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neocortical epilepsy has comprised only a minor portion of epilepsy surgical series until now.1 it has not homogenous clinical manifestations .
widely different seizure semiologies could be found depending on the location of the epileptogenic foci .
scalp eeg often misleads or falsely localizes the ictal onset zone due to inaccessible location or widespread ictal onset .
a focal structural lesion on mri is a reliable indicator of the seizure onset.24 concordance of electrophysiological studies and mri findings has high predictive value for good surgical outcome.5,6 however , mri is ineffective in many partial epilepsy patients , even patients with cortical dysplasia .
surgical outcome has usually been known to be less well satisfactory.1,7,8 in the context of all these issues , determining the prognostic factors for good surgical outcome and suggesting the guideline of successful epilepsy surgery are very important for the this special group of patients .
extratemporal resections are known to be generally less successful than are temporal lobe resections . by multivariate analysis ,
the median proportion of long - term seizure - free patients ( more than five years ) was 66% with temporal lobe epilepsy ( tle ) , 46% with occipital and parietal lobe epilepsies ( ole and ple ) , and 27% with frontal lobe epilepsy ( fle).9 however , the recent series suggested that many neocortical epilepsy patients are likely to benefit from surgical treatment , and 57.5% became seizure free more than two years after surgery ( 41.0% in fle , 71.2% in neocortical tle , 57.5% in ple , and 68.1% in ole).10 it has been a consistent finding that the presence of focal mri lesion indicated good surgical outcome in epilepsy surgery including temporal and extratemporal lobe epilepsies.1,1114 the seizure free rate ranged from 20 to 50% in nonlesional epilepsy depending on the lobes ( table 1 ) .
a meta - analysis to assess predictors of epilepsy surgery found out that a good outcome was related with presence of febrile convulsion , mesial temporal sclerosis , tumors , abnormal mri , eeg / mri concordance , and extensive surgical resection.15 the resection of an epileptognic lesion with an ictal onset zone is recognized as the most important factor for a good surgical outcome .
the majority of surgical series suggested that the identification of a specific lesion usually leads to a favorable outcome.24 our recent study using multivariate analysis of a large series of patients also showed that a focal lesion on mri , focal hypometabolism on pet , and localized ictal onset on eeg were independent significant positive prognostic factors.10 in another large series of fle , mri - negative malformation of cortical development , any extrafrontal mri abnormality , generalized / non - localized ictal eeg patterns , occurrence of acute post - operative seizures , and incomplete surgical resection were correlated with poor surgical outcome.16 mri is also an important factor to make the patients have epilepsy surgery after presurgical evaluations . up to 30% of patients who underwent presurgical evaluations for respective surgery
ultimately did not have this form of surgery.17 localized mri abnormality and consistently localized eeg findings were most strongly associated with leading to surgery.17,18
there is inherent difficulty in identifying the epileptogenic zone in nonlesional neocortical epilepsy , which leads to the incomplete resection . until recently ,
only a minority of patients have been known to be seizure free after the resection .
however , the seizure free rate of the patients even with normal mri who underwent surgery was significantly superior to that of the patients who did not have surgery.18 seizure free outcome ranged from 31 to 70% for nonlesional temporal lobe epilepsy , and from 17% to 57% for extratemporal nonlesional epilepsy ( table 2).1924 two recent large studies including ours demonstrated that seizure free outcomes were 47 and 55% for nonlesional tle , and 41 and 43% for nonlesional extratemporal lobe epilepsy patients.23,24 our study also showed that localization by fdg - pet or interictal eeg was correlated with a seizure - free outcome .
concordance with two or more presurgical evaluations among interictal eeg , ictal eeg , fdg - pet , and ictal spect was significantly related to a seizure - free outcome.24 as for long - term outcome of patients with mri negative tle , only non - congruent fdg - pet results could be identified to be associated with poor surgical outcome.25 in another study with the tle patients with normal mri , the absence of contralateral or extratemporal interictal spikes and concordant subtraction ictal spect were associated with good surgical outcome.26 with careful interpretation of other studies including functional neuroimaging and the presence of concordant results , surgical treatment can benefit selected patients with nonlesional neocortical epilepsy .
ictal spect and fdg - pet can be critical for localizing the seizure focus , especially in nonlesional patients .
we analyzed 33 consecutive patients with nonlesional neocortical epilepsy who had a scalp ictal onset zone localized in the temporal lobe and good surgical outcome after focal neocortical resection.27 epileptogenic zone outside the temporal lobe was not infrequently encountered in the patients who were diagnosed as non - lesional lateral temporal lobe epilepsy on long - term scalp video - eeg monitoring .
some studies demonstrated that fdg - pet and subtraction spect were valuable in the diagnosis of non - lesional neocortical epilepsy.2833 however , in the patients with intracranial ictal onset zones outside the temporal lobe , their values were limited .
fdg - pet and subtraction spect had localizing value in no more than half of patients , which even showed false localizations occasionally .
careful placement of intracranial electrodes on the presumed epileptogenic zone and adjacent areas should be needed for these patients .
accurate localization of ictal onset zone by intracranial electrodes is one of the most sensitive and important method in the success of epilepsy surgery.3437 in the presurgical evaluation of non - lesional neocortical epilepsy , intracranial monitoring is indispensable but has the limitation of the possibility of insufficient sampling .
one recent study showed that stereo - eeg was equally effective in the presurgical evaluation of both mri - negative and lesional epilepsies .
