these associations were attenuated after adjustment for baseline markers of lv systolic and diastolic dysfunction , but type 2 diabetes was still significantly associated with a higher lv mass index ( 3.41 g / m ) and e / e ( 1.43 ) .
individuals with igm compared with those with ngm had a 2.93 g / m higher lv mass index , independent of baseline lv mass index .
markers of lv systolic and diastolic dysfunction at follow - up according to baseline glucose status ( ngm = reference ) associations of type 2 diabetes with markers of lv systolic and diastolic dysfunction were largely independent of blood pressure , lipid levels , renal function , c - reactive protein , and coronary artery disease ( supplementary data table a ) .
adjustment for hba1c levels or insulin resistance diminished the associations of type 2 diabetes with lv ejection fraction and la volume index .
associations between type 2 diabetes and lv mass index were attenuated after adjustment for waist circumference .
interaction analyses showed that type 2 diabetes was particularly associated with a lower lv ejection fraction in individuals with prior cvd . a longer diabetes duration ( per year )
was associated with a 0.59 ml / m higher la volume index at follow - up but not with other markers of lv systolic and diastolic function .
greater stiffness of carotid , brachial , and femoral arteries , measured as lower distensibility coefficients , was associated with higher levels of la volume index , lv mass index , and e / e ( table 3 ) .
after adjustment for age and sex , every 10 kpa lower carotid artery distensibility coefficient was associated with a 0.31 ml / m higher la volume index and a 0.58 g / m higher lv mass index .
every 10 kpa lower brachial artery distensibility coefficient was associated with a 0.43 ml / m higher la volume index and a 0.09 higher e / e. every 10 kpa lower femoral artery distensibility coefficient was associated with a 0.80 ml / m higher la volume index , a 0.91 g / m higher lv mass index , and a 0.09 higher e / e. these associations were independent of mean arterial pressure , except for associations with e / e. after adjustment for baseline markers of lv diastolic dysfunction , 10 kpa lower distensibility coefficients of carotid , brachial , and femoral arteries remained significantly associated with a 0.29 , 0.38 , and 0.73 ml / m higher la volume index , respectively .
markers of lv systolic and diastolic dysfunction at follow - up according to baseline arterial distensibility coefficients associations of femoral distensibility coefficients with markers of lv systolic and diastolic dysfunction were partly dependent on body weight and systolic blood pressure ( supplementary data table a ) .
lower compliance coefficients of brachial and femoral arteries , as well as a higher young elastic modulus or pulse pressure , were similarly associated with more severe lv diastolic dysfunction ( supplementary data table b ) .
a higher aortic augmentation index was the only marker of arterial stiffness that was significantly associated with lv systolic dysfunction : lv ejection fraction was 0.21% lower with every percentage point higher aortic augmentation index .
carotid - femoral transit time was measured in a subset of 178 individuals , and lv ejection fraction was 0.14% lower with every second shorter carotid - femoral transit time , though not significantly ( p = 0.13 ) .
there was no interaction between glucose status and arterial distensibility coefficients in their associations with lv systolic and diastolic dysfunction ( p values for interaction > 0.10 ) .
figure 1 shows the associations of glucose status and sd scores of arterial stiffness ( measured as femoral artery distensibility coefficients ) with sd scores for all four markers of lv systolic and diastolic dysfunction , adjusted for age and sex .
the right half of each histogram shows that values hardly changed if glucose status and arterial stiffness were combined in one model .
for instance , the presence of type 2 diabetes was associated with a 0.26 sd lower lv ejection fraction after adjustment for age and sex .
after subsequent adjustment for arterial stiffness , this value remained similar , despite a loss of statistical significance : 0.25 ( p = 0.08 ) .
associations of glucose status with la volume index , lv mass index , and e / e hardly changed either after adjustment for arterial stiffness .
furthermore , each sd higher arterial stiffness score was associated with a 0.14 sd higher la volume index after adjustment for age and sex .
after subsequent adjustment for glucose status , this value was 0.12 ( p = 0.02 ) .
associations of arterial stiffness with lv mass index and e / e did not change significantly either after adjustment for glucose status , despite a loss of statistical significance .
standardized associations of impaired glucose metabolism ( black bars ) , type 2 diabetes ( light gray bars ) , and lower femoral artery distensibility coefficients ( dark gray bars ) with markers of lv systolic and diastolic dysfunction , adjusted for age and sex ( left half of each histogram ) and , additionally , for each other ( right half of each histogram ) . *
there was no significant effect modification by sex , use of ace inhibitors , or lipid - lowering , glucose - lowering , or blood pressure lowering medication at follow - up on the associations of glucose status or arterial stiffness with markers of lv systolic and diastolic dysfunction ( p > 0.10 , data not shown ) .
the presence of type 2 diabetes was associated with more severe levels of all markers of lv systolic and diastolic dysfunction : lv ejection fraction , la volume index , lv mass index , and e / e ( table 2 ) .
after adjustment for age and sex , lv ejection fraction at follow - up was 2.98% lower in individuals with type 2 diabetes at baseline compared with that in subjects with ngm .
individuals with type 2 diabetes had a 3.71 ml / m higher la volume index , 5.86 g / m higher lv mass index , and 1.64 higher e / e. these associations were attenuated after adjustment for baseline markers of lv systolic and diastolic dysfunction , but type 2 diabetes was still significantly associated with a higher lv mass index ( 3.41 g / m ) and e / e ( 1.43 ) . individuals with igm compared with those with ngm had a 2.93 g / m higher lv mass index , independent of baseline lv mass index .
markers of lv systolic and diastolic dysfunction at follow - up according to baseline glucose status ( ngm = reference ) associations of type 2 diabetes with markers of lv systolic and diastolic dysfunction were largely independent of blood pressure , lipid levels , renal function , c - reactive protein , and coronary artery disease ( supplementary data table a ) .
adjustment for hba1c levels or insulin resistance diminished the associations of type 2 diabetes with lv ejection fraction and la volume index . associations between type 2 diabetes and lv mass index were attenuated after adjustment for waist circumference .
interaction analyses showed that type 2 diabetes was particularly associated with a lower lv ejection fraction in individuals with prior cvd .
a longer diabetes duration ( per year ) was associated with a 0.59 ml / m higher la volume index at follow - up but not with other markers of lv systolic and diastolic function .
greater stiffness of carotid , brachial , and femoral arteries , measured as lower distensibility coefficients , was associated with higher levels of la volume index , lv mass index , and e / e ( table 3 ) .
after adjustment for age and sex , every 10 kpa lower carotid artery distensibility coefficient was associated with a 0.31 ml / m higher la volume index and a 0.58 g / m higher lv mass index .
every 10 kpa lower brachial artery distensibility coefficient was associated with a 0.43 ml / m higher la volume index and a 0.09 higher e / e. every 10 kpa lower femoral artery distensibility coefficient was associated with a 0.80 ml / m higher la volume index , a 0.91 g / m higher lv mass index , and a 0.09 higher e / e. these associations were independent of mean arterial pressure , except for associations with e / e. after adjustment for baseline markers of lv diastolic dysfunction , 10 kpa lower distensibility coefficients of carotid , brachial , and femoral arteries remained significantly associated with a 0.29 , 0.38 , and 0.73 ml / m higher la volume index , respectively .
markers of lv systolic and diastolic dysfunction at follow - up according to baseline arterial distensibility coefficients associations of femoral distensibility coefficients with markers of lv systolic and diastolic dysfunction were partly dependent on body weight and systolic blood pressure ( supplementary data table a ) .
lower compliance coefficients of brachial and femoral arteries , as well as a higher young elastic modulus or pulse pressure , were similarly associated with more severe lv diastolic dysfunction ( supplementary data table b ) .
a higher aortic augmentation index was the only marker of arterial stiffness that was significantly associated with lv systolic dysfunction : lv ejection fraction was 0.21% lower with every percentage point higher aortic augmentation index .
carotid - femoral transit time was measured in a subset of 178 individuals , and lv ejection fraction was 0.14% lower with every second shorter carotid - femoral transit time , though not significantly ( p = 0.13 ) .
there was no interaction between glucose status and arterial distensibility coefficients in their associations with lv systolic and diastolic dysfunction ( p values for interaction > 0.10 ) .
figure 1 shows the associations of glucose status and sd scores of arterial stiffness ( measured as femoral artery distensibility coefficients ) with sd scores for all four markers of lv systolic and diastolic dysfunction , adjusted for age and sex .
the right half of each histogram shows that values hardly changed if glucose status and arterial stiffness were combined in one model .
for instance , the presence of type 2 diabetes was associated with a 0.26 sd lower lv ejection fraction after adjustment for age and sex .
after subsequent adjustment for arterial stiffness , this value remained similar , despite a loss of statistical significance : 0.25 ( p = 0.08 ) .
associations of glucose status with la volume index , lv mass index , and e / e hardly changed either after adjustment for arterial stiffness . furthermore , each sd higher arterial stiffness score was associated with a 0.14 sd higher la volume index after adjustment for age and sex .
after subsequent adjustment for glucose status , this value was 0.12 ( p = 0.02 ) .
associations of arterial stiffness with lv mass index and e / e did not change significantly either after adjustment for glucose status , despite a loss of statistical significance .
standardized associations of impaired glucose metabolism ( black bars ) , type 2 diabetes ( light gray bars ) , and lower femoral artery distensibility coefficients ( dark gray bars ) with markers of lv systolic and diastolic dysfunction , adjusted for age and sex ( left half of each histogram ) and , additionally , for each other ( right half of each histogram ) .
there was no significant effect modification by sex , use of ace inhibitors , or lipid - lowering , glucose - lowering , or blood pressure lowering medication at follow - up on the associations of glucose status or arterial stiffness with markers of lv systolic and diastolic dysfunction ( p > 0.10 , data not shown ) .
this study shows that both glucose status and arterial distensibility coefficients were prospectively associated with more severe lv diastolic dysfunction .
furthermore , associations of glucose status and arterial stiffness with lv diastolic dysfunction were largely independent of each other and indicated a deterioration of lv diastolic dysfunction compared with baseline .
strengths include the population - based design and comprehensive assessment of glucose status , arterial stiffness , and lv systolic and diastolic dysfunction .
these data provide a unique insight into many different aspects of arterial stiffness and their influence on lv systolic and diastolic dysfunction .
lv diastolic dysfunction at baseline was therefore based on la volume index only , and this might have been subject to some misclassification .
furthermore , differences between attendees and nonattendees suggest that a healthy cohort bias may have occurred in this study .
we also had missing data for some echocardiographic markers , predominantly in individuals with high bmi .
our findings that lv systolic and diastolic dysfunction was more severe in type 2 diabetic patients than in individuals with ngm are in line with previous data reporting associations between glucose metabolism and lv systolic and diastolic dysfunction ( 20,21 ) . the type 2 diabetes associated lv diastolic dysfunction at follow - up could not completely be explained by an already more severe lv diastolic dysfunction at baseline .
these results imply that lv diastolic dysfunction deteriorates more in individuals with than in those without type 2 diabetes .
there was a trend toward more severe lv systolic and diastolic dysfunction in individuals with igm as well , but this was not statistically significant .
individuals with a longer duration of type 2 diabetes appeared to have a higher la volume index at follow - up .
peripheral markers of arterial stiffness , like arterial distensibility and compliance coefficients , and brachial pulse pressure were most consistently associated with markers of lv diastolic dysfunction .
associations of arterial distensibility coefficients with lv diastolic dysfunction were not due to elevated blood pressure , since adjustment for mean arterial pressure only resulted in a small reduction of the associations .
associations of arterial distensibility coefficients with la volume index and e / e at follow - up were largely independent of baseline la volume index .
this might imply that 1 ) arterial stiffness precedes deterioration of lv diastolic dysfunction or 2 ) stiffening of arteries and lv walls occurs simultaneously .
the first might be due to arterial wave reflections that lead to an increased lv load and decreased coronary perfusion ( 6 ) .
the second might be due to coinciding structural changes in arterial and lv walls ( 9 ) . in the current study
, wave reflections seemed especially harmful to lv systolic function , since augmentation index was the only marker of arterial stiffness that was significantly associated with lv ejection fraction , followed by carotid - femoral transit time .
lv diastolic dysfunction seemed to be more coherent to peripheral artery stiffening than to central artery stiffness .
this might support the second theory : that stiffening of arteries and lv walls occurs simultaneously .
type 2 diabetes and arterial stiffness were largely independently associated with lv systolic and diastolic dysfunction in the current study .
more severe lv systolic and diastolic dysfunction in type 2 diabetes therefore can not be explained or can or just partly be explained by increased arterial stiffness
. other mechanisms that might play a role in the development of lv systolic and diastolic dysfunction in type 2 diabetes include an altered cardiac metabolism or increased stiffening of the lv due to myocardial fibrosis or an elevated cardiomyocyte resting tension ( 11,2224 ) .
this might be reflected by the lowered associations between type 2 diabetes and markers of lv systolic and diastolic dysfunction after adjustment for hba1c .
overweight and obesity are known to be associated with a higher lv mass index , and indeed , waist circumference seemed to play a role in type 2 diabetes related higher lv mass index ( 25 ) .
lv systolic function was particularly worsened in type 2 diabetic patients with prior cardiovascular disease . since individuals with type 2 diabetes already had a worse cardiovascular profile at baseline , the influence of changes in cardiovascular risk factors earlier in life on lv systolic and diastolic dysfunction may also be worth examining .
to conclude , the presence of type 2 diabetes and the presence of arterial stiffness are both associated with deterioration of lv diastolic dysfunction .
it is likely that type 2 diabetes and arterial stiffness relate to lv diastolic dysfunction through different pathways , since these associations were independent of each other .
a better understanding of the mechanisms behind those different pathways in changes in lv diastolic dysfunction could help identify targets for prevention of heart failure .
