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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 .
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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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