38 to get the appropriate information from intracranial monitoring , many intracranial electrodes may be needed .
nevertheless , because these electrodes cover only limited portion of brain , the true ictal onset zone can sometimes be missed3941 and sometimes the repositioning or additional electrode can be needed . to avoid missing true ictal osnet zone , it is important to make strong hypothesis derived from congruent results of presurgical evaluation . sometimes , the repositioning or adding intracranial electrodes is helpful to find the true ictal onset zone after the failure of identifying it at the initial evaluation .
our group recruited 18 cases underwent a second invasive study consisting of repositioning or additional implantation of intracranial electrodes performed a week after the initial invasive study.42 the repositioning of intracranial electrodes identified a new ictal onset zone in 13 patients . in another four cases
seven of 11 patients who were ultimately found to have focal ictal onset zone by the second evaluation became seizure free after the operation .
these results support consideration of one - week interval repositioning of intracranial electrodes in selected patients .
the objectives of intracranial eeg are to define interictal abnormalities and the ictal onset zone , and to map the cortical function . based on these results
the surgical outcome depends strongly on the identification and complete resection of a well - defined epileptogenic zone .
therefore , the extent of the resection may contribute to the surgical outcome.43 intracranial eegs were analyzed in 177 consecutive patients who had undergone resective epileptic surgery.44 based on the results of invasive evaluations , slow propagation and focal or regional ictal onset were associated with a seizure - free outcome .
a seizure - free outcome was significantly associated with a resection that included the area showing ictal spreading rhythm during the first three seconds or included all the electrodes showing pathological delta waves or frequent interictal spikes more than 0.2 hz .
presurgical evaluations other than mri including functional neuroimagings can correctly localize the epileptogenic lobe in these patients .
we should know the usefulness and limitations of various presurgical diagnostic modalities and their meanings in combination including intracranial eegs .
the strength of hypothesis based on the results of non - invasive evaluation is a key to successful epilepsy surgery for these patients . | there is inherent difficulty in identifying the epileptogenic zone in nonlesional neocortical epilepsy , which leads to the incomplete resection .
however , with careful interpretation of other studies including functional neuroimaging and the presence of concordant results , surgical treatment can benefit selected patients with nonlesional neocortical epilepsy .
two recent large studies including ours demonstrated that seizure free outcomes were 47 and 55% for nonlesional tle , and 41 and 43% for nonlesional extratemporal lobe epilepsy patients .
concordance with two or more presurgical evaluations among interictal eeg , ictal eeg , fdg - pet , and ictal spect was significantly related to a seizure - free outcome .
however , we should be cautious to the possibility of false localization of ictal eeg or functional neuroimaging in nonlesional neocortical epilepsy .
careful placement of intracranial electrodes on the presumed epileptogenic zone and the adjacent areas should be needed for these patients .
the repositioning of intracranial electrodes after the failure in identifying ictal onset zone at the initial intracranial study might identify a new ictal onset zone .
consideration of one - week interval repositioning of intracranial electrodes could be helpful in selected patients .
intracranial eeg is one of the most important procedures in planning surgery and achieving a good surgical outcome in resective epilepsy surgery .
slow propagation and focal or regional ictal onset rather than widespread onset were associated with a seizure - free outcome .
complete resection including the area with initial three second ictal rhythm and interictal abnormalities predicts a good surgical outcome . |
fresh heparinized blood samples were obtained from 50 patients with newly diagnosed type 1 diabetes ( age range 1843 years ) ( duration of type 1 diabetes 20 weeks ) and 30 healthy control subjects ( age range 2048 years ; summarized in table 1 and detailed in supplementary table 1 ) .
these studies were carried out with the approval of the local research ethics committee , and informed consent was obtained from all participants .
summary of clinical characteristics of patients with type 1 diabetes and healthy nondiabetic control subjects peptides based on sequences of naturally processed and hla - dr4 presented ia2 ( 709736 , 752775 , and 853872 ) , proinsulin ( c19-a3 ) , and gad65 epitopes ( 335352 and 554575 ) , as well as overlapping regions of the insulin b ( 120 , 625 , and 1130 ) and a chain ( 121 ) , were synthesized by 9-fluorenylmethyl carbamate chemistry and purified by high - performance liquid chromatography ( thermo hybaid , ulm , germany ) , a peptide of the cs protein of plasmodium falciparum ( 363377 ) was used as a negative control .
pediacel , a penta - vaccine consisting of purified diphtheria toxoid , purified tetanus toxoid , acellular pertussis vaccine , inactivated poliovirus , and haemophilus influenzae type b polysaccharide , was obtained from sanofi pasteur ( berkshire , u.k . ) and used at 1 l / ml as a positive control .
detection of ifn- production by cd4 t - cells in response to peptide stimulation was carried out using an enzyme - linked immunospot ( elispot ) assay that has significant discriminative ability for type 1 diabetes in blinded proficiency testing , as described previously ( 11,12 ) .
the elispot platform was adapted to a direct format to detect il-17secreting cd4 t - cells using plates precoated with monoclonal anti
il-17 capture antibody 72-h incubation and biotinylated anti il-17 detector antibody ( r&d systems , abingdon , u.k . ) .
data are expressed as the mean number of spots per triplicate and compared with the mean spot number in the presence of diluent alone ( stimulation index ) .
a response is considered positive when the stimulation index is 3 using cutoffs determined using receiver - operator characteristic plots as previously described .
interassay coefficient of variation for the spot number in the il-17 elispot assay was 8.6% when evaluated by measuring spot number elicited when fresh peripheral blood mononuclear cells from the same donor were obtained on five separate occasions over a 3-month period and cultured with a low dose of pediacel ( 0.2 l / ml ) to mimic the small number of spots observed in autoreactive responses .
islet and pancreas samples were obtained from two patients who died at different stages of type 1 diabetes ( 5 days and 10 years after diagnosis ) as described ( 13 ) .
rna from cryostat sections obtained from pancreatic blocks or from fresh frozen purified islets was extracted with the rneasy micro kit ( qiagen , hilden , germany ) .
a total of 1 g rna ( total pancreas ) or 200 ng rna ( purified islets ) was reverse - transcribed using oligo - dt ( 0.5 g/l ; genelink , hawthorne , ny ) and moloney murine leukemia virus reverse - transcriptase ( promega , madison , wi ) .
quantitative pcr assays were performed on a lightcycler 480 ( roche , mannheim , germany ) using taqman universal assay conditions and using the following taqman assays on demand : il17a ( hs00174383_m1 ) , il22 ( hs01574154_m1 ) , ifng ( hs00989291_m1 ) , rorc ( hs01076112_m1 ) , tbx21 ( hs00894392_m1 ) , and hprt ( hs99999909_m1 ) as housekeeping genes .
relative values were determined by normalizing the expression for each gene of interest to the housekeeping gene ( hprt ) and to a calibrator sample as described in the 2 method ( 14 ) .
the calibrator sample was a pool of all of the samples included in the study ( pancreases and islets ) .
for il22 , a different calibrator sample was used because it was not detectable using the standard calibrator .
this second calibrator was a pool of samples from type 1 diabetic patients ( pancreata and islets ) .
male wistar rats ( charles river laboratories , brussels , belgium ) were housed and used according to the guidelines of the belgian regulations for animal care .