our findings that lv systolic and diastolic dysfunction was more severe in type 2 diabetic patients than in individuals with ngm are in line with previous data reporting associations between glucose metabolism and lv systolic and diastolic dysfunction ( 20,21 ) . the type 2 diabetes associated lv diastolic dysfunction at follow - up could not completely be explained by an already more severe lv diastolic dysfunction at baseline .
these results imply that lv diastolic dysfunction deteriorates more in individuals with than in those without type 2 diabetes .
there was a trend toward more severe lv systolic and diastolic dysfunction in individuals with igm as well , but this was not statistically significant .
individuals with a longer duration of type 2 diabetes appeared to have a higher la volume index at follow - up .
peripheral markers of arterial stiffness , like arterial distensibility and compliance coefficients , and brachial pulse pressure were most consistently associated with markers of lv diastolic dysfunction .
associations of arterial distensibility coefficients with lv diastolic dysfunction were not due to elevated blood pressure , since adjustment for mean arterial pressure only resulted in a small reduction of the associations .
associations of arterial distensibility coefficients with la volume index and e / e at follow - up were largely independent of baseline la volume index .
this might imply that 1 ) arterial stiffness precedes deterioration of lv diastolic dysfunction or 2 ) stiffening of arteries and lv walls occurs simultaneously .
the first might be due to arterial wave reflections that lead to an increased lv load and decreased coronary perfusion ( 6 ) .
the second might be due to coinciding structural changes in arterial and lv walls ( 9 ) . in the current study
, wave reflections seemed especially harmful to lv systolic function , since augmentation index was the only marker of arterial stiffness that was significantly associated with lv ejection fraction , followed by carotid - femoral transit time .
lv diastolic dysfunction seemed to be more coherent to peripheral artery stiffening than to central artery stiffness .
this might support the second theory : that stiffening of arteries and lv walls occurs simultaneously .
type 2 diabetes and arterial stiffness were largely independently associated with lv systolic and diastolic dysfunction in the current study .
more severe lv systolic and diastolic dysfunction in type 2 diabetes therefore can not be explained or can or just partly be explained by increased arterial stiffness
. other mechanisms that might play a role in the development of lv systolic and diastolic dysfunction in type 2 diabetes include an altered cardiac metabolism or increased stiffening of the lv due to myocardial fibrosis or an elevated cardiomyocyte resting tension ( 11,2224 ) .
this might be reflected by the lowered associations between type 2 diabetes and markers of lv systolic and diastolic dysfunction after adjustment for hba1c .
overweight and obesity are known to be associated with a higher lv mass index , and indeed , waist circumference seemed to play a role in type 2 diabetes related higher lv mass index ( 25 ) .
lv systolic function was particularly worsened in type 2 diabetic patients with prior cardiovascular disease . since individuals with type 2 diabetes already had a worse cardiovascular profile at baseline , the influence of changes in cardiovascular risk factors earlier in life on lv systolic and diastolic dysfunction may also be worth examining .
to conclude , the presence of type 2 diabetes and the presence of arterial stiffness are both associated with deterioration of lv diastolic dysfunction .
it is likely that type 2 diabetes and arterial stiffness relate to lv diastolic dysfunction through different pathways , since these associations were independent of each other .
a better understanding of the mechanisms behind those different pathways in changes in lv diastolic dysfunction could help identify targets for prevention of heart failure .
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objectiveto investigate relative contributions of glucose status and arterial stiffness to markers of left ventricular ( lv ) systolic and diastolic dysfunction after 8 years of follow-up.research design and methodsin the population - based prospective hoorn study , 394 individuals with preserved lv systolic and diastolic function participated , of whom 87 had impaired glucose metabolism and 128 had type 2 diabetes .
measurements including arterial ultrasound and echocardiography were performed according to standardized protocols.resultsthe presence of type 2 diabetes was associated with more severe lv systolic and diastolic dysfunction 8 years later : lv ejection fraction was 2.98% ( 95% ci 0.465.51 ) lower , and left atrial ( la ) volume index , lv mass index , and tissue doppler - derived e / e were 3.71 ml / m2 ( 1.206.22 ) , 5.86 g / m2.7 ( 2.948.78 ) , and 1.64 units ( 0.952.33 ) higher , respectively .
furthermore , presence of impaired glucose metabolism or type 2 diabetes was associated with 8-year increases in lv mass index .
more arterial stiffness ( measured as a lower distensibility ) was associated with lv diastolic dysfunction 8 years later : la volume index , lv mass index , and e / e at follow - up were higher .
subsequent adjustments for baseline mean arterial pressure and/or lv diastolic dysfunction did not eliminate these associations .
associations of type 2 diabetes and arterial stiffness with markers of lv diastolic dysfunction were largely independent of each other.conclusionsboth glucose status and arterial distensibility are independently associated with more severe lv diastolic dysfunction 8 years later and with deterioration of lv diastolic dysfunction .
therefore , type 2 diabetes and arterial stiffness may relate to lv diastolic dysfunction through different pathways .
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RESEARCH DESIGN AND METHODS
Echocardiography
Arterial stiffness
Reproducibility
Other baseline measurements
Statistical analysis
RESULTS
Glucose status
Arterial stiffness
Combined influence of glucose status and arterial stiffness
CONCLUSIONS
Glucose status
Arterial stiffness
Combined influence of glucose status and arterial stiffness
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female sex workers ( fsws ) are at high risk of hiv infection and transmission .
a systematic review conducted in over 25 countries with medium and high hiv prevalence indicated that 36.9% of sex workers in sub - saharan africa were hiv positive with some countries having hiv prevalence as high as 70.7% . despite this high burden of hiv infection ,
only 58% of sex workers have access to hiv prevention and care programmes and less than half of hiv positive sex workers are enrolled into hiv care .
unaids reported that , in 2013 , art averted 6.3 million aids related deaths worldwide as a result of the increased number of people receiving art . in 2015 , who released revised guidelines for hiv treatment and recommended initiation of art among all hiv infected individuals irrespective of their cd4 count , for improved treatment outcomes and prevention of hiv transmission .
sex workers are among the priority populations for expansion of hiv services including art . however , despite the existence of established targeted sex worker interventions , lots of challenges still exist that have hindered many from enrolling into care .
several studies have highlighted barriers to linkage to care among female sex workers including discrimination by hospital staff [ 3 , 7 ] , delays at the health facility [ 8 , 9 ] , lack of money for transport and food , fear of drugs , stigma , use of drugs and alcohol , fear to be seen by clients , lack of knowledge about the treatment center [ 10 , 11 ] , and delays in accessing treatment among those who felt they were still healthy .
however , while these barriers have been reported , there is still limited information on linkage to hiv care among sex workers in sub - saharan africa , particularly on effective linkage models to inform scale up of hiv interventions among sex workers . in 2012 , reach out mbuya hiv / aids initiative ( rom ) , a faith - based community non - government organization in uganda , introduced mobile outreach services to promote hiv testing and treatment among fsws and their clients .
although the program has over 200 fsws enrolled in hiv care , some of the fsws who tested hiv positive did not enrol for treatment .
we conducted a study to identify the facilitators and barriers to linkage to hiv care among the fsws who tested positive in order to design appropriate hiv interventions for this key population group .
this was a cross - sectional qualitative study that involved in - depth and key informant interviews .
this study was part of a larger study that explored the facilitators and barriers to linkage to hiv care among fsws in kampala and wakiso districts in central uganda .
rom promotes hiv prevention through peer educators and provides hiv counselling and testing ( hct ) services through established outreach clinics that provide care and treatment for hiv positive fsws .
mobile hct outreaches are conducted in locations surrounding mbuya parish in kampala and wakiso districts in central uganda .
in - depth interview ( idi ) participants were selected from 301 hiv positive female sex workers who were registered in the hct registers at the different outreach points between may 2012 and december 2013 .
of the 301 registered clients , 144 were traced and interviewed as part of the larger study where 28 of these were purposively selected to participate in the in - depth interviews .
these included 14 fsws who were in hiv care and 14 who had not yet enrolled into care .
the selected fsws were contacted by counsellors by phone to explain the study and to ask them to participate in the study .
fsws with missing phone contact information or those who were unavailable after a minimum of three telephone attempts were tracked through their peer educators and friends .
key informant interview ( kii ) participants were selected from peer educators who were trained by rom and were involved in following up fsws to encourage them to access hiv care .
in addition , five rom staffs who were involved in the implementation of the outreach program were interviewed as key informants .
rom provides static and bi - weekly outreach clinics for fsws using staff specifically trained on how to deal with key populations including fsws . at the time of the study ,
hiv testing and linkage to hiv care services were offered using bi - weekly mobile outreach clinics , brothel based testing , and nocturnal mobile vans at places where the fsws reside or conduct business .
hiv testing was conducted following the national rapid testing algorithm and posttest counselling was provided to hiv infected women who were advised on the available health facilities for follow - up care .
hiv infected women were advised to begin care immediately either at the static or mobile clinics operated by rom or other partners and were followed up with phone calls or by peer educators a week after testing and once every quarter thereafter , to ascertain linkage to care .
those who were enrolled at the static clinic were subjected to a home assessment by the community art and tb treatment supporters ( catts ) to enable adequate follow - up .
the information collected using the home assessment tool included the names and number of household members , water and sanitation , economic status , property owned , distance to the facility , and acceptable mode for communication .
sex workers , who agreed to participate in the study , provided written informed consent and were interviewed in the language of their choice using unstructured , pretested questionnaires .
the pretesting was conducted in the nearby project area two weeks before the commencement of the study . to ensure anonymity ,
data were collected on the facilitators and barriers to linkage to hiv care by trained research assistants with experience in handling fsws .
data were collected on age , education , and linkage status to aid proper categorization of the study participants ( linked or not linked to care ) .
we defined linkage to care as having registered within an hiv clinic . the interview themes ( i.e. , sociodemographic , socioeconomic , structural , and hiv - related factors )
were informed by theoretical constructs from the health belief model and the socio - ecological model and further informed by literature on barriers and facilitators to hiv service access among fsws .
figure 1 presents a summary of the key variables and their interrelationships in informing linkage to hiv care among fsws .
interviews were conducted at selected brothels , fsws homes , and static and outreach clinics .
key informant interviews were conducted at the workplace or residence of the respondents , in english and luganda , using unstructured pretested questionnaires .
the themes for the key informant interviews included hiv treatment seeking behaviour of the fsws , the challenges of enrolling into care , and the interventions to enhance linkage to hiv care in this population .
additional data , including age , education , job category , residence , and the time allocated to working with fsws , was collected .
the review of transcripts was guided by a priori themes pertaining to linkage to care , loss to follow - up , stopping treatment , and facilitators and barriers to linkage .
issues that cut across the different themes which were captured by both authors were coded and categorized while those that were captured by one but not the other were subjected to further analysis until they were resolved and used to support the overriding themes or dropped .
the study was approved by makerere university school of public health higher degrees research and ethics committee ( irb # : irb00011353 ) and the uganda national council of science and technology .
of the 28 fsws interviewed , 14 ( 50% ) were enrolled in hiv care ( nine at rom ) , 8 had not yet enrolled , and 6 had previously enrolled but were either lost to follow - up or had stopped treatment .
of the 28 women interviewed , 24 ( 85% ) were aged 2030 years while the rest where > 30 years .
half ( 14 ) had at least secondary or higher education and the other half ( 14 ) had at least primary education .
the fsws who were lost to follow - up or had stopped treatment were aged between 15 and 36 years with their education level ranging from none to secondary .
the respondents included fsws who operate in bars ( 6 ) , on the streets ( 8) , at home ( 3 ) , and in brothels ( 11 ) ( table 1 ) .
the findings have been grouped into two themes : ( a ) facilitators of linkage to hiv care and ( b ) barriers to linkage to hiv care among fsws , as presented below .
the facilitators have been further grouped into two main themes : health system and social network factors ( table 2 ) .
majority of the participants associated their linkage to care with the good quality of the services including good attitude and friendliness exhibited by the counsellors towards them .
they noted that the counsellors talked to them with ease and were not judgmental : the counsellors are good , they talk to us very well and are almost like our parents .
when i was told my results i got so scared as though i would die there and then .
i failed to breath for 30 minutes ; the counsellor got me some cold water to drink and the care she showed me encouraged me to enrol for treatment .
( fsw , 29 years , enrolled in care ) the counsellors are good , they talk to us very well and are almost like our parents .
when i was told my results i got so scared as though i would die there and then .
i failed to breath for 30 minutes ; the counsellor got me some cold water to drink and the care she showed me encouraged me to enrol for treatment .
( fsw , 29 years , enrolled in care ) participants reported that the counsellors provided detailed information and encouragement to help them start treatment .
the counsellors told them that testing hiv positive was not the end of life and that with medication they would be helped to stay healthy and live longer .
in addition , participants appreciated the good follow - up support for those who tested positive .
some said the counsellors called them and followed them up to their homes , which made them feel cared for : i started treatment due to counselor x 's advice , she was so caring and always followed me up , she could visit me ; otherwise on my own i had even disappeared but she was on my case .
( fsw , 24 years , enrolled in care ) i started treatment due to counselor x 's advice , she was so caring and always followed me up , she could visit me ; otherwise on my own i had even disappeared but she was on my case .
( fsw , 24 years , enrolled in care ) some participants acknowledged that it was easy for them to enrol in care because of the same - day results and immediate initiation of treatment .
the peer educators and fellow sex workers enabled them to start treatment by encouraging them and helping to allay their fears regarding hiv treatment and occasionally escorting them to the clinics to start medication .
some respondents said they would forget the clinic days because the clinic did not run every day but the peer educators reminded them . during the key informant interviews the peer educators mentioned how tirelessly they worked to follow up the hiv positive fsws ; they were sometimes abused by the sex workers but they never gave up on them : i feel it is my calling to help these people and even though i am not paid i make sure i have brought them to get medicine .