-cells were purified by autofluorescence - activated cell sorting ( facsaria ; bd bioscience , san jose , ca ) ( 15,16 ) .
-cells were cultured for 2 days in ham s f-10 medium containing 10 mmol / l glucose , 2 mmol / l glutamax , 50 mol / l 3-isobutyl-1-methylxanthine , 5% fbs , 0.5% charcoal - absorbed bsa ( boehringer , indianapolis , in ) , 50 units / ml penicillin , and 50 g / ml streptomycin ( 16 ) .
during cytokine exposure , cells were cultured in the same medium but without serum ( 17 ) .
we have previously shown ( 18 ) that human islet exposure to proinflammatory cytokines induces apoptosis in the presence or absence of serum . the rat insulin - producing ins-1e cell line ( a gift from dr . c. wollheim , centre medical universitaire , geneva , switzerland ) was cultured as previously described ( 19 ) .
human islets were obtained from nine donors ( two donors at the diabetes research unit at king s college london and seven isolated in the department of endocrinology and metabolism , metabolic unit , university of pisa , as previously described ) ; of note , similar effects of il-17 were observed in islets isolated in london and pisa and sent to brussels [ data not shown ] ) , where they were cultured in complete medium as described above .
-cell proportion was evaluated by insulin staining in each individual preparation and represented 4872% of the dispersed islets ( mean [ sem ]
-cell purity 62 4.7% ) ; these dispersed islets or whole islets were exposed to cytokines for 24 and 48 h , as indicated in the figures .
the following sirnas were used in this study : block - it stealth select sirna sistat1 : 5-cccuagaagacuuacaagaugaaua-3 ( invitrogen , paisley , u.k . ) ; allstars negative control sirna ( sictrl ) was used for control - transfected conditions ( qiagen , venlo , the netherlands ; sequence not provided ) .
the transfection of sirnas in ins-1e cells and primary fluorescence activated cell - sorted rat -cells was done as previously described , with an efficiency of transfection > 90% ( 20 ) .
nuclear factor ( nf)-b inhibition was carried out using an adenovirus encoding a super - repressor ib that blocks nf-b activation as previously described ( 21 )
. the following cytokine concentrations were used , based on previous dose - response experiments by our group ( 19,22,23 ) : recombinant human il-1 ( specific activity [ sa ] 1.8 10 units / mg ; a gift from c.w .
reinolds , national cancer institute , bethesda , md ) at 10 or 50 units / ml as indicated ; recombinant rat ifn- ( sa 2 10 units / mg ; r&d systems ) at 100 units / ml ; recombinant human ifn- ( r&d systems ) at 1,000 units / ml ; recombinant human tumor necrosis factor ( tnf)- ( r&d systems ) at 1,000 units / ml ; and recombinant rat il-17a ( biovision , mountain view , ca ) and recombinant human il-17a ( r&d systems ) at 20 ng / ml .
when cells were treated with cytokines , culture supernatants were collected for nitrite determination ( nitrite is a stable product of nitric oxide [ no ] oxidation ) at od using the griess method . in some experiments , the inducible nitric oxide synthase inhibitor n - methyl - l - arginine ( l - nma ) ( sigma , st .
the percentage of viable , apoptotic , and necrotic cells was determined after a 15-min incubation with the dna - binding dyes propidium iodide ( pi , 5 g / ml ; sigma - aldrich ) and hoechst dye 33342 ( ho , 5 g / ml ; sigma - aldrich ) ( 16 ) .
viability was evaluated by two independent observers , one of them being unaware of sample identity .
results are expressed as percent apoptosis , calculated as number of ( apoptotic cells / total number of cells ) 100 .
poly(a ) mrna was isolated from ins-1e cells , rat primary -cells , or human islets using the dynabeads mrna direct kit ( invitrogen ) and was reverse - transcribed and amplified by pcr as previously described ( 16,24 ) , using sybr green and compared with a standard curve ( 25 ) .
we have previously shown that mrna expression of these genes is not affected by the present experimental conditions ( 20 ) .
frequency data between patient and control groups were compared with the two - sided fisher exact test .
peptides based on sequences of naturally processed and hla - dr4 presented ia2 ( 709736 , 752775 , and 853872 ) , proinsulin ( c19-a3 ) , and gad65 epitopes ( 335352 and 554575 ) , as well as overlapping regions of the insulin b ( 120 , 625 , and 1130 ) and a chain ( 121 ) , were synthesized by 9-fluorenylmethyl carbamate chemistry and purified by high - performance liquid chromatography ( thermo hybaid , ulm , germany ) , a peptide of the cs protein of plasmodium falciparum ( 363377 ) was used as a negative control .
pediacel , a penta - vaccine consisting of purified diphtheria toxoid , purified tetanus toxoid , acellular pertussis vaccine , inactivated poliovirus , and haemophilus influenzae type b polysaccharide , was obtained from sanofi pasteur ( berkshire , u.k . ) and used at 1 l / ml as a positive control .
detection of ifn- production by cd4 t - cells in response to peptide stimulation was carried out using an enzyme - linked immunospot ( elispot ) assay that has significant discriminative ability for type 1 diabetes in blinded proficiency testing , as described previously ( 11,12 ) .
the elispot platform was adapted to a direct format to detect il-17secreting cd4 t - cells using plates precoated with monoclonal anti
data are expressed as the mean number of spots per triplicate and compared with the mean spot number in the presence of diluent alone ( stimulation index ) .
a response is considered positive when the stimulation index is 3 using cutoffs determined using receiver - operator characteristic plots as previously described .
interassay coefficient of variation for the spot number in the il-17 elispot assay was 8.6% when evaluated by measuring spot number elicited when fresh peripheral blood mononuclear cells from the same donor were obtained on five separate occasions over a 3-month period and cultured with a low dose of pediacel ( 0.2 l / ml ) to mimic the small number of spots observed in autoreactive responses .
islet and pancreas samples were obtained from two patients who died at different stages of type 1 diabetes ( 5 days and 10 years after diagnosis ) as described ( 13 ) .
rna from cryostat sections obtained from pancreatic blocks or from fresh frozen purified islets was extracted with the rneasy micro kit ( qiagen , hilden , germany ) .
a total of 1 g rna ( total pancreas ) or 200 ng rna ( purified islets ) was reverse - transcribed using oligo - dt ( 0.5 g/l ; genelink , hawthorne , ny ) and moloney murine leukemia virus reverse - transcriptase ( promega , madison , wi ) .