( peer educator , 29 years ) they come and tell me that on such and such a date i will be going to the clinic so remind me , i will note this in my diary and when the date approaches i go to them and tell them it is the day for the clinic .
( peer educator , 25 years ) i feel it is my calling to help these people and even though i am not paid i make sure i have brought them to get medicine .
( peer educator , 29 years ) they come and tell me that on such and such a date i will be going to the clinic so remind me , i will note this in my diary and when the date approaches i go to them and tell them it is the day for the clinic .
( peer educator , 25 years ) the proximity of the clinic to their residence was another enabling factor for linkage to care .
some fsws did not want to move long distances for hiv care and others did not have transport so they thought that enrolling at rom would help reduce such burdens . across all interviews fsws who were enrolled in savings and
they noted that the members of the support groups like village savings groups started by rom for sex workers only or mixed with other community members helped them to enrol into care .
the members of the savings group reportedly advised them to start on treatment and live longer .
being part of a savings group also created hope that money would be available for food and they would have capital to start their own business and live longer : i thought i was alone but when i realized we were many , i stopped fearing and also started medicine .
my group members say that everyone is sick and so we should use our money well to care for ourselves and look nice to get customers and save much more .
( fsw , 25 years , enrolled in care ) i thought i was alone but when i realized we were many , i stopped fearing and also started medicine .
my group members say that everyone is sick and so we should use our money well to care for ourselves and look nice to get customers and save much more .
( fsw , 25 years , enrolled in care ) across all the interviews the participants cited the need to remain healthy as one of their motivators for linkage to care . in order to continue doing their work , they wanted to start medication to remain healthy and not show any signs of opportunistic infections , which would scare away their customers .
others wanted to remain healthy so as to live longer and take care of their children : it is because of my children ; i have to live for them because i am a widow , if i do n't take care of myself and die who will take care of them .
( fsw with 3 children , 24 years , in care ) it is because of my children ; i have to live for them because i am a widow , if i do n't take care of myself and die who will take care of them .
( fsw with 3 children , 24 years , in care ) several participants reported that seeing their colleagues improve after starting treatment motivated them to start on treatment while others did so because many of their friends were dying , which compelled them to start treatment so as to survive : i had a friend who stopped treatment and died , when we took her to the hospital the nurses said if only she continued with treatment she would be alive .
when they ( providers ) came looking for sex workers to be enrolled into care i was the first on the list to go for treatment .
( fsw 20 years , enrolled in care ) i had a friend who stopped treatment and died , when we took her to the hospital the nurses said if only she continued with treatment she would be alive .
when they ( providers ) came looking for sex workers to be enrolled into care i was the first on the list to go for treatment .
( fsw 20 years , enrolled in care ) barriers were similarly categorized into two themes : health systems and social network factors ( table 3 ) .
the fsws enrolled at the government facilities mentioned discrimination from the health workers who openly exhibited a negative attitude towards sex workers .
they said the health workers reacted differently after realizing that they were dealing with a sex worker and , upon telling their friends , those who had not yet registered for care feared to enrol : the counselor asked me what i do and i said sex work .
she called four other counselors to ask me why i do that kind of work ; i wanted to ask them ,
what do you expect me to do ? ' but the counselor stood up and asked me rudely ; is that also work to mention among people ? '
that counselor annoyed me so much i walked off and never wanted to see her again .
( peer educator who is a fsw , 29 years ) the counselor asked me what i do and i said sex work .
she called four other counselors to ask me why i do that kind of work ; i wanted to ask them , what do you expect me to do ? ' but the counselor stood up and asked me rudely ; is that also work to mention among people ? '
that counselor annoyed me so much i walked off and never wanted to see her again .
( peer educator who is a fsw , 29 years ) the four fsws who were enrolled at government facilities noted that they were discouraged by the requirement to present a treatment supporter coupled with the long procedures for enrolment that involved several visits to the facility : it was not easy to start arvs because i was told that unless i went with someone i would not be started on arvs and this person was always moving and too hard to get .
may be i do not want someone else to know that i am positive .
( fsw , 29 years , enrolled in care ) it was not easy to start arvs because i was told that unless i went with someone i would not be started on arvs and this person was always moving and too hard to get .
i felt this was not right because i must go for treatment by myself may be i do not want someone else to know that i am positive .
( fsw , 29 years , enrolled in care ) almost all the participants cited stigma as a major barrier to linkage to care .
the majority feared to be seen by their clients at the clinic . although the close proximity of the clinic was mentioned as a motivator , for some fsws it was a hindrance .
participants were afraid of being seen at the hiv clinic by customers , their friends , and other people in the village and were particularly concerned about rumours : i can not go to that clinic because everyone here goes there and they will see me which will spoil my business . right now
the competition is much and the other girls will make me a topic because they want to take away my customers yet they are also sick .
( fsw 22 years , not enrolled ) i can not go to that clinic because everyone here goes there and they will see me which will spoil my business . right now
the competition is much and the other girls will make me a topic because they want to take away my customers yet they are also sick .
( fsw 22 years , not enrolled ) due to lack of information , many participants had many myths about arvs ( e.g. , that they kill fast and need a lot of care ) .
they avoided the arvs by not going to the clinic while some opted to wait until they got other jobs because of the perception that arvs could weaken them and disrupt their work .
some respondents feared drugs and could not imagine swallowing them for life so they opted out . on the other hand , some participants feared death after testing and lost hope for living ; they did not see the need for starting on art : madam is n't there any danger with the arvs because friends tell me that when i start taking arvs they will burn my kidney and lungs ( fsw , 20 years , not in care and never went to school ) madam is n't there any danger with the arvs because friends tell me that when i start taking arvs they will burn my kidney and lungs ( fsw , 20 years , not in care and never went to school ) some fsws did not know what hiv is while others encountered difficulties in identifying an hiv clinic because they lacked proper information about the location of the clinics .
this was mainly a challenge to the young girls ( aged between 20 and 25 years ) who lived in the brothels as indicated by the project staff : some were testing for the first time and others had ever tested but they were not even shocked when told that they were positive because they were just starting to understand what hiv is .
( key informant ) some were testing for the first time and others had ever tested but they were not even shocked when told that they were positive because they were just starting to understand what hiv is .
( key informant ) some fsws mentioned that they could not enrol in an hiv clinic since they did not believe their hiv results .
some thought it was just a prick performed hurriedly in the night and wondered how it translated into an hiv positive result so they waited and retested many times : when i reached home , i started doubting the results and decided to go for another test in a different place .
i continued living in denial but after some time i had to admit that i was hiv positive and started taking septrin .
( fsw 29 yrs , not enrolled in care ) when i reached home , i started doubting the results and decided to go for another test in a different place .
i continued living in denial but after some time i had to admit that i was hiv positive and started taking septrin .
( fsw 29 yrs , not enrolled in care ) a third of the fsws mentioned that they were using herbs as a substitute for arvs and therefore saw no need of being registered in an hiv clinic .
besides many of the fsws said they used local herbs from traditional healers to attract customers and did not have to line up for herbs like they did in the hiv clinic : no .
i have not received treatment for hiv except local herbs which i prefer to take and i do not want to be forced to take medication [ meaning arvs ] . even with the herbs now am on medication ; i do take the local herbs and am fine with the treatment , i do n't want to be forced to take arvs .
( fsw , 24 yrs , not enrolled ) no . i have not received treatment for hiv except local herbs which i prefer to take and i do not want to be forced to take medication [ meaning arvs ] . even with the herbs now am on medication ; i do take the local herbs and am fine with the treatment , i do n't want to be forced to take arvs .
( fsw , 24 yrs , not enrolled ) some participants bought painkillers from general clinics instead of enrolling in an hiv clinic ( self - medication ) .
even when the nurses in these clinics suspected that they were hiv infected , they did not tell them because they wanted business through treating their on and off sickness . because they are always travelling in different places looking for clients , fsws cited lack of time as one of the barriers to linkage to care : i am still very busy right now looking for rent and food .
i can not lose any customer at this difficult moment but when i have raised some money to care for myself i will start medicine without any worries .
( fsw 23 years , not enrolled ) i am still very busy right now looking for rent and food .
i can not lose any customer at this difficult moment but when i have raised some money to care for myself i will start medicine without any worries .
( fsw 23 years , not enrolled ) the kis felt that the street based sex workers had more challenges with linkage to hiv care compared to those in brothels because they were difficult to trace given that they only relied on phone numbers which were off most of the time : those on the streets do not want to access care .
( key informant ) those on the streets do not want to access care .
( key informant ) across all interviews , the fsws mentioned lack of finances as a major hindrance to linkage to care .
they were concerned about requirements such as good food and frequent transport to the health facility : it 's the financial challenges and hunger because sometimes i go without food and my friends tell me that taking medication requires eating and drinking .
( fsw 22 years , not enrolled ) it 's the financial challenges and hunger because sometimes i go without food and my friends tell me that taking medication requires eating and drinking .
our study of facilitators and barriers to linkage to hiv care among female sex workers in kampala and wakiso districts in uganda shows that health system and social network factors are the major facilitators and barriers to linkage to hiv care .
good quality health services ( especially polite and caring providers , strong follow - up structures using peer educators , and provider telephone calls ) and social network factors ( encouragement from peers and membership of savings group and the need to maintain good health ) were the primary facilitators of linkage to hiv care among fsws . on the other hand , perceived stigma ,
various forms of misinformation , and prohibitive clinic policies were major barriers to linkage to hiv care .
contrary to the experience of fsws at rom , those who were registered in the public health facilities encountered negative staff attitudes , a major barrier that has been documented in several studies [ 8 , 16 , 17 ] .
these findings suggest that efforts to improve linkage and retention in hiv care among fsws should focus on quality of services and improving social support networks . ensuring good quality services , particularly providers who can deliver services in a respectful and no - judgmental manner without undue delays and restrictions and adequate community education and mobilization as well as support structures for retention are key health system issues that can improve the quality of services . using peers to educate communities and follow - up of those who need treatment reduce the burden on health workers and strengthens community engagement . as reported elsewhere , the requirement of a treatment supporter before initiating care and treatment was a barrier to treatment that should no longer be strictly enforced .
the training of providers and peer educators prior to implementation of this program could have contributed to delivery of quality services at rom and are a key intervention area .
given the overriding concerns about being seen at hiv facilities , training for providers should emphasize the need for privacy and confidentiality .
further , providing an integrated package of services for sex workers could reduce stigma around accessing hiv and other services .
training should also emphasize flexibility with certain clinic policies that could hinder access to hiv services among sex workers .
social network issues were also strong facilitators and barriers to linkage and require interventions particularly targeted at enhancing support by peers and reducing misinformation and stigma . enacted stigma , myths , and fears about arvs coupled with lack of correct information about hiv continue to be major barriers to linkage to hiv care .
financial constraints were a prominent barrier to linkage to care . the savings groups were a good avenue for social support and financial stability and could be integrated into health programs for low - income sex workers [ 20 , 21 ] .
collectively , these findings suggest that programs targeting fsws should integrate continuous sensitization and provision of tailor made services that address the individual level , community , and health system needs of fsw .
this study is based on interviews with 14 fsws who were in hiv care and 14 others who were not . by all counts ,
the numbers are too small to represent the views of all fsws who are enrolled or not enrolled in hiv care .
however , our study findings provide good insights into the facilitators and barriers to linkage that can inform interventions to improving linkage for fsws .
future research is needed to further understand how the fsws can be maintained in care once they have been linked .
our study shows that fsws ability to be linked to hiv care is largely influenced by good quality friendly services and community support systems especially from their peers .
hiv programs for fsw should focus on enhancing these as well as dealing with the barriers that mainly stem from stigma , ignorance , and work - related challenges .
these findings call for a need to design interventions that utilize a multichannel , multipronged approach to increase access to hiv services among fsws .
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introduction .
while four in ten female sex workers ( fsws ) in sub - saharan africa are infected with hiv , only a small proportion is enrolled in hiv care .
we explored facilitators and barriers to linkage to hiv care among fsws receiving hiv testing services at a community - based organization in periurban uganda .
methods .
the cross - sectional qualitative study was conducted among 28 hiv positive fsws from may to july 2014 .
key informant interviews were conducted with five project staff and eleven peer educators .
data were collected on facilitators for and barriers to linkage to hiv care and manually analyzed following a thematic framework approach . results .
facilitators for linkage to hiv care included the perceived good quality of health services with same - day results and immediate initiation of treatment , community peer support systems , individual 's need to remain healthy , and having alternative sources of income .
linkage barriers included perceived stigma , fear to be seen at outreach hiv clinics , fear and myths about antiretroviral therapy , lack of time to attend clinic , and financial constraints .
conclusion .
linkage to hiv care among fsws is influenced by good quality friendly services and peer support .
hiv service delivery programs for fsws should focus on enhancing these and dealing with barriers stemming from stigma and misinformation .
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1. Introduction
2. Methods
3. Results
4. Discussion
5. Conclusion
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for patients with operable breast cancer , the major prognostic determinant is whether there has or has not been spread to the axilla and the number of involved axillary nodes .
initially it was suggested that breast cancer first spreads locoregionally via lymphatics to the axillary lymph nodes and then metastasises more distantly . in accordance with this concept ,
subsequently fisher postulated that the extent of micrometastases at diagnosis of breast cancer is an indicator of outcome , with biological behaviour of cancer predetermining the likelihood of progression of the disease . nowadays gene expression profiling arrays can delineate tumour types with different prognoses .
the surgical approach for breast cancer treatment evolved from the extensive radical mastectomy and the patey modified radical mastectomy to breast conserving and minimally invasive techniques .
traditionally the surgical management of breast cancer comprised wide local resection of the primary tumour and axillary lymph node dissection ( alnd ) .
axillary status is the most important prognostic factor in breast cancer providing staging information and therefore largely defining treatment strategy .
diagnostic imaging modalities such as ultrasound , magnetic resonance mammography , positron emission tomography , and 99 m technetium ( tc ) sestamibi scintimammography are not reliable for staging the axilla , particularly with lymph node metastases < 0.5 cm [ 8 , 9 ] .
clinically palpable lymph nodes prove to be false positive in 2530% of patients and about 40% have positive results after ultrasound with or without fine - needle aspiration node negativity .
studying 24,740 women with invasive breast cancer , carter et al . showed that approximately 80% with tumour size < 1 cm , 50% with up to 5 cm , and 30% with > 5% had negative axilla , a fact suggesting that metastases do not occur exclusively via the axillary lymph nodes , but rather lymph node status serves as an indicator of the tumour 's ability to spread . additionally , it has been recently shown that the molecular profile of the primary tumour is a more significant prognostic indicator in terms of disease - free survival ( dfs ) and overall survival ( os ) than lymph node metastases .
the combination of the introduction of population - based mammographic screening for breast cancer , modern imaging methods , and increased public awareness resulted in patients being diagnosed more often with smaller - size tumours and less likelihood of axillary lymph node metastases .
it is evident now that 6070% of patients with early breast cancer are node negative at the time of diagnosis and alnd puts them at significant risk of short- and long - term morbidities without benefit .
alnd is associated with acute complication rates of 2030% including seroma formation , local swelling , numbness , impaired shoulder movement , neuropathy , infection , and chronic lymphoedema rates of 737% . in a prospective study by petrek et al . evaluating a cohort of 923 women with 20 years
follow up , it was shown that breast - cancer - related lymphoedema following alnd occurred maximally in the first 3 years following surgery ; however , up to 23% of patients may still develop arm swelling during the rest of their lives .
several randomised studies have established that sentinel node biopsy ( snb ) is a safe and accurate procedure for detecting tumour cells in sln and predicting the status of the other axillary nodes ( non - sln ) .
although accuracy and appropriateness of snb were disputed by the finding of 510% false - negative cases when snb was followed by axillary dissection at high - risk patients for axillary nodal disease [ 13 , 18 ] , false - negative snb results seem to have decreased with the increasing experience of surgeons , and it is expected that the utilisation of snb in the future will be increased . a meta - analysis of seven prospective randomised controlled trials by kell et al . demonstrated that snb is equivalent to alnd for the detection of lymph node metastasis with the additional advantage of reduction of up to 75% in morbidity in patients with early stage breast cancer .
furthermore , a trend towards an improved detection of ln metastases was shown when snb is used .
patients undergoing snb have a 22% higher odds ratio of having a positive sln , due to the more intensive pathological examination which utilises multiple sections and immunohistochemistry ( ihc ) .
in contrast , the false - negative cases seen after axillary dissection are probably due to the inability of the pathologist to perform serial sections and ihc on the 2030 lymph nodes found in a complete axillary clearance specimen .
studies have shown that sln is the only positive lymph node in 3867% of patients when alnc followed .
interestingly , it has been reported that only 48% of patients with negative alns have internal mammary lymph node involvement ( imn ) whereas 2550% of patients with affected alns have also imn metastases [ 23 , 24 ] .
dissection of imn is not recommended because of the high morbidity and the uncertain benefit on survival .
the recently published outcomes of nsabp b-32 trial established the efficacy of sln biopsy alone with no further alnd in 5611 breast cancer patients with clinically negative lymph nodes .
women with invasive breast cancer who were randomly assigned to either sln resection plus alnd ( group 1 ) or to sln resection alone with alnd only if the slns were positive ( group 2 ) , after 8 years of followup , showed statistically equivalent overall survival , disease - free survival , and regional control .
patient followup is still continuing for longer - term assessment of survival and regional control .
moreover , a closer look into mature studies focused on axillary relapses and overall survival is in agreement with current findings favouring slb .
the national surgical adjuvant breast and bowel project ( nsabp ) b04 randomised study compared breast cancer patients with clinically negative alns managed either by radical mastectomy , total mastectomy with axillary radiation , or total mastectomy alone .
the results clearly defined that alnd decreases the risk of locoregional relaps ; however , no significant differences in survival were found among the treatment groups .
this study has been criticised because of the variability of numbers of lymph nodes resected in the total mastectomy alone arm and the lack of statistical power to detect a small difference in outcome . indeed , a meta - analysis has suggested that inadequate axillary treatment may lead to not only an increased risk of local relapse but also a 5% reduction in survival . as a result of increasing detection of early breast cancer and the high rate of micrometastases and itcs ( itcs ) found in the detailed pathological examination of sln
, a new debate has opened about the consequent necessity of alnd in these patients .
this has arisen because of better understanding of breast cancer behaviour and improved efficacy of combined therapeutic modalities . in this paper
we report the current guidelines concerning the management of the axilla after slnb and review the different aspects arising from recent studies on the role of micrometastases and itc clusters in sln on decision making .
the american society of clinical oncology ( asco ) expert panel conducted a systematic review of the literature available through february 2004 on the use of snb in early - stage breast cancer in order to develop guidelines for the management of the axilla ( http://jop.ascopubs.org/content/1/4/134 ) , and these are similar to those of the national institute of health and clinical excellence ( nice ) recommendations in uk ( http://www.nice.org.uk/nicemedia/live/12132/43413/43413.pdf ) .
alnd is the standard of care in those with a macrometastatic or micrometastatic positive sln to maximise local control .
if the sln is negative , a calnd ( calnd ) is not necessary .
itcs detected by ihc are of unknown clinical significance , and when identified , the sln is regarded as negative and no further alnd is required . although ihc is often used , it is not included in routine sln evaluation for breast cancer at this time .
asco and nice recommendations for slnb , alnd alone and managing of the axilla after slnb are summarised in table 1 .
in contrast the german guidelines do not recommend axillary clearance for 1 - 2 sln positive in patients with t1 and t2 tumours ( http://www.ago-online.de ) .
it has been suggested that slnb should be carried out by an experienced team in order to minimise false negativity and improve the predictive value of the procedure .
the american joint committee on cancer ( ajcc ) in the sixth edition of the cancer staging manual defined a lymph node metastatic tumour with maximum diameter > 2 mm as macrometastasis ( pn1 ) , when the diameter of deposit is 0.22 mm as micrometastasis ( pnmi ) , and a lesion of single tumour cells or small cell clusters with diameter < 0.2 mm as itcs [ pn0(i+ ) ] .
itcs are not distinguishable by h&e staining but detected only with immunohistochemistry ( ihc ) or molecular methods .
moore et al . suggested that the presence of itcs was unrelated to known prognostic variables and partly the result of instrumentation and manipulation of the tumour .
the management of patients with minimal sln involvement is problematic . in a meta - analysis of 25 studies of patients with sln micrometastases , in approximately 20% there was nonsentinel node disease falling to 9% when the sln involvement was detected by ihc .
furthermore , the consequent effect on dfs and os remains controversial , so the biological relevance and clinical significance is a matter of debate .
amaros investigates the benefit of a calnd in comparison to treatment with axillary radiotherapy ( art ) in patients with sln - positive breast cancer .
a recently published substudy evaluated the identification rate and the nodal involvement of the first 2,000 patients between 2001 and 2005 who entered from 26 european institutions .
the sentinel node identification rate was 97% which is high considering the relatively early days of this procedure .
34% were sln positive of whom 63% had macrometastases , 25% had micrometastases , and 12% had itcs . in the calnd arm non - sln involvement
was identified in 41% of patients with macrometastases and in 18% of patients with either micrometastases or itcs .
several studies have investigated the significance of occult metastases , such as micrometastases or small clusters of tumour cells in association with non - sln involvement and the impact of calnd on disease - free survival and overall survival , and the larger ones are summarised in table 2 [ 3743 ] .
although the majority show no prognostic impact of itcs in the sentinel node , a large dutch investigation with 5-year followup indicated that women with itcs who received adjuvant chemotherapy had a significantly better event - free survival compared with untreated cases with itcs .
furthermore , a finnish study showed a worse 5-year breast - cancer - specific survival for those with itc compared with node negative cases . in the
largest published multicenter retrospective study of 187 sln - itcs patients undergoing calnd , houvenaeghel et al . reported an incidence of 16% non - sln involvement .
the difference in the risk of non - sln involvement between sentinel nodes with itcs ( 16% ) and those with micrometastases ( 14% ) was not statistically significant .
however it was not apparent whether the presence of non - sln metastases should affect the therapeutic decision in these patients .
the authors proposed that calnd could be avoided in patients with tubular , colloid , or medullary small primary tumours ( pt1 ) with a risk of non - sln involvement approximately 5% .
mirror is a large dutch cohort retrospective study which assesses the impact of sln - itcs and micrometastases on 5-year disease - free survival in patients with favourable primary tumour characteristics . according to recent published data , both patients with snb micrometastases and those with itcs who did not undergo calnd experienced a far higher 5-year axillary recurrence rate , 6% in comparison to 1% of snb - negative patients who did not undergo calnd . additionally , both patients with sln micrometastases and itcs had approximately 5-year disease - free survival improved by 10% with adjuvant systemic therapy .
mirror findings support an aggressive treatment approach in patients with either sln micrometastases or itcs .
many investigators have studied the incidence of non - sln involvement in patients with sln micrometastases to define which patients may need further axillary treatment . wada and imoto . collected 22 studies from 1999 until 2006 referring to the frequency of sln micrometastases in patients with breast cancer and the prevalence of non - sln involvement in those patients after alnd .
the frequency of sln micrometastases was 38% with non - sln micrometastases ranging from 0 to 57% .
additionally , a wide range of non - sln macrometastases was found ( 018% ) . because the prevalence of non - sln micrometastases was low , the prognostic impact was unclear .
the wide range of results arose from the different numbers of patients involved , variations in number of pathological sections examined , and differences in tumour stage and grade .
results of studies in which patients with micrometastases in snb and who were not treated by completion axillary node clearance are summarised in table 3 [ 38 , 4854 ] .
most of the studies had small numbers and relatively short followup and tended to conclude that there was no benefit from completion axillary node clearance .
the largest study ; however , found a significantly worse disease - free survival for women with micrometastases who did not undergo calnd . .
de boer et al . conducted a systematic review of 58 studies conducted from 1977 to 2008 included 297,533 patients , aiming to define the prognostic relevance of micrometastases and itcs in patients with breast cancer . using random - effect meta - analysis they showed that the presence of aln metastases < 2 mm in diameter detected on single - section examination was associated with poorer overall survival .
moreover the presence of occult metastases on retrospective examination of aln - negative patients by step sectioning and/or immunohistochemistry ( n = 7740 patients ) was associated with poorer 5-year disease - free and overall survival .
outcomes from sentinel lymph node biopsy studies were not assessable due to small patient groups and short followup .
the international breast cancer study group trial ibcsg-23 - 01 is a randomised multicentre study designed to determine the significance of minimal ln metastasis in patients with breast cancer .
the trial was initiated in april 2001 , and it compares survival between patients with sln micrometastases who undergo slnb alone with those who receive calnd .
it is known that the extent of macrometastases in sln is strongly correlated with non - sln involvement .
the long - term effect of the residual axillary disease in the sentinel - lymph - node - positive patient on local and systemic recurrence has not been clearly defined for patients receiving modern radiotherapy and chemotherapy .
older studies of patients with symptomatic breast cancers have shown that inadequate axillary surgery does lead to reduced overall survival [ 5760 ] .
the study set out to randomise 1900 women with breast cancer and 15 involved snln to either calnd or observation .
all had a lumpectomy and tangential breast irradiation , but systemic therapy was at the discretion of the treating centre .
after a median followup of 6.3 years , the relapse - free survival for the alnd group was 82% compared with 84% for the observation group , and the overall survival was 92% in both groups .
unfortunately the trial stopped accrual after 891 cases had been entered which makes it underpowered to detect a 5% difference in outcome .
an additional aspect to be considered is that the guy 's wide excision studies showed no difference between the wide excision group and the radical mastectomy group at 10 years whereas after 25 years there was a significantly worse relapse - free and overall survival in the wide excision group with inadequately treated axillae . in a retrospective study ,
takei et al . confirmed the importance of calnd in sln - positive patients with high nuclear grade and hormone - negative breast cancer .
it was noticed that of 459 patients with macrometastatic disease treated with calnd , after a median follow - up period of 34 months , the axillary recurrence rate was only 0.6% .
bilimoria et al . studied a cohort of 403,167 patients with clinically node - negative breast cancer that underwent slnb from the us national cancer data base ( 19982005 ) .
of the 97,314 ( 24% ) patients identified with nodal metastases , 28% had no further surgical intervention in the axilla and 72% underwent calnd .
after a median followup of 63 months , it was found that in all patients and separately in those with macroscopic and microscopic nodal disease , the unadjusted axillary recurrence rate and overall survival were comparable .
after adjustment for clinicopathological differences , there was a trend towards a lower risk of axillary recurrence and death in patients with macroscopic nodal involvement undergoing calnd . for those with micrometastases , recurrence rates were similar in those undergoing either slnb alone or calnd .
of 26,986 patients with slnb - positive breast cancer from the seer database ( surveillance , epidemiology , and end results ) , 16% had no further axillary treatment and 84% had calnd .
after a median followup of 50 months , although a higher rate of ipsilateral regional recurrence was noticed in patients who underwent slnb alone , no statistically significant differences in overall survival ( os ) between patients who underwent slnb alone versus complete alnd were found .
the investigators suggested that in patients with small , low - grade primary tumours , positive er status , older age and who have received segmental mastectomy , calnd may be omitted .
hwang et al . reviewed the outcome of 3,366 patients with invasive breast cancer who underwent slnb from 1993 to 2005 .
of 750 sln - positive patients , 65% , 45.9% , and 34.2% were pn1 , pn1mi , and pn0 ( i+ ) , respectively .