quantitative pcr assays were performed on a lightcycler 480 ( roche , mannheim , germany ) using taqman universal assay conditions and using the following taqman assays on demand : il17a ( hs00174383_m1 ) , il22 ( hs01574154_m1 ) , ifng ( hs00989291_m1 ) , rorc ( hs01076112_m1 ) , tbx21 ( hs00894392_m1 ) , and hprt ( hs99999909_m1 ) as housekeeping genes .
relative values were determined by normalizing the expression for each gene of interest to the housekeeping gene ( hprt ) and to a calibrator sample as described in the 2 method ( 14 ) .
the calibrator sample was a pool of all of the samples included in the study ( pancreases and islets ) .
for il22 , a different calibrator sample was used because it was not detectable using the standard calibrator .
this second calibrator was a pool of samples from type 1 diabetic patients ( pancreata and islets ) .
male wistar rats ( charles river laboratories , brussels , belgium ) were housed and used according to the guidelines of the belgian regulations for animal care .
-cells were purified by autofluorescence - activated cell sorting ( facsaria ; bd bioscience , san jose , ca ) ( 15,16 ) .
-cells were cultured for 2 days in ham s f-10 medium containing 10 mmol / l glucose , 2 mmol / l glutamax , 50 mol / l 3-isobutyl-1-methylxanthine , 5% fbs , 0.5% charcoal - absorbed bsa ( boehringer , indianapolis , in ) , 50 units / ml penicillin , and 50 g / ml streptomycin ( 16 ) . during cytokine exposure , cells were cultured in the same medium but without serum ( 17 ) . we have previously shown ( 18 ) that human islet exposure to proinflammatory cytokines induces apoptosis in the presence or absence of serum . the rat insulin - producing ins-1e cell line ( a gift from dr . c. wollheim , centre medical universitaire , geneva , switzerland ) was cultured as previously described ( 19 ) .
human islets were obtained from nine donors ( two donors at the diabetes research unit at king s college london and seven isolated in the department of endocrinology and metabolism , metabolic unit , university of pisa , as previously described ) ; of note , similar effects of il-17 were observed in islets isolated in london and pisa and sent to brussels [ data not shown ] ) , where they were cultured in complete medium as described above .
-cell proportion was evaluated by insulin staining in each individual preparation and represented 4872% of the dispersed islets ( mean [ sem ] -cell purity 62 4.7% ) ; these dispersed islets or whole islets were exposed to cytokines for 24 and 48 h , as indicated in the figures .
the following sirnas were used in this study : block - it stealth select sirna sistat1 : 5-cccuagaagacuuacaagaugaaua-3 ( invitrogen , paisley , u.k . ) ; allstars negative control sirna ( sictrl ) was used for control - transfected conditions ( qiagen , venlo , the netherlands ; sequence not provided ) .
the transfection of sirnas in ins-1e cells and primary fluorescence activated cell - sorted rat -cells was done as previously described , with an efficiency of transfection > 90% ( 20 ) .
nuclear factor ( nf)-b inhibition was carried out using an adenovirus encoding a super - repressor ib that blocks nf-b activation as previously described ( 21 ) .
the following cytokine concentrations were used , based on previous dose - response experiments by our group ( 19,22,23 ) : recombinant human il-1 ( specific activity [ sa ] 1.8 10 units / mg ; a gift from c.w .
reinolds , national cancer institute , bethesda , md ) at 10 or 50 units / ml as indicated ; recombinant rat ifn- ( sa 2 10 units / mg ; r&d systems ) at 100 units / ml ; recombinant human ifn- ( r&d systems ) at 1,000 units / ml ; recombinant human tumor necrosis factor ( tnf)- ( r&d systems ) at 1,000 units / ml ; and recombinant rat il-17a ( biovision , mountain view , ca ) and recombinant human il-17a ( r&d systems ) at 20 ng / ml .
when cells were treated with cytokines , culture supernatants were collected for nitrite determination ( nitrite is a stable product of nitric oxide [ no ] oxidation ) at od using the griess method . in some experiments , the inducible nitric oxide synthase inhibitor n - methyl - l - arginine ( l - nma ) ( sigma , st .
louis , mo ) was used at a concentration of 5 mmol / l to inhibit no production .
the percentage of viable , apoptotic , and necrotic cells was determined after a 15-min incubation with the dna - binding dyes propidium iodide ( pi , 5 g / ml ; sigma - aldrich ) and hoechst dye 33342 ( ho , 5 g / ml ; sigma - aldrich ) ( 16 ) .
viability was evaluated by two independent observers , one of them being unaware of sample identity .
results are expressed as percent apoptosis , calculated as number of ( apoptotic cells / total number of cells ) 100 .
poly(a ) mrna was isolated from ins-1e cells , rat primary -cells , or human islets using the dynabeads mrna direct kit ( invitrogen ) and was reverse - transcribed and amplified by pcr as previously described ( 16,24 ) , using sybr green and compared with a standard curve ( 25 ) .
expression values were corrected for the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase ( gapdh ) . we have previously shown that mrna expression of these genes is not affected by the present experimental conditions ( 20 ) .
frequency data between patient and control groups were compared with the two - sided fisher exact test .
we examined peripheral blood responses of 50 patients with new - onset type 1 diabetes and 30 age- , hla- , and sex - matched healthy nondiabetic control subjects against a well - defined panel of -cell autoantigenic peptides using a sensitive elispot to detect il-17 secretion .
il-17 responses were observed in 27 of 50 patients ( 54% ) compared with 3 of 30 ( 10% ) healthy control subjects ( p < 0.0001 ) ( fig .
1a ) . among patients , 21 of 50 ( 42% ) had responses against two or more -cell autoantigenic peptides compared with 2 of 30 ( 8% ) healthy control subjects ( p = 0.0007 ; median responses 1 and 0 peptides , respectively ) .
a : il-17 responses to -cell autoantigens in patients with type 1 diabetes ( ; n = 50 ) are significantly more prevalent than in healthy control subjects ( ; n = 30 ) , whereas the prevalence of reactivity to recall antigens is similar .
b : for comparison , ifn- responses to -cell autoantigens in patients with type 1 diabetes are also significantly more prevalent than in healthy control subjects , and the prevalence of reactivity to recall antigens is similar ( * * * p < 0.0001 ) .
c : graph shows number of peptides provoking il-17 production in five patients tested in the original sampling near diagnosis and again a median of 12 months ( range 914 ) later . there is a nonsignificant trend for the number of peptides testing positive to decline over time ( paired t test ; p = 0.09 ) .
d : frequency of responses to individual -cell autoantigenic peptides among type 1 diabetic patients as measured by production of il-17 ( ) and ifn- ( ) .
e : photomicrographs of wells in which cells have been cultured with negative control ( peptide diluent ) , -cell autoantigen , and recall stimulus ( pediacel vaccine ) for il-17 ( upper row ) and ifn- ( lower row ) .
it is noteworthy that the frequency of ifn- responder cells for recall antigens is severalfold higher than for il-17 cells . in keeping with our previous findings , ifn- production against the same peptide panel
was also observed and was significantly more frequent in patients than in control subjects ( 31 of 50 [ 62% ] compared with 4 of 30 [ 13% ] [ p < 0.0001 ; fig .
il-17 responses were attributable to cd4 t - cells , since reactivity is lost after immunomagnetic depletion of these cells ( data not shown ) .
five patients were retested for il-17 responsiveness a median of 12 months later ( range 914 months ) against the entire peptide panel . in three patients ,
the number of peptides provoking an il-17 response declined . in each of these , the responses detected in the second sample had the same peptide specificities as in the original sample .
in two patients , the number of peptides ( and specific peptides targeted ) remained the same ( fig .
1c ) . the pattern of il-17 reactivity against -cell autoantigenic peptides did not differ markedly from the ifn- response ( fig .
1d ) , suggesting complementarities of the priming and maintenance stimuli for these two types of responses ; however , the number of il-17 autoreactive and recall antigen - specific responder cells was typically fewer than for ifn- ( fig .
all but one patient had received insulin therapy before testing , and it is conceivable that the responses we observed against overlapping peptides of insulin were induced as a result . however , il-17 responses to overlapping peptides of insulin ( a121 , b120 , b625 , and b1130 ) were no more frequent overall than responses to proinsulin , ia-2 , and gad65 epitopes , and the single patient tested before insulin therapy commenced showed il-17 responses to insulin peptides a121 and b1130 as well as gad 554575 , ia-2 752775 , and ia-2 85372 .
studies on at - risk subjects will be required to provide conclusive evidence that insulin treatment naive subjects make il-17 responses against this autoantigen . in 12 of the patients ( 24% ) ,
both il-17 and ifn- responses were observed for the same -cell peptide , although no single peptide emerged as dominant .
these assays were unable to discriminate dual - secreting cells from populations of single - secretors .
there was no significant relationship between age of onset of type 1 diabetes , duration of disease , the presence of islet cell autoantibodies or hla genotype , and the detection of an il-17 response .
the frequency of hla - dr3/dr4 heterozygosity was 13 of 46 ( 28% ) patients compared with 9 of 28 ( 32% ) control subjects ( p = 0.79 ) .
there was no relationship between possession of either hla - dr4 or hla - dr3 and the presence of an il-17 response to -cell peptides . among patients ,
the frequency of il-17 responses was similar in dr3/dr4 heterozygous and nonheterozygous individuals ( 6/13 [ 54% ] vs. 17/33 [ 53% ] , respectively ) .
il-17 responses were observed more frequently in males ( 19/32 , 59% ) than females ( 8/18 , 44% ) but this was not significant ( p = 0.38 ) .
patients and control subjects did not differ in the frequency of response to recall antigens ( present in the pentavalent vaccine pediacel ) or an irrelevant peptide of plasmodium falciparum ( circumsporozoite 363377 ) .
collectively , these data indicate that , at diagnosis , type 1 diabetes is characterized by the presence of circulating -cell autoantigen - specific cd4 t - cells secreting the proinflammatory cytokine il-17 .
to examine whether the diabetes - related il-17 signature that we detected in peripheral blood near diagnosis is relevant to the process of inflammation that leads to -cell damage , we obtained purified islets of langerhans from two patients , one of whom died within 5 days of diagnosis ( designated t1d1 ) and one after 10 years of disease ( t1d2 ) as well as from three nondiabetic control subjects ( 13 ) .
as previously reported ( 13 ) , the islets in the diseased organs showed infiltration by mononuclear cells ( insulitis ) , a characteristic pathological feature of type 1 diabetes .
notably , cd4 t - cells were detected in a higher proportion of islets when studied near diagnosis ( 69 vs. 28% islets positive in t1d1 and t1d2 , respectively ) and were present in greater number ( approximately threefold higher ) , compared with longstanding disease .
quantitative rt - pcr analysis of these samples showed a relative sixfold higher expression of il17a over the control samples in the islets obtained close to diagnosis , whereas il17a was not detectable in the longstanding patient ( fig .
a similar pattern was seen for ifng expression , which showed a relative 32-fold higher level of transcripts in t1d1 but was barely detectable in t1d2 ( fig .
we next examined whether the transcription factors associated with th17 and th1 differentiation , rorc and tbx21 , respectively , were detectable .
transcripts for both th17- and th1-associated transcription factors were detected at elevated levels in t1d1 islets , and tbx21 was also detected in t1d2 islets ( fig . 2c and d ) .
further support for the presence of cells with a th17 differentiation pathway in islets near to diagnosis is demonstrated by the detection of il22 in t1d1 but not t1d2 ( fig .
. quantitative rt - pcr analysis of pancreas blocks from the same samples was less informative , presumably as a result of dilutional effects ( islets represent < 1% of pancreatic cells ) .
il17a and ifng transcripts were elevated in t1d1 but not t1d2 pancreas , whereas those for the transcription factors were similar to control subjects ( data not shown ) . real - time quantitative pcr analysis of expression of il17 ( a ) , ifng ( b ) , rorc ( c ) , tbx21 ( d ) , and il22 ( e ) in islets of langerhans obtained from two patients with type 1 diabetes ( t1d1 , who died within 5 days of diagnosis , and t1d2 , who died 10 years after diagnosis ) .
mean ( sem ) values ( technical replicates ) are also shown for three control subjects ( organ donors ) .
levels of target gene mrna transcripts are normalized to the housekeeping gene hprt and compared with an in - house calibrator sample as described .
hc , healthy controls . the finding that il-17secreting cd4 t - cells specific for -cell autoantigens are present in the circulation and that il17a transcripts are elevated in the pancreatic islets near to diagnosis of type 1 diabetes prompted us to examine the effects of il-17 on human -cells .
it is well established that human -cells are highly sensitive to the action of the proinflammatory cytokines il-1 and tnf- , which mediate a proapoptotic effect in combination with ifn- ( 22,26 ) . whereas il-17a alone had no proapoptotic effect on cultured human islets , its addition significantly exacerbated the apoptosis induced by the combination of il-1 and ifn- or tnf- and ifn- ( fig .
this step was associated with significantly increased release of nitrite , suggesting increased no production by human islets exposed to these cytokine combinations .
the addition of the inhibitor of nitric oxide synthase ( nos ) , l - nma , substantially decreased nitrite accumulation in the medium , indicating diminished no production by the cytokine - treated islets ( fig .
l - nma , however , did not prevent cytokine - induced islet cell apoptosis ( fig .