of these patients , 196 had no further axillary surgery due to clinician and patient preference . according to clinicopathological variables ,
adjuvant treatment was applied and locoregional and distant recurrence and survival were studied . after a median followup of 29.5 months , no patient had an axillary recurrence , one had supraclavicular lymph node recurrence , and three patients developed metastatic disease to the lung or bone . the median time to recurrence was 32 months .
notably the patients with distant metastases had t3 grade iii invasive carcinoma . despite the low axillary recurrence rate
, authors suggested that it is not possible from these results to conclude definitively that calnd should be abandoned for these patients .
several factors correlated with the likelihood of additional non - sln metastasis have been investigated in an effort to distinguish which patients could avoid extensive axillary surgery .
characteristics of the primary tumour , such as size [ 40 , 66 ] , grade , hormone receptor and her2 profile , tumour type , multifocality , mean proliferative fraction , and lymphovascular invasion [ 68 , 69 ] , have all been studied .
additional features of the involved slns , such as size of metastases , number of positive slns , ratio of positive to resected slns , and the extracapsular spread have been also examined [ 21 , 61 , 7072 ] .
particularly patients with minimal sln metastases are at a significantly lower risk to have further non - sln invasion than those with sln macrometastases ( 1324% versus 4579% ) . however
, none of these characteristics individually can determine a subset of patients for whom alnd is unnecessary .
molecular profiling of metastatic foci different from the primary tumour could be used as indicator for the selection of patients who might benefit of completion axillary dissection .
the most important prognostic factors for the presence of non - sln metastases in patients with minimal sln involvement are presented in table 4 .
several mathematical models have been developed to predict the risk of non - sln involvement in patients with sln - positive breast cancer .
these include four nomograms : the memorial sloan - kettering cancer center ( mskcc ) , ( https://www.mskcc.org/mskcc/html/15938.cfm ) , mayo ( https://www.mayoclinic.org/breast-cancer/sentinelbiopsy.html ) , cambridge , and stanford ( https://www3-hrpdcc.stanford.edu/nsln-calculator/ ) .
there are three scoring systems , the tenon , mda , and saidi , and two recursive partitioning ( rp ) tools developed by kohrt et al . .
the institut curie studied 588 consecutive patients with positive slns who underwent alnd to compare the actual rate of non - sln metastases with those predicted by breast cancer nomogram of memorial sloan - kettering cancer center ( mskcc ) . while the predicted rate in non - sln macrometastases was relatively accurate , when the nomogram was applied to the 213 slns that contained only micrometastases , the predicted rate 59% was far away from the actual rate 44% of non - sln micrometastases detected by ihc .
consequently , the authors concluded that a different predictive model should be created for patients with micrometastases .
molecular tests based on technology such as oncotype dx ( genomic health , redwood city , calif , usa ) or other multigene arrays developed prognostic and predictive markers aiming to personalize surgical and adjuvant treatment of early breast cancer .
an accurate , intraoperative sentinel lymph node test probably could help in avoidance of delayed axillary dissections .
molecular tests may be proved more sensitive than current intraoperative tests but have not yet been validated .
osna is an automated assay for the detection of cytokeratin message , ck19 mrna , present in approximately 98% of breast cancers .
it provides an opportunity to make an intraoperative diagnosis of sentinel node involvement within 30 minutes , avoiding frozen section and allowing a one - stage procedure .
larger verification studies in which half of the bisected sentinel node was sent for histology and the other half homogenised for osna are shown in table 5 [ 71 , 72 , 7981 ] .
the usual reason for discordance between histopathology and osna was an uneven distribution of nodal metastases ( tissue allocation bias ) .
the studies indicate a good concordance between results of histopathology and osna . indeed in a study conducted by osaka , comparing osna with frozen section ,
it is likely that in time osna will replace histopathological examination of sentinel lymph nodes because of its ease , accuracy , and potential for enabling almost all patients to have a one - stage operation for early breast cancer .
recent studies aiming to determine if calnd is beneficial in patients with sln - positive breast cancer and even more in patients with minimal sln involvement have reached contrary conclusions .
limitations in the published studies include the methods of pathological evaluation of lymph nodes and that the number of additional non - sln - positive nodes is usually not known
thus lymph node step sectioning and ihc lead to increased identification of minimal metastases upstaging 9% to 25% of patients who initially were considered node negative [ 83 , 84 ] and did not have further axillary surgery .
the different rates of recurrence may be due to the molecular type of cancers and so that patients with sln metastases may also have different risks of metastatic involvement .
finally it is possible that not all minor tumour foci in axillary lymph nodes progress to local recurrences . according to al - hajj et al .
, only a minority of cancer cells potentially give metastases and most itcs are not viable and do not have the ability to form new tumours .
there may be two different breast cancer cell populations , true stem cells that have the capacity to develop metastases and the nonstem cells that never grow and are finally destroyed . at the level of everyday clinical practice , with both promising and disappointing results of the published studies
, most breast surgeons will hardly ever take the risk of avoiding completion axillary dissection in breast cancer patients even with minimal sentinel lymph node metastases .
many are seeking to find a balance between the needs of the majority to have minimal axillary surgery with minimal postoperative morbidity against the possibility that a minority will suffer relapse , morbidity , and possible increased mortality from undertreatment .
results from ongoing phase iii trials will perhaps provide new guidelines for the treatment of patients with micrometastases or itcs .
a predictive model which estimates accurately the likelihood of additional disease in the axilla might help tailor surgical therapy to the needs of the individual patient and identify those most likely to benefit from completion or alnd . genetic assays defining prognostic markers and new intraoperative tests detecting accurately sln involvement will help in early therapeutic decision making in the future .
it is important that premature decisions to restrict axillary surgery are not made on a basis of early results from underpowered clinical trials .
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sentinel lymph node biopsy ( slnb ) is a safe and accurate minimally invasive method for detecting axillary lymph node ( aln ) involvement in the clinically negative axilla thereby reducing morbidity in patients who avoid unnecessary axillary lymph node dissection ( alnd ) .
although current guidelines recommend completion alnd when macro- and micrometastatic diseases are identified by slnb , the benefit of this surgical intervention is under debate .
additionally , the management of the axilla in the presence of isolated tumour cells ( itcs ) in slnb is questioned .
particularly controversial is the prognostic significance of minimal slnb metastasis in relation to local recurrence and overall survival .
preliminary results of the recently published z0011 trial suggest similar outcomes after snb or alnd when the sn is positive , but this finding has to be interpreted with caution .
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1. Introduction
2. Current Guidelines
3. Micrometastases and Isolated Tumour Cells (ITCs)
4. Completion ALND and Micrometastases
5. Completion Axillary Lymph Node Dissection in SLN Macrometastases
6. Predictive Models
7. One-Step Nucleic Acid Amplification (OSNA)
8. Conclusions
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the construct of unintended pregnancies ( ups ) is multifactorial and broadly encompasses pregnancies that are either unwanted or mistimed.1 in many instances , ups are likely to end by induced abortion ; worldwide estimates suggest that 50% will be voluntarily terminated.2 in 2008 , it was estimated that 43.8 million abortions occurred worldwide , of which 86% occurred in low-/middle - income countries.3 furthermore , between 2003 and 2008 , the number of induced abortions was found to have decreased in high - income countries but to have increased in low-/middle - income countries . during this same period of time , the proportion of unsafe abortions increased and they were believed to account for 13% of maternal deaths , with the majority of these concentrated in countries with restrictive laws on abortion.4 elective abortion in brazil is considered non - legal , unless the pregnancy resulted from a rape , would cause a life - threatening condition to the mother , or the fetus has anencephaly or any other malformation that is incompatible with the extrauterine life . as a result ,
data on prevalence and associated costs of abortion are limited and remain a challenge to collect .
however , it is recognized worldwide that direct health costs of up and resulting abortions can significantly impact local health services and the families affected.5 health system and societal costs can also arise from ups that do not end in abortion and are carried to parturition . in the united states ,
the public health costs attributed to unintended births in a single year cost taxpayers $ 11.1 billion ( 2006).6 furthermore , previous studies have reported that ups are more likely to result in preterm births and low - birth - weight babies , which would increase health costs for neonatal care and costs associated with long - term disabilities.7 this is particularly relevant for ups in adolescence , which are more likely to result in low - birth - weight births , which can increase health costs.8 over the past few decades , brazil , like many fast - emerging economies , has experienced a fertility transition noted by a dramatic reduction in the total fertility rate , which currently sits at 1.8 births per woman.9,10 furthermore , during the fertility transition , the rate of contraception has also increased.11 despite increased contraception use , the cumulative rate of spontaneous and induced abortion changed very little between 1996 and 2006 , for which population survey estimates are available.12,13 furthermore , a survey of more than 1,000 women aged 1024 years in a single postnatal unit in brazil reported that more than 50% of all births were unintended , suggesting that possibly significant unmet contraception need still exists.8 similarly , for the whole country , the data from the last demographic and health survey ( dhs ) performed in 2006 indicates that 55% of all births were unintended.14 prevention of up using publicly funded programs in high - income countries has proven to generate significant cost savings for health services and public services.15 previous economic studies evaluating the consequences of up have mostly been performed in high - income countries where publicly funded fertility services and abortion legislation are different from those in low-/middle - income countries.15 the aim of this study was to evaluate the cost consequences of up in brazil , an emerging economy with vastly different fertility planning services than most advanced economies and with restrictive abortion laws . to the best of our knowledge ,
this is the first published study that describes the economics of up , and it is believed that this work can aid decision makers regarding access to contraceptives .
a model was developed to evaluate the humanistic burden and financial impact of ups in brazil .
the analysis factored in costs and outcomes for 1 year post - delivery of ups .
possible birth outcomes of up included induced abortion , miscarriage , and ongoing pregnancy resulting in birth .
all birth deliveries were assumed to have taken place in a hospital within the brazil public health system as reported by datasus , the data system for the ministry of health in brazil .
available data was further separated into vaginal ( 61.0% ) and cesarean section ( c - section ) deliveries ( 38.4%).16,17 outcomes and resulting costs for infants were also followed . these included stillbirths and infant survival and complications from term and preterm deliveries .
complications included admission to a neonatal intensive care unit ( icu ) , hospitalization during the first year , and cerebral palsy .
data from datasus were used when possible to populate the model to reflect the reality of the public health care system . however , for parameters not identified in datasus , other relevant sources were used .
a decision tree model was constructed in microsoft excel ( microsoft corporation , redmond , wa , usa ) from the perspective of the brazilian public health system ( figure 1 ) .
the total annual number of pregnancies was estimated using the reported number of live births for the year 2010 and adjusted with estimated percentages of induced abortion and miscarriage.18 the percentages of induced abortions and miscarriage were derived from the population - based 2006 dhs and found to be 1.5% and 8.9% , respectively.19 all induced abortions in the model were assumed to be from unintended pregnancies resulting in an adjusted abortion rate of 2.7% .
the up rate was estimated to represent 55% of all pregnancies.20 maternal mortality rates of 20 , ten , and 30 deaths per 100,000 births were used for miscarriage , vaginal delivery , and c - section delivery , respectively.2123 in addition , a case fatality rate of 100 deaths per 100,000 abortions was assumed.24 for both delivery methods , it was estimated that 7.8% of all deliveries were preterm births and 0.85% were stillbirths.25 the remainder of the deliveries was assumed to be term births .
differential infant mortality rates were used for term and preterm births , with 8.5 per 1,000 live births for the former and 68 per 1,000 live births for the latter.25,26 the model assessed infant complications , including admissions to neonatal icu , hospitalization during the first year , and cerebral palsy .
all preterm birth infants were assumed to require neonatal intensive care , compared to 7.6% of term birth infants.27 for preterm infants , the average number of hospitalizations during the first year was 1.7 , mostly from respiratory issues.28 for term birth infants , hospitalization was calculated as a relative risk reduction.29 cerebral palsy occurred in 0.1% and 1.7% of term and preterm births , respectively.30 resource use and unit costs were identified from published sources and the dhs in brazil . in brazil
the public national health system ( sus ) pays for around 70%75% of all reproductive procedures .
however , in this model , it is assumed that the costs are those from the public system , although , in approximately one - quarter of ups , the costs are probably higher when related to women s care covered by insurance or privately .
costs of elective abortion were not included in the analysis because these procedures rarely meet the legal requirements and would not be covered by the public health system .
costs of miscarriage were related to the following procedures : pelvic exam , blood test , ultrasound , and dilation and curettage .
the cost components for each method included antenatal care and labor and delivery.31,32 unit cost for hospitalization readmission during the first year was r$2,794.33 this unit cost was also assumed for neonatal care admission , while cerebral palsy was estimated to be r$19,854.33 cost parameters are summarized in table 1 .
a model was developed to evaluate the humanistic burden and financial impact of ups in brazil .
the analysis factored in costs and outcomes for 1 year post - delivery of ups .
possible birth outcomes of up included induced abortion , miscarriage , and ongoing pregnancy resulting in birth .
all birth deliveries were assumed to have taken place in a hospital within the brazil public health system as reported by datasus , the data system for the ministry of health in brazil .
available data was further separated into vaginal ( 61.0% ) and cesarean section ( c - section ) deliveries ( 38.4%).16,17 outcomes and resulting costs for infants were also followed . these included stillbirths and infant survival and complications from term and preterm deliveries .
complications included admission to a neonatal intensive care unit ( icu ) , hospitalization during the first year , and cerebral palsy .
data from datasus were used when possible to populate the model to reflect the reality of the public health care system . however , for parameters not identified in datasus , other relevant sources were used .
a decision tree model was constructed in microsoft excel ( microsoft corporation , redmond , wa , usa ) from the perspective of the brazilian public health system ( figure 1 ) .
the total annual number of pregnancies was estimated using the reported number of live births for the year 2010 and adjusted with estimated percentages of induced abortion and miscarriage.18 the percentages of induced abortions and miscarriage were derived from the population - based 2006 dhs and found to be 1.5% and 8.9% , respectively.19 all induced abortions in the model were assumed to be from unintended pregnancies resulting in an adjusted abortion rate of 2.7% .