3b ) , in keeping with our previous observation that cytokine - induced human -cell death is not mediated by no formation ( 18,27 ) .
furthermore , transcripts for il17ra , but not il17rc , were significantly upregulated by il-1 and ifn- treatment of human islets ( fig .
i ) , indicating that conditioning of -cells by these cytokines renders them susceptible to the apoptosis - promoting actions of il-17a via specific cytokine - receptor induction .
a : human islets were treated with il-1 ( 50 units / ml ) , tnf- , and ifn- ( both at 1,000 units / ml ) in the presence of 20 ng / ml ril-17a for 48 h. apoptosis was evaluated using ho / pi staining .
results are means sem of six to nine independent experiments ( * * * p < 0.001 vs. untreated cells ;
p < 0.001 vs. il-1 + ifn-treated cells ; p < 0.001 vs. tnf- and ifn-treated cells )
b : human islets were treated with il-1 ( 50 units / ml ) and ifn- ( 1,000 units / ml ) in the presence of 20 ng / ml ril-17a for 48 h with or without the addition of l - nma .
results are means sem of three independent experiments ( * * * p < 0.001 vs. untreated cells ; p < 0.01 vs. il-1 + ifn-treated cells ;
p < 0.05 vs. il-1 + ifn- + l - nma treated cells ) ( anova with bonferroni correction ) .
c f : panels show representative images of cell death in whole human islets without cytokine ( c ) , islets cultured with il-17a ( 20 ng / ml ) ( d ) , islets cultured with il-1 ( 50 units / ml ) + ifn- ( 1,000 units / ml ) ( e ) , and islets stimulated with il-1 ( 50 units / ml ) + ifn- ( 1,000 units / ml ) + il-17a ( 20 ng / ml ) ( f ) .
g : human islets were treated with cytokines as described above for 48 h in the presence of l - nma , and supernatants were assayed for nitrite content .
nitrite is significantly raised in the presence of il-1 ( 50 units / ml ) + ifn- ( 1,000 units / ml ) and further increased when supplemented with 20 ng / ml ril-17a . in the presence of l - nma
results are means sem of three independent experiments ( * p < 0.05 vs. untreated cells ;
p < 0.05 vs. il-1 + ifn- + il-17a treated cells ) ( anova with bonferroni correction ) .
h : the combination of cytokines il-1 and ifn- upregulate il-17ra mrna expression in human islets after 24 h. human islets were treated with il-1 ( 50 units / ml ) and ifn- ( 1,000 units / ml ) for 24 h ; il-17ra mrna expression was assayed by rt - pcr and normalized for the housekeeping gene -actin ( actb ) ; * p < 0.05 vs. untreated ( student t test ) .
i : in contrast , under the same conditions as in h , il-1 and ifn- do not increase il-17rc mrna expression in human islets after 24 h. results are means sem of six independent experiments .
( a high - quality color representation of this figure is available in the online issue . )
we next examined the transcriptional pathways involved in il-1/ifn-mediated il-17ra induction using either sirna targeting of stat1 or a super - repressor of nf-b activity .
we first confirmed that the il-1/ifn- cytokine combination induces il-17ra in the rat insulin - producing ins-1e cells and in primary rat -cells , in association with a proapoptotic signal that is significantly enhanced by addition of il-17a ( supplementary figs .
ins-1e cells treated with sirna targeting stat1 showed significantly reduced il17ra induction after treatment with the il-1/ifn- combination ( fig .
similarly , ins-1e cells infected with an adenovirus encoding a super - repressor ib to inhibit nf-b activity showed significantly reduced il17ra induction compared with control infected cells ( fig .
these data indicate that the induction of il-17ra expression in -cells depends on both stat1 and nf-b transcriptional pathways .
il-17ra expressed in rat insulin - producing ins-1e cells is upregulated by cytokines via stat1 and nf-b activation .
nmol / l of either a control sirna ( ) or a sirna targeting stat1 ( ) .
after 1 day of recovery , cells were left untreated or were treated with il-1 ( 10 units / ml ) + ifn- ( 100 units / ml ) for 24 h. il-17ra mrna expression was assayed by rt - pcr and normalized for the housekeeping gene gapdh .
cytokine treatment significantly upregulates il-17ra ( * * p < 0.01 vs. untreated cells ; student t test ) .
sirna targeting of stat1 significantly reduces il-17ra induction ( p < 0.05 vs. control sirna ) .
results are means sem of five independent experiments . in another series of experiments ,
ins-1e cells were infected with an adenovirus encoding a super - repressor ib to inhibit nf-b activity ( ) or with adenovirus encoding luciferase as control ( ) .
cells were left untreated or treated with il-1 ( 10 units / ml ) + ifn- ( 100 units / ml ) for 24 h. cytokine treatment significantly upregulates il-17ra ( * * * p < 0.001 and * p < 0.05 vs. relevant untreated cells ) .
adenovirus - mediated inhibition of nf-b significantly reduces il-17ra induction . p < 0.05 vs. control .
we examined peripheral blood responses of 50 patients with new - onset type 1 diabetes and 30 age- , hla- , and sex - matched healthy nondiabetic control subjects against a well - defined panel of -cell autoantigenic peptides using a sensitive elispot to detect il-17 secretion .
il-17 responses were observed in 27 of 50 patients ( 54% ) compared with 3 of 30 ( 10% ) healthy control subjects ( p < 0.0001 ) ( fig .