the up rate was estimated to represent 55% of all pregnancies.20 maternal mortality rates of 20 , ten , and 30 deaths per 100,000 births were used for miscarriage , vaginal delivery , and c - section delivery , respectively.2123 in addition , a case fatality rate of 100 deaths per 100,000 abortions was assumed.24 for both delivery methods , it was estimated that 7.8% of all deliveries were preterm births and 0.85% were stillbirths.25 the remainder of the deliveries was assumed to be term births .
differential infant mortality rates were used for term and preterm births , with 8.5 per 1,000 live births for the former and 68 per 1,000 live births for the latter.25,26 the model assessed infant complications , including admissions to neonatal icu , hospitalization during the first year , and cerebral palsy .
all preterm birth infants were assumed to require neonatal intensive care , compared to 7.6% of term birth infants.27 for preterm infants , the average number of hospitalizations during the first year was 1.7 , mostly from respiratory issues.28 for term birth infants , hospitalization was calculated as a relative risk reduction.29 cerebral palsy occurred in 0.1% and 1.7% of term and preterm births , respectively.30
resource use and unit costs were identified from published sources and the dhs in brazil . in brazil
the public national health system ( sus ) pays for around 70%75% of all reproductive procedures .
however , in this model , it is assumed that the costs are those from the public system , although , in approximately one - quarter of ups , the costs are probably higher when related to women s care covered by insurance or privately .
costs of elective abortion were not included in the analysis because these procedures rarely meet the legal requirements and would not be covered by the public health system .
costs of miscarriage were related to the following procedures : pelvic exam , blood test , ultrasound , and dilation and curettage .
the cost components for each method included antenatal care and labor and delivery.31,32 unit cost for hospitalization readmission during the first year was r$2,794.33 this unit cost was also assumed for neonatal care admission , while cerebral palsy was estimated to be r$19,854.33 cost parameters are summarized in table 1 .
based on the modeled parameters we estimate 1.79 million annual ups and 1.47 million annual planned pregnancies .
we estimate annual maternal deaths of 351 , of which 49 ( 14% ) are attributed to abortions and 302 to complications from miscarriages and deliveries .
the number of infant deaths within the 12 months following birth was estimated at 32,864 .
the number of estimated neonatal admissions for the year 2010 associated with up was 224,631 which included all preterm deliveries and 7.6% of all term deliveries .
the disaggregated values for different pregnancy outcomes attributed to up are described in table 2 .
costs of abortions are not reimbursed in the public health system and are not included in the analysis .
the total costs attributed to up are estimated to be r$4.1 billion annually , of which approximately r$32.9 million ( 0.8% ) was attributed to miscarriage and r$4.07 billion ( 99.2% ) to births and resulting complications . from the direct birth - related costs , antenatal care accounted for approximately r$233 million ( 5.7% ) of birth costs , with labor and delivery costs responsible for r$988 million ( 24.3% ) .
the remainder of birth costs were attributed to infant complications , estimated to be r$2.8 billion ( 70.0% ) , with neonatal costs accountable for approximately r$628 million ( 15.4% ) and hospital readmission in the first year costs of r$2.1 billion ( 52.9% ) of all birth costs annually . based on the national cost estimates and the number of annual ups
our analysis estimates the costs and outcomes associated with unintended pregnancies to derive a cost per up of r$2,293 .
the cost per up factors in a range of health - related costs attributed to those resulting in abortion and resulting live births for those carried to parturition .
this figure is broadly aligned with previous analyses in the united states of $ 1,609 per up after factoring in the purchasing power parity between the two countries.35 it is also worth noting that the estimates provided here are likely an underestimate of all costs .
for example , the analysis described here focused on the costs that occur within the public health systems .
consequently , costs of elective abortions paid for by individuals are not represented in this analysis .
furthermore , because elective abortion is illegal in brazil except in extreme and rare cases , hidden costs from elective abortions may filter into the public health system .
for instance , costs associated with post - abortive care are likely to be classified as miscarriages when reported to national authorities . additionally , the long - term societal costs that arise from up and reduced educational attainment and lost productivity of young mothers has not been accounted for in our analysis.3639 our analysis highlights that considerable cost savings can be achieved by reducing up , which are thought to represent 55% of all pregnancies in brazil.20 the analysis accounted for both untimed and unwanted births to estimate the up rate and associated costs , in which live births resulting from unwanted pregnancies represented the largest share of health costs , representing 99% of all costs .
this is also attributed to the fact that abortion is a relatively inexpensive procedure that is paid for outside of the health service . because a substantial proportion of pregnancies are attributed to mistimed pregnancies , ie , pregnancies that would have occurred at some point in the future , but occurred
sooner due to mistimed pregnancy , the actual cost savings to be realized by reducing up is less than described here and would mostly be associated with averting those pregnancies considered to be unwanted . to put the potential cost savings into perspective
, an analysis conducted for the state of california , usa estimated that preventing many up in a single year will offer savings of $ 1.1 billion up to 2 years of age of the child , and that every dollar spent on averting up offers $ 2.76 of saving in 2 years and $ 5.33 at 5 years post - delivery.15 a national - level analysis that focused on the federally financed medicaid program in the united states noted that taxpayer savings of $ 4.7$6.2 billion could be achieved by reducing unintended pregnancies.40 based on the cost savings attributed to up , it is anticipated that future health researchers can build on the research described here to better understand the cost savings that may be achieved through improved contraceptive use .
the analysis sought to incorporate a broad range of costs associated with up to illustrate that costs extend beyond the point of delivery .
for instance , ups often occur in adolescent and young adults and can be predictive of low birth weight , preterm birth , and neonatal admission.8,4143 the precise mechanism by which up influences preterm birth is likely to be multifactorial .
however , some studies have observed that women with ups are less likely to receive adequate prenatal care44 and are also more likely to smoke and drink alcohol during pregnancy , which contributes to adverse outcomes.44,45 it is important to highlight these costs because it suggests that ups place unnecessary demand on health services that are often stretched to capacity . since up , for the most part , can be avoided , this illustrates the health service benefits and health service capacity that could be released by reducing up .
much of the humanistic burden attributed to up relates to maternal mortality and morbidity of women who pursue unsafe abortions . in our analysis
, we estimate 49 annual deaths from abortions representing 13% of the estimated maternal deaths .
it has been suggested that access to improved contraceptive services could prevent maternal mortality by 25%35%.46 furthermore , estimates from the world health organization reported that unsafe abortion disables approximately 5 million women each year , suggesting reductions in morbidity could also be achieved through improved contraception use.4 empirical evidence and clinical guidelines agree that the most effective approach to preventing up is through education and contraceptive use , of which long - acting contraceptive ( larc ) methods are the most effective intervention.4751 in particular , among adolescents , prevailing evidence suggests that education and contraception are the main interventions for reducing up.49 in advanced economies , the percentage of women using larc methods has been steadily increasing .
a recent study noted that the proportion of women using larc methods has increased from 2.4% of women aged 1544 years since 2002 to 8.5% of women in 2009.52 in the united states , the american college of obstetricians and gynecologists ( acog ) advocates use of intrauterine contraceptives ( both copper - intrauterine device and the levonorgestrel - releasing intrauterine system ) and implants as first - line therapy for adolescent women because they contribute to reduce the number of ups.48 larc has also proven to be cost - saving compared with combined oral contraceptives in the united kingdom .
investigators reported that this finding was influenced almost entirely by the lower failure rates associated with larc.50,53 an analysis conducted in the united states also noted that cost savings could be achieved by switching women from oral contraceptives to larcs , and approximately 50% of costs of up were attributed to contraceptive adherence.54 despite the positive benefits of larc , use in brazil lags behind that of other countries . a survey conducted in 2008 in northern brazil reported that only 1% of women attending a post - abortion clinic elected for an intrauterine device , despite 93% of women having knowledge of the product.55 several barriers have been identified , which often limit uptake of larc methods.56 these include health care provider lack of knowledge of risks , myths , misconceptions , and lack of training for insertion of the various devices .
also , misinformation and fear of pain at insertion among women can limit demand for larc methods .
furthermore , larc can often involve considerable up - front expense , although evidence does suggest that these methods are cost - effective compared with alternative contraceptive methods .
because of the expense , reimbursement can influence the rate of uptake . even recognizing the importance of abortion , and especially induced and unsafe abortion , for maternal mortality and morbidity in brazil , there are no reliable and available data confirming a pandemic of such conditions as commonly reported by media and gray literature .
data extracted from the 2006 brazilian dhs indicated that only 1.5% self - reported induced abortions among all pregnancies for the whole period of the survey.14,19 a probable contribution for this scenario is the high prevalence of and easy access to contraceptive use among women in reproductive age in the country during the last 2 decades .
although all induced abortions are technically classified as unsafe , this is not the impression of the majority of obstetricians working in the field .
there is a general belief that complications due to unsafe induced abortions are less and less frequent and that there was recently a marked drop in their occurrence . with no direct information to confirm this , one indirect point of evidence is the decrease in the proportional contribution of abortion as cause of maternal mortality in the country .
in fact , official brazilian data confirm that it was 16.4% in 1990 ( representing the third cause of maternal mortality ) , while only 8.9% in 2000 and 9.0% in 2010 ( the fifth cause , behind even the indirect obstetric causes).20 in the meantime , there was a spread of the use of misoprostol as an agent for inducing abortion , not only in health facilities for situations where abortion is allowed , but mainly for situations not legally permitted , wherein the women obtain misoprostol in the black market and self - administer it vaginally .
this is believed to be the current most common way of inducing medical abortion and also to be associated with the low rates of severe complications due to abortion , especially infection and hemorrhage , in brazil .
there are also at least two possible limitations for the external validity of the current cost estimates .
the first refers to the assumption that all miscarriages are really diagnosed and treated with dilation and curettage .
however , it is possible that a proportion of individuals will never reach health facilities and receive no intervention .
currently , in brazil , the majority of medical abortions are performed by the woman through self - administration of misoprostol acquired off - label or in the black market . with these procedures ,
the rate of complications due to medical abortions has dropped significantly during the last decade , with some of these cases arriving at hospitals already as complete abortions that need no complementary procedures .
although insufficient data are available to support this scenario , this would probably impact strongly on the estimates of post - abortion care , taking into account the correspondent reduced morbidity and mortality .
the cost consequences of live births attributed to up in adolescents can extend beyond the observed costs of increased prenatal care , preterm births , and abortions .
studies have noted that adolescent women are more likely to dropout from school following pregnancy .
furthermore , others have suggested that future generations will follow the reproductive pattern of their parents , which could perpetuate the likelihood of up in the offspring of these mothers.57 as reported , education has been shown to break the association of adolescent fertility across generations , suggesting the longer - term benefits to be achieved by reducing up .
our analysis illustrates the cost consequences associated with up in brazil . in the past 2 decades , brazil has made considerable progress toward reducing abortion rates , but , despite these efforts , the humanistic and direct economic costs associated with up remain high.58 to achieve economic savings from reducing the up rate will require education , training of providers , and improved access to effective contraceptive measures .
it is believed that the research described herein can inform the burden of up and the benefits of improved fertility planning .
the cost consequences associated with up in brazil could be investigated in other settings using the same methodology used in this report .
it is important to estimate the cost associated to up to initiate actions to reduce the high rate observed in many countries .
the cost consequences associated with up in brazil could be investigated in other settings using the same methodology used in this report .
it is important to estimate the cost associated to up to initiate actions to reduce the high rate observed in many countries .
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backgroundunintended pregnancy ( up ) is an unmet medical need with consequences worldwide .
we evaluate the costs of up based on pregnancies in brazil from for the year 2010.methodsthe consequences of up were evaluated using decision analysis based on pregnancy rates and outcomes as miscarriage , induced abortion , and live birth , which were factored into the analysis . the model discriminated between maternal and child outcomes and accounted for costs ( in brazilian currency [ real$ , r$ ] ) within the brazilian public health service attributed to preterm birth , neonatal admission , cerebral palsy , and neonatal and maternal mortality .
event probabilities were obtained from local resources.resultswe estimate that 1.8 million ups resulted in 159,151 miscarriages , 48,769 induced abortions , 1.58 million live births , and 312 maternal deaths , including ten ( 3% ) attributed to unsafe abortions .
the total estimated costs attributed to up are r$4.1 billion annually , including r$32 million ( 0.8% ) and r$4.07 billion ( 99.2% ) attributed to miscarriages and births and complications , respectively .
direct birth costs accounted for approximately r$1.22 billion ( 30.0% ) , with labor and delivery responsible for most costs ( r$988 million ; 24.3% ) for the year 2010 .
the remainder of costs were for infant complications ( r$2.84 billion ; 72.3% ) with hospital readmission during the first year accounting for approximately r$2.15 billion ( 52.9% ) .
based on the national cost , we estimate the cost per up to be r$2,293.conclusiondespite weaknesses in precise estimates in annual pregnancies and induced abortions , our estimates reflect the costs of up for different pregnancy outcomes .
the main costs associated with up are in those carried to parturition .
the health cost of abortion represents a small proportion of total costs as these are paid for outside of the public health system .
consequently , reductions in up will generate not only cost savings , but reductions in woman and child morbidity and mortality .
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Introduction
Methods
Model description
Data source and analysis
Cost resources
Results
Discussion
Conclusion
Implications
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approximately 30% of women have sustained at least 1 vertebral fracture by the age of 75 .
these fractures are an important and common cause of morbidity in osteoporotic patients ; moreover , fractures evidenced both clinically and at radiographic examination are associated with an increased mortality rate .
back pain owing to vertebral fractures has a significant affect on osteoporotic patients [ 46 ] , and the number and severity of these fractures also increases the risk of developing chronic back pain .
this has a marked negative impact on the quality of life ( qol ) and functional impairment of the affected patients .
conventional treatments for osteoporosis including bisphosphonates , selective estrogen receptor modulators ( serms ) , and estrogen have been shown to reduce the rate of bone resorption and to preserve bone mass .
another therapeutic option includes rhpth , an agent that has been shown to increase both bone mass and bone strength .
when injected , teriparatide ( rdna origin ) , the amino - terminal fragment of human pth ( rhpth 134 ) , is a potent bone formation agent for the treatment of severe osteoporosis .
it increases osteoblast production / growth and prevents osteoblast apoptosis ; at the same time , it enhances absorption of calcium from the intestine , renal reabsorption of calcium , while decreasing the excretion of phosphates in the kidney [ 1114 ] .
when administered once daily by subcutaneous injection , rhpth increases bone density and improves trabecular architecture , cortical geometry , and strength .
not only does rhpth increase trabecular bone density by stimulating bone formation , but it also stimulates osteoclastic bone resorption [ 1719 ] . in several studies , rhpth has been shown to increase bone mineral density in postmenopausal osteoporosis , in senile osteoporosis in men , and in glucocorticoid - induced osteoporosis .
teriparatide acts via the pth-1 receptor on osteoblasts and bone marrow stromal cells to induce osteoblastic bone formation , that is , osteoid synthesis and accelerated mineralization .
this , in turn , results in reductions in skeletal fractures to levels equivalent to , or over , those obtained by using antiresorptive agents .
the increase in bone mineral density induced by rhpth is substantial , ranging 10% to 15% over 2 to 3 years in most studies [ 10,2024 ] . moreover , rhpth can cause demonstrable increases in bone mineral density and changes in markers of bone turnover within 3 months since the start of treatment .
quality of life ( qol ) can be measured to compare the effect of different treatments for osteoporosis .
measuring pain scores only for these patients would be inadequate , because apart from acute and chronic back pain , patients with vertebral fractures also experience anxiety , constant fear of falling , or suffering another fracture while their daily living activities are impaired .
most information has been collected thanks to the efforts made by some researchers to develop specific instruments to test the physical and emotional disability generated by the disease .
generic tools available for measuring qol are useful to evaluate general health but they lack disease specificity .
more recently , some specific instruments have been developed to measure the qol in osteoporosis more accurately .
the most widely used are the osteoporosis quality of life questionnaire ( oqlq ) , the osteoporosis assessment questionnaire ( opaq ) , the osteoporosis - targeted quality of life questionnaire ( optqol ) , and the quality of life questionnaire of the european foundation for osteoporosis ( qualeffo-41 ) .
one of the first ones was qualeffo-41 , which has been translated and validated in different languages including italian .
this questionnaire has proved to be repeatable , coherent , and able to discriminate between patients and controls . in the last few years , other specific questionnaires
have been developed , but not all of them have been as extensively used and validated in different countries as qualeffo-41 .
the goal of this study was firstly to assess the validity of rhpth treatment in a cohort of postmenopausal women with severe osteoporosis ; secondly , to evaluate the improvement in qol and pain symptoms after several months of rhpth therapy .
a follow - up qualeffo-41 questionnaire was used to quantify the patient s pain and the affect on qol after rhpth therapy .
the study was a 18 months , randomized prospective cohort study conducted at department of molecular and clinical endocrinology and oncology , university of naples federico ii , naples , italy .
inclusion criteria for this study consisted of back pain , postmenopausal osteoporosis ( t - score 2.5 at lumbar spine or femoral neck ) , the presence of 2 osteoporotic vertebral fractures , previous treatment for osteoporosis .
the exclusion criteria were : an increased risk of osteosarcoma ( ie , patients with paget disease bone , previous skeletal exposure to external beam radiotherapy , or previous malignant neoplasm involving the skeleton ) , hypercalcemia , malignant neoplasms , impaired renal function , liver disease , history of diseases other than postmenopausal osteoporosis that affect bone metabolism , nephrolithiasis , alcohol or drug abuse .
secondary osteoporosis was excluded in order to avoid the interference of the primitive disease with the patient s quality of life .
informed consent was obtained from all subjects and the study protocol was approved by the hospital / science s ethical committee .
eighty - one postmenopausal women were enrolled and divided in two groups with no statistically significant differences in any of the considered variables : group a forty - two women ( mean age 659 yrs ; mean body mass index
bmi 24.52.6 kg / m ) , with severe postmenopausal osteoporosis ( mean lumbar bmd 3.880.70 , mean femoral neck bmd 3.070.60 and with 2 vertebral atraumatic fractures ) , persistent back pain and previous treatment with biphosphonates for osteoporosis ; group b thirty - nine women matched for age ( 6014.4 yrs ) , bmi ( 22.88.8 kg / m ) , menopausal status , affected by back pain , severe postmenopausal osteoporosis ( lumbar spine bmd 3.900.73 , mean femoral neck bmd 3.020.61 and with 2 vertebral atraumatic fractures ) previously treated for osteoporosis with biphosphonates .
bmi , age at menopause , lifestyle habits ( i.e. , smoking , drinking , nutrition style ) , nutrition anamnesis ( calcium intake ) , history of diseases other than osteoporosis , family history of osteoporosis were considered .
groups were randomized to daily treatment with 20 g s.c . of recombinant human parathyroid hormone ( rhpth 134 ) , self - administered injections ( group a ) or 70 mg per os of alendronate every week ( group b ) .
all women received 1,000 mg elemental calcium daily and 800 iu of vitamin d daily for 18 months .
biochemical markers of bone turnover were dosed in all the selected female patients : alkaline phosphatase ( alp : 35104
u / l ) , n - terminal propeptide of type i procollagen ( pinp : 19102 g / l ) , n - telopeptide crosslinks ( ntx : 565 nmol / mmol crea ) were assessed at baseline , 3 , 12 and 18 months ( t0 , t3 , t12 , t18 ) .
the bmd of the lumbar spine and the proximal femur was measured by dual - energy x - ray absorptiometry ( dual - energy x - ray absorptiometry qdr 1000 ; hologic , waltham , ma , usa ) at baseline and at 18 months ( t0 , t18 ) .
all women underwent anteroposterior and lateral radiography of thoracic and lumbar spine at t0 and t18 .
the qol questionnaire of the european foundation for osteoporosis ( qualeffo ) was administered at baseline and at the end of the study to evaluate the impact of rhpth on health - related qol .
originally , the questionnaire consisted of 48 questions and 6 visual analogue scales . in the qualeffo validation study ,
this resulted in the qualeffo-41 , which consisted of 41 questions in 5 domains : pain , physical function , social function , general health perception , and mental function .
all answers were recorded so that all items range from 1 to 5 , and all answers were standardized so that 1 represents the best and 5 the worst qol , with the exception of questions e23 - 25 ( questions with 3 answer options ) , questions e24 - 26 - 27 - 28 ( 4 answer options ) , and questions g33 - 34 - 35 - 37 - 39 - 40 ( answer with reverse scores : 1 is the worst while 5 is the best ) .
domain scores are calculated by averaging the answers of 1 domain and transforming the scores to a score from 0 to 100 .
data are expressed as mean sd or percentage ; moreover , to assess the affect of teriparatide treatment on markers of bone turnover , bone mineral density , and on health - related qol , pearson correlation coefficients were computed in the 2 study groups .
therefore , percentage changes of biomarkers , bone mineral density , and qualeffo results were calculated .
there was no significant difference in these characteristics between the 2 groups . in the group a , 39 ( 93% ) out of the 42 recruited women completed the study : 2 patients discontinued the drug therapy because of lack of compliance with the study treatment ; 1 patient discontinued because of a new vertebral atraumatic fracture .
the aim of this study was to evaluate percentage changes from baseline in biochemical markers of bone turnover , values of bone mineral density ( measured at lumbar spine and proximal femur ) , and measurements of qol .
follow - up checks , where we also evaluated clinical conditions , adverse events , compliance to treatment and use of non - steroid anti - inflammatory drugs , were carried out at 3 , 12 and 18 months since the beginning of treatment . in group a serum levels of pinp increased of 90% , 145% and 127% at t3 , t12 , t18 , respectively ; bone alp levels increased of 57% , 79% and 65% ; ntx levels increased of 53% at t3 , of 100% at t12 , of 110% at t18 . in group
b percentage changes from baseline of serum levels of pinp were 50% , 70% and 74% at t3 , t12 , t18 , respectively ; bone alp levels decreased of 30% , 48% and 41% ; ntx levels were reduced by 55% at t3 , of 69% at t12 , of 72% at t18 .
mean percentage changes from baseline are shown over time for all 3 biochemical markers in figures 1 and 2 .
mean pinp values were 426 g / l at t0 , 8012 g / l at t3 , 10334 g / l at t12 , 9526 g / l at t18 in group a ( t0 vs t3 r : 0.81 , p<0.001 ; t0 vs t12 r : 0.88 , p<0.001 ; t0 vs t18 r : 0.86 , p<0.001 ) and
7816 g / l , 395 g / l , 2313 g / l , 208 g / l at t0 , t3 , t12 , t18 respectively ( t0 vs t3 r : 0.67 , p<0.001 ; t0 vs t12 r : 0.76 , p<0.001 ; t0 vs t18 r : 0.82 , p<0.001 ) . in group
u / l , 10720 u / l at t3 , 12245 u / l at t12 and
11235 u / l at t18 ( t0 vs t3 r : 0.72 , p<0.001 ; t0 vs t12 r : 0.46 , p<0.01 ; t0 vs t18 r : 0.85 , p<0.001 ) ; instead , in group b mean alp was 72 15 u / l at baseline , 5015 after 3 months , 3717 u / l at t12 and 427 u / l at the end of the study ( t0 vs t3 r : 0.91 , p<0.001 ; t0 vs t12 r : 0.53 , p<0.001 ; t0 vs t18 r : 0.65 , p<0.001 ) .
ntx mean values in group a were 317 nmol / mmol crea , 4721 nmol / mmol crea , 6211 nmol / mmol crea , 6523 nmol / mmol crea at t0 , t3 , t12 and t18 respectively ( t0 vs t3 r : 0.22 , p>0.1 ; t0 vs t12 r : 0.57 , p<0.001 ; t0 vs t18 r : 0.49 , p<0.01 ) while in group b ntx mean values were 534 nmol / mmol crea at t0 , 29 11 nmol / mmol crea at t3 , 2013 nmol / mmol crea at t12 and 158 nmol / mmol crea at t18 ( t0 vs t3 r : 0.74 , p<0.001 ; t0 vs t12 r : 0.58 , p<0.001 ; t0 vs t18 r : 0.69 , p<0.001 ) . by the end of the study period ,
the bmd values expressed in terms of t - score in our total pool displayed important changes ( figure 3 ) . at month 18 ,
lumbar spine bmd increased by 12.4% in group a compared with group b in which it increased by 3.85% .
specifically , in group a mean t - score at t0 was 3.870.71 and mean t - score at t18 was 3.390.72 ( r : 0.88 ; p<0.001 ) ; instead , in group b , mean t - score at t0 was 3.900.73 and mean t - score at t18 : 3.750.72 ( r : 0.98 ; p<0.001 ) .
the bmd in the femur increased from baseline at month 18 , in group a , by 5.2% and by 1.99% in group b. in group a , mean femoral neck t - score was 3.070.60 at baseline and mean t - score at t18 was 2.910.63 at the end of the study ( r : 0.87 ; p<0.001 ) ; in group b , mean femoral t - score was 3.020.61 at t0 and 2.960.64 at t18 ( r : 0.99 ; p<0.001 ) . at t0 ,
patients treated with teriparatide were more protected against new fractures , compared with patients treated with bisphosphonates ; in fact , only 1 new vertebral fracture occurred in group a ( 2.4% ) at study endpoint ( t18 ) vs 6 new vertebral fractures that occurred in group b ( 15.7% ) at t18 .
the most - common reported adverse effects were back pain worsened in the first month of treatment that was reported by 14% of women ; nausea , reported by 10% ; headache and dizziness that were reported by only 1 and 2 women .
the reported adverse effects were abdominal pain in 9 patients , arthralgia in 4 patients , and dyspepsia in 1 patient .
we evaluated the teriparatide impact on several aspects of qol by administering the patients qualeffo-41 test at t0 and t18 ( figure 4 ) .
first domain ( domain a ) result indicated a serious reduction in pain ( 22% ) after treatment with rhpth : mean scores measured was 729.2 at start and 5614 after 18 months ( r : 0.61 ; p<.001 ) compared with group b ( 9.7% ; 718.7 at t0 and 6511 at t18 ; r : 0.53 ; p<.001 ) .
for everyday activities ( domain b ) , an average of 5513.6 was measured at t0 and of 4022.9 at t18
( r : 0.44 ; p<.01 ) in group a which had a total improvement of 27.3% , while the improvement was of 11% in group b ( 61.118 at t0 and 54.315.7 at t18 ; r : 0.68 ; p<.001 ) .
the performed domestic job domain ( domain c ) showed an improvement of 29% in group a ( 6010.7 at t0 ; 42.613.3 at t18 ; r : 0.68 ; p<.001 ) and of 2.9% in group b ( 62.113.3 at t0 ; 60.313.7 at t18 ; r : 0.88 ; p<.001 ) .
the mean score of domain d ( locomotor function ) was of 48.410 at baseline and 30.214.3 at the end of the study that indicates a percentage change of 37.8% in group a compared to group b where the change was of 11.5% ( 46.88.6 at t0 ; 41.410.05 at t18 ; r : 0.69 ; p<.001 ) .
the quality of free time and of the social activities ( domain e ) at the 2 time points they were 36.28.6 and 25.98.6 , indicating a percentage change of 28.4% in group a ( r : 0.50 ; p<.01 ) ; the values of group b were 38.214 at t0 and 34.214.3 at t18 reaching a percentage change of 10.5% only ( r : 0.87 ; p<.001 ) .