1a ) . among patients , 21 of 50 ( 42% ) had responses against two or more -cell autoantigenic peptides compared with 2 of 30 ( 8% ) healthy control subjects ( p = 0.0007 ; median responses 1 and 0 peptides , respectively ) .
a : il-17 responses to -cell autoantigens in patients with type 1 diabetes ( ; n = 50 ) are significantly more prevalent than in healthy control subjects ( ; n = 30 ) , whereas the prevalence of reactivity to recall antigens is similar .
b : for comparison , ifn- responses to -cell autoantigens in patients with type 1 diabetes are also significantly more prevalent than in healthy control subjects , and the prevalence of reactivity to recall antigens is similar ( * * * p < 0.0001 ) .
c : graph shows number of peptides provoking il-17 production in five patients tested in the original sampling near diagnosis and again a median of 12 months ( range 914 ) later . there is a nonsignificant trend for the number of peptides testing positive to decline over time ( paired t test ; p = 0.09 ) .
d : frequency of responses to individual -cell autoantigenic peptides among type 1 diabetic patients as measured by production of il-17 ( ) and ifn- ( ) .
e : photomicrographs of wells in which cells have been cultured with negative control ( peptide diluent ) , -cell autoantigen , and recall stimulus ( pediacel vaccine ) for il-17 ( upper row ) and ifn- ( lower row ) .
it is noteworthy that the frequency of ifn- responder cells for recall antigens is severalfold higher than for il-17 cells . in keeping with our previous findings , ifn- production against the same peptide panel
was also observed and was significantly more frequent in patients than in control subjects ( 31 of 50 [ 62% ] compared with 4 of 30 [ 13% ] [ p < 0.0001 ; fig .
il-17 responses were attributable to cd4 t - cells , since reactivity is lost after immunomagnetic depletion of these cells ( data not shown ) .
five patients were retested for il-17 responsiveness a median of 12 months later ( range 914 months ) against the entire peptide panel . in three patients ,
the number of peptides provoking an il-17 response declined . in each of these , the responses detected in the second sample had the same peptide specificities as in the original sample .
in two patients , the number of peptides ( and specific peptides targeted ) remained the same ( fig .
1c ) . the pattern of il-17 reactivity against -cell autoantigenic peptides did not differ markedly from the ifn- response ( fig .
1d ) , suggesting complementarities of the priming and maintenance stimuli for these two types of responses ; however , the number of il-17 autoreactive and recall antigen - specific responder cells was typically fewer than for ifn- ( fig .
all but one patient had received insulin therapy before testing , and it is conceivable that the responses we observed against overlapping peptides of insulin were induced as a result . however , il-17 responses to overlapping peptides of insulin ( a121 , b120 , b625 , and b1130 ) were no more frequent overall than responses to proinsulin , ia-2 , and gad65 epitopes , and the single patient tested before insulin therapy commenced showed il-17 responses to insulin peptides a121 and b1130 as well as gad 554575 , ia-2 752775 , and ia-2 85372 .
studies on at - risk subjects will be required to provide conclusive evidence that insulin treatment naive subjects make il-17 responses against this autoantigen . in 12 of the patients ( 24% ) ,
both il-17 and ifn- responses were observed for the same -cell peptide , although no single peptide emerged as dominant .
these assays were unable to discriminate dual - secreting cells from populations of single - secretors .
there was no significant relationship between age of onset of type 1 diabetes , duration of disease , the presence of islet cell autoantibodies or hla genotype , and the detection of an il-17 response .
the frequency of hla - dr3/dr4 heterozygosity was 13 of 46 ( 28% ) patients compared with 9 of 28 ( 32% ) control subjects ( p = 0.79 ) .
there was no relationship between possession of either hla - dr4 or hla - dr3 and the presence of an il-17 response to -cell peptides . among patients ,
the frequency of il-17 responses was similar in dr3/dr4 heterozygous and nonheterozygous individuals ( 6/13 [ 54% ] vs. 17/33 [ 53% ] , respectively ) .
il-17 responses were observed more frequently in males ( 19/32 , 59% ) than females ( 8/18 , 44% ) but this was not significant ( p = 0.38 ) .
patients and control subjects did not differ in the frequency of response to recall antigens ( present in the pentavalent vaccine pediacel ) or an irrelevant peptide of plasmodium falciparum ( circumsporozoite 363377 ) .
collectively , these data indicate that , at diagnosis , type 1 diabetes is characterized by the presence of circulating -cell autoantigen - specific cd4 t - cells secreting the proinflammatory cytokine il-17 .
to examine whether the diabetes - related il-17 signature that we detected in peripheral blood near diagnosis is relevant to the process of inflammation that leads to -cell damage , we obtained purified islets of langerhans from two patients , one of whom died within 5 days of diagnosis ( designated t1d1 ) and one after 10 years of disease ( t1d2 ) as well as from three nondiabetic control subjects ( 13 ) .
as previously reported ( 13 ) , the islets in the diseased organs showed infiltration by mononuclear cells ( insulitis ) , a characteristic pathological feature of type 1 diabetes .
notably , cd4 t - cells were detected in a higher proportion of islets when studied near diagnosis ( 69 vs. 28% islets positive in t1d1 and t1d2 , respectively ) and were present in greater number ( approximately threefold higher ) , compared with longstanding disease .
quantitative rt - pcr analysis of these samples showed a relative sixfold higher expression of il17a over the control samples in the islets obtained close to diagnosis , whereas il17a was not detectable in the longstanding patient ( fig .
a similar pattern was seen for ifng expression , which showed a relative 32-fold higher level of transcripts in t1d1 but was barely detectable in t1d2 ( fig .
we next examined whether the transcription factors associated with th17 and th1 differentiation , rorc and tbx21 , respectively , were detectable .
transcripts for both th17- and th1-associated transcription factors were detected at elevated levels in t1d1 islets , and tbx21 was also detected in t1d2 islets ( fig . 2c and d ) .
further support for the presence of cells with a th17 differentiation pathway in islets near to diagnosis is demonstrated by the detection of il22 in t1d1 but not t1d2 ( fig .