patients taking teriparatide showed also an improvement of 33.9% in the self - perception of their health ( domain f ) ( 5925 at t0 ; 3916.7 at t18 ; r : 0.84 ; p<.001 ) versus 12.8% improvement of group b ( 63.228.2 at t0 ; 55.124.8 at t18 ; r : 0.90 ; p<.001 ) . in the mood domain ( domain g ) ,
the qualeffo - test revealed a mean value of 20.67.2 at t0 and 29.39.7 at t18 in group a ( r : 0.71 ; p<.001 ) .
these data demonstrated a considerable improvement ( 29.7% ) compared with group b ( 1.8% ; 32.59.8 at t0 ; 33.19.9 at t18 ; r : 0.18 ; p>.1 ) .
as a consequence of pain relief , the consumption of nonsteroidal anti - inflammatory drugs also decreased in 29 women of group a , while it did not decrease in group b.
in group a serum levels of pinp increased of 90% , 145% and 127% at t3 , t12 , t18 , respectively ; bone alp levels increased of 57% , 79% and 65% ; ntx levels increased of 53% at t3 , of 100% at t12 , of 110% at t18 . in group
b percentage changes from baseline of serum levels of pinp were 50% , 70% and 74% at t3 , t12 , t18 , respectively ; bone alp levels decreased of 30% , 48% and 41% ; ntx levels were reduced by 55% at t3 , of 69% at t12 , of 72% at t18 .
mean percentage changes from baseline are shown over time for all 3 biochemical markers in figures 1 and 2 .
mean pinp values were 426 g / l at t0 , 8012 g / l at t3 , 10334 g / l at t12 , 9526 g / l at t18 in group a ( t0 vs t3 r : 0.81 , p<0.001 ; t0 vs t12 r : 0.88 , p<0.001 ; t0 vs t18 r : 0.86 , p<0.001 ) and
7816 g / l , 395 g / l , 2313 g / l , 208 g / l at t0 , t3 , t12 , t18 respectively ( t0 vs t3 r : 0.67 , p<0.001 ; t0 vs t12 r : 0.76 , p<0.001 ; t0 vs t18 r : 0.82 , p<0.001 ) .
u / l , 10720 u / l at t3 , 12245 u / l at t12 and
11235 u / l at t18 ( t0 vs t3 r : 0.72 , p<0.001 ; t0 vs t12 r : 0.46 , p<0.01 ; t0 vs t18 r : 0.85 , p<0.001 ) ; instead , in group b mean alp was 72 15 u / l at baseline , 5015 after 3 months , 3717 u / l at t12 and 427 u / l at the end of the study ( t0 vs t3 r : 0.91 , p<0.001 ; t0 vs t12 r : 0.53 , p<0.001 ; t0 vs t18 r : 0.65 , p<0.001 ) .
ntx mean values in group a were 317 nmol / mmol crea , 4721 nmol / mmol crea , 6211 nmol / mmol crea , 6523 nmol / mmol crea at t0 , t3 , t12 and t18 respectively ( t0 vs t3 r : 0.22 , p>0.1 ; t0 vs t12 r : 0.57 , p<0.001 ; t0 vs t18 r : 0.49 , p<0.01 ) while in group b ntx mean values were 534 nmol / mmol crea at t0 , 29 11 nmol / mmol crea at t3 , 2013 nmol / mmol crea at t12 and 158 nmol / mmol crea at t18 ( t0 vs t3 r : 0.74 , p<0.001 ; t0 vs t12 r : 0.58 , p<0.001 ; t0 vs t18 r : 0.69 , p<0.001 ) .
by the end of the study period , the bmd values expressed in terms of t - score in our total pool displayed important changes ( figure 3 ) . at month 18 ,
lumbar spine bmd increased by 12.4% in group a compared with group b in which it increased by 3.85% .
specifically , in group a mean t - score at t0 was 3.870.71 and mean t - score at t18 was 3.390.72 ( r : 0.88 ; p<0.001 ) ; instead , in group b , mean t - score at t0 was 3.900.73 and mean t - score at t18 : 3.750.72 ( r : 0.98 ; p<0.001 ) .
the bmd in the femur increased from baseline at month 18 , in group a , by 5.2% and by 1.99% in group b. in group a , mean femoral neck t - score was 3.070.60 at baseline and mean t - score at t18 was 2.910.63 at the end of the study ( r : 0.87 ; p<0.001 ) ; in group b , mean femoral t - score was 3.020.61 at t0 and 2.960.64 at t18 ( r : 0.99 ; p<0.001 ) .
at t0 , all patients of groups a and b showed baseline vertebral fractures . patients treated with teriparatide were more protected against new fractures , compared with patients treated with bisphosphonates ; in fact , only 1 new vertebral fracture occurred in group a ( 2.4% ) at study endpoint ( t18 ) vs 6 new vertebral fractures that occurred in group b ( 15.7% ) at t18 .
the most - common reported adverse effects were back pain worsened in the first month of treatment that was reported by 14% of women ; nausea , reported by 10% ; headache and dizziness that were reported by only 1 and 2 women .
the reported adverse effects were abdominal pain in 9 patients , arthralgia in 4 patients , and dyspepsia in 1 patient .
we evaluated the teriparatide impact on several aspects of qol by administering the patients qualeffo-41 test at t0 and t18 ( figure 4 ) . first domain ( domain
a ) result indicated a serious reduction in pain ( 22% ) after treatment with rhpth : mean scores measured was 729.2 at start and 5614 after 18 months ( r : 0.61 ; p<.001 ) compared with group b ( 9.7% ; 718.7 at t0 and 6511 at t18 ; r : 0.53 ; p<.001 ) . for everyday activities ( domain b ) , an average of 5513.6 was measured at t0 and of 4022.9 at t18 ( r : 0.44 ; p<.01 ) in group a which had a total improvement of 27.3% , while the improvement was of 11% in group b ( 61.118 at t0 and 54.315.7 at t18 ; r : 0.68 ; p<.001 ) .
the performed domestic job domain ( domain c ) showed an improvement of 29% in group a ( 6010.7 at t0 ; 42.613.3 at t18 ; r : 0.68 ; p<.001 ) and of 2.9% in group b ( 62.113.3 at t0 ; 60.313.7 at t18 ; r : 0.88 ; p<.001 ) .
the mean score of domain d ( locomotor function ) was of 48.410 at baseline and 30.214.3 at the end of the study that indicates a percentage change of 37.8% in group a compared to group b where the change was of 11.5% ( 46.88.6 at t0 ; 41.410.05 at t18 ; r : 0.69 ; p<.001 ) .
the quality of free time and of the social activities ( domain e ) at the 2 time points they were 36.28.6 and 25.98.6 , indicating a percentage change of 28.4% in group a ( r : 0.50 ; p<.01 ) ; the values of group b were 38.214 at t0 and 34.214.3 at t18 reaching a percentage change of 10.5% only ( r : 0.87 ; p<.001 ) .
patients taking teriparatide showed also an improvement of 33.9% in the self - perception of their health ( domain f ) ( 5925 at t0 ; 3916.7 at t18 ; r : 0.84 ; p<.001 ) versus 12.8% improvement of group b ( 63.228.2 at t0 ; 55.124.8 at t18 ; r : 0.90 ; p<.001 ) . in the mood domain ( domain g ) , the qualeffo - test revealed a mean value of 20.67.2 at t0 and 29.39.7 at t18 in group a ( r : 0.71 ; p<.001 ) .
these data demonstrated a considerable improvement ( 29.7% ) compared with group b ( 1.8% ; 32.59.8 at t0 ; 33.19.9 at t18 ; r : 0.18 ; p>.1 ) .
as a consequence of pain relief , the consumption of nonsteroidal anti - inflammatory drugs also decreased in 29 women of group a , while it did not decrease in group b.
our results demonstrate that daily injections of rhpth for 18 months are an efficacious and generally well - tolerated therapy in postmenopausal women with severe osteoporosis . more importantly , in our experience rhpth therapy results in decreased fractures and pain symptoms in patients with osteoporosis , and
teriparatide is a bone - forming agent and its effect is demonstrated by increases in biochemical markers of bone turnover over the study period : bone formation markers showed more rapid and higher increases than resorption ones , suggesting an early imbalance of bone turnover in favor of formation ; these data are in agreement with previous studies .
treatment with teriparatide resulted in a greater increase in bone mineral density ( bmd ) .
after 18 months of therapy with rhpth , bone mineral density in the lumbar spine and of the proximal femur increased by 12.4% and 5.2% ; these reported percentage increases are consistent with results of other studies . at month 18 in patients treated with bisphosphonates , instead , lumbar spine increased only by 3.85% and the bone mineral density in the femur increased by 1.99% .
the differences in the percentage increases between the 2 groups of patients can be explained with substantial differences between antiresorptive and anabolic therapeutic effects on bone mass and architecture as well as on bone mineral density .
moreover , rhpth increases trabecular connectivity , whereas the majority of bone mineral density increases observed with bisphosphonate treatment are a result of increased mineralization of existing bone matrix .
teriparatide has been referred to be an efficacious treatment against new fractures , making a significant change in the course of severe osteoporosis , which can lead rapidly not only to varying degrees of disability , the loss of self - sufficiency , and to institutionalization , but also to death [ 3436 ] . in this study , we have confirmed that patients with osteoporosis treated with teriparatide experience improvements in pain symptoms .
in addition , use of teriparatide caused a considerable decrease of pain , accompanied by a consequent reduction of the need for nonsteroidal anti - inflammatory drugs by the patients ; this was the main factor that assured an absolute compliance of the patients .
in severe osteoporosis vertebral fractures are an important cause of back pain owing to muscle weakness and altered posture , resulting in serious acute and chronic pain that contributes to further disabilities ; in fact , vertebral fractures are the top health condition accounting for length of hospitalization , and added significantly to the length of hospitalization in patients admitted for other medical problems . apart from the physical disabilities had by these patients
in fact , patients with vertebral fractures often experience impaired physical functions , limited activities of daily living , limited leisure and recreational activities , and significant emotional distress with loss of self - esteem and depression [ 3436 ] . in this study
, we have confirmed that patients with osteoporosis treated with teriparatide experience improvements in pain symptoms . to analyze all these variables and to measure the values of qol at baseline and at the end of the study , we used the qualeffo - test , developed by lips and associates in 1997 .
specifically , we used qualeffo-41 , that represents a qol questionnaire which is brief , easy to administer , and with adequate preliminary psychometric properties .
we preferred this test to the generic ones ( such as nhp , sip , sf-36 , eq - sd ) because of its specificity in the evaluation of qol in people affected by osteoporosis with vertebral deformities ; in fact , disadvantages of generic instruments are that they usually include irrelevant questions and that these questionnaires are less able to investigate those aspects that mostly influence qol of osteoporotic patients [ 2628 ] .
this makes the qualeffo-41 potentially useful during routine clinical practice and research for the treatment and the follow - up of postmenopausal women with severe osteoporosis . by using this test
, our study evidences that the use of rhpth influences all considered domains of qol ; in fact , pain , physical and social functions , mood significantly improved in nearly all patients treated with rhpth at the end of the study .
thus , the reduction of back pain observed from baseline of active treatment through posttreatment follow - up is consistent with the reduction of new vertebral painful fractures , that means improved qol for this kind of patients .
our data clearly demonstrate that rhpth is more effective than bisphosphonates in acting on back pain and all the domains of qol .
adverse events ( back pain , headache , nausea , dizziness ) in our patients receiving rhpth were mild and transient , with a percentage lower than in previous studies .
the adverse effects attributed to teriparatide did not stop the patients from continuing with the treatment .
our results demonstrate that rhpth increases bone mineral density considerably , reduces the occurrence of new fractures , and the need for analgesic therapy .
therefore , this study evidences that this anabolic agent represents a valid therapeutic option in severe postmenopausal osteoporosis ; moreover , patients after this treatment experience , improvements not only in pain relief , but also in certain aspects of emotional functioning , activities of daily living , and leisure activities , improve qol considerably .
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summarybackgroundwe studied the use of teriparatide in postmenopausal women with severe osteoporosis.material/methodstwo groups ( a and b ) of patients affected by severe osteoporosis ( t - score 2.5 at bone mineral density were analyzed and 2 vertebral fractures on radiograph).group a was treated for 18 months with 20 g / day of teriparatide .
group b was treated with bisphosphonates 70 mg / week .
every woman assumed 1 g of calcium and 800 iu of vitamin d3 daily .
we evaluated the effects of therapy after 18 months ( t18 ) from the beginning with bone turnover markers ( alkaline phosphatase , procollagen type 1 n - terminal propeptide , and n - telopeptide cross - links ) and dual - energy x - ray absorptiometry.resultsgroup a , at t18 procollagen type 1 n - terminal propeptide levels , increased 127% ; bone alkaline phosphatase levels increased to 65% ; n - telopeptide cross - links levels increased to 110%.group b , at t18 procollagen type 1 n - terminal propeptide levels , decreased to 74% ; bone alkaline phosphatase levels decreased to 41% ; n - telopeptide cross - links levels decreased to 72%.after 18 months , lumbar bone mineral density increased to 12.4% and femoral bone mineral density increased to 5.2% in group a. group b lumbar bone mineral density increased to 3.85% and femoral bone mineral density increased to 1.99% .
only a new vertebral fracture occurred in group a ( 2.4% ) , whereas 6 fractures occurred in group b ( 15.7%).the quality of life questionnaire of the european foundation for osteoporosis ( qualeffo ) revealed a significant improvement in daily living , performed domestic jobs , and locomotor function in groups a and b.conclusionsthe use of rhpth in patients with severe osteoporosis offers more protection against fractures and improves the qol more than bisphosphonates .
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Background
Material and Methods
Results
Markers of bone turnover
Bone mineral density
X-ray evaluation
Adverse events
Quality of life
Discussion
Conclusions
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