. quantitative rt - pcr analysis of pancreas blocks from the same samples was less informative , presumably as a result of dilutional effects ( islets represent < 1% of pancreatic cells ) . il17a and ifng transcripts
were elevated in t1d1 but not t1d2 pancreas , whereas those for the transcription factors were similar to control subjects ( data not shown ) .
real - time quantitative pcr analysis of expression of il17 ( a ) , ifng ( b ) , rorc ( c ) , tbx21 ( d ) , and il22 ( e ) in islets of langerhans obtained from two patients with type 1 diabetes ( t1d1 , who died within 5 days of diagnosis , and t1d2 , who died 10 years after diagnosis ) .
mean ( sem ) values ( technical replicates ) are also shown for three control subjects ( organ donors ) .
levels of target gene mrna transcripts are normalized to the housekeeping gene hprt and compared with an in - house calibrator sample as described .
the finding that il-17secreting cd4 t - cells specific for -cell autoantigens are present in the circulation and that il17a transcripts are elevated in the pancreatic islets near to diagnosis of type 1 diabetes prompted us to examine the effects of il-17 on human -cells .
it is well established that human -cells are highly sensitive to the action of the proinflammatory cytokines il-1 and tnf- , which mediate a proapoptotic effect in combination with ifn- ( 22,26 ) . whereas il-17a alone had no proapoptotic effect on cultured human islets , its addition significantly exacerbated the apoptosis induced by the combination of il-1 and ifn- or tnf- and ifn- ( fig .
this step was associated with significantly increased release of nitrite , suggesting increased no production by human islets exposed to these cytokine combinations .
the addition of the inhibitor of nitric oxide synthase ( nos ) , l - nma , substantially decreased nitrite accumulation in the medium , indicating diminished no production by the cytokine - treated islets ( fig .
l - nma , however , did not prevent cytokine - induced islet cell apoptosis ( fig .
3b ) , in keeping with our previous observation that cytokine - induced human -cell death is not mediated by no formation ( 18,27 ) .
furthermore , transcripts for il17ra , but not il17rc , were significantly upregulated by il-1 and ifn- treatment of human islets ( fig .
i ) , indicating that conditioning of -cells by these cytokines renders them susceptible to the apoptosis - promoting actions of il-17a via specific cytokine - receptor induction . mechanisms through which il-17 promotes cytokine - induced -cell apoptosis .
a : human islets were treated with il-1 ( 50 units / ml ) , tnf- , and ifn- ( both at 1,000 units / ml ) in the presence of 20 ng / ml ril-17a for 48 h. apoptosis was evaluated using ho / pi staining .
results are means sem of six to nine independent experiments ( * * * p < 0.001 vs. untreated cells ;
p < 0.001 vs. il-1 + ifn-treated cells ; p < 0.001 vs. tnf- and ifn-treated cells ) ( anova with bonferroni correction ) .
b : human islets were treated with il-1 ( 50 units / ml ) and ifn- ( 1,000 units / ml ) in the presence of 20 ng / ml ril-17a for 48 h with or without the addition of l - nma .
results are means sem of three independent experiments ( * * * p < 0.001 vs. untreated cells ;
p < 0.05 vs. il-1 + ifn- + l - nma treated cells ) ( anova with bonferroni correction ) .
c f : panels show representative images of cell death in whole human islets without cytokine ( c ) , islets cultured with il-17a ( 20 ng / ml ) ( d ) , islets cultured with il-1 ( 50 units / ml ) + ifn- ( 1,000 units / ml ) ( e ) , and islets stimulated with il-1 ( 50 units / ml ) + ifn- ( 1,000 units / ml ) + il-17a ( 20 ng / ml ) ( f ) .
g : human islets were treated with cytokines as described above for 48 h in the presence of l - nma , and supernatants were assayed for nitrite content .
nitrite is significantly raised in the presence of il-1 ( 50 units / ml ) + ifn- ( 1,000 units / ml ) and further increased when supplemented with 20 ng / ml ril-17a . in the presence of l - nma
results are means sem of three independent experiments ( * p < 0.05 vs. untreated cells ; p < 0.05 vs. il-1 + ifn- + il-17a treated cells ) ( anova with bonferroni correction ) .
h : the combination of cytokines il-1 and ifn- upregulate il-17ra mrna expression in human islets after 24 h. human islets were treated with il-1 ( 50 units / ml ) and ifn- ( 1,000 units / ml ) for 24 h ; il-17ra mrna expression was assayed by rt - pcr and normalized for the housekeeping gene -actin ( actb ) ; * p < 0.05 vs. untreated ( student t test ) .
i : in contrast , under the same conditions as in h , il-1 and ifn- do not increase il-17rc mrna expression in human islets after 24 h. results are means sem of six independent experiments .
( a high - quality color representation of this figure is available in the online issue . )
we next examined the transcriptional pathways involved in il-1/ifn-mediated il-17ra induction using either sirna targeting of stat1 or a super - repressor of nf-b activity .
we first confirmed that the il-1/ifn- cytokine combination induces il-17ra in the rat insulin - producing ins-1e cells and in primary rat -cells , in association with a proapoptotic signal that is significantly enhanced by addition of il-17a ( supplementary figs .
ins-1e cells treated with sirna targeting stat1 showed significantly reduced il17ra induction after treatment with the il-1/ifn- combination ( fig .
similarly , ins-1e cells infected with an adenovirus encoding a super - repressor ib to inhibit nf-b activity showed significantly reduced il17ra induction compared with control infected cells ( fig .
4 ) . these data indicate that the induction of il-17ra expression in -cells depends on both stat1 and nf-b transcriptional pathways .
il-17ra expressed in rat insulin - producing ins-1e cells is upregulated by cytokines via stat1 and nf-b activation .
nmol / l of either a control sirna ( ) or a sirna targeting stat1 ( ) .
after 1 day of recovery , cells were left untreated or were treated with il-1 ( 10 units / ml ) + ifn- ( 100 units / ml ) for 24 h. il-17ra mrna expression was assayed by rt - pcr and normalized for the housekeeping gene gapdh .
cytokine treatment significantly upregulates il-17ra ( * * p < 0.01 vs. untreated cells ; student t test ) .
sirna targeting of stat1 significantly reduces il-17ra induction ( p < 0.05 vs. control sirna ) .
results are means sem of five independent experiments . in another series of experiments ,
ins-1e cells were infected with an adenovirus encoding a super - repressor ib to inhibit nf-b activity ( ) or with adenovirus encoding luciferase as control ( ) .
cells were left untreated or treated with il-1 ( 10 units / ml ) + ifn- ( 100 units / ml ) for 24 h. cytokine treatment significantly upregulates il-17ra ( * * * p < 0.001 and * p < 0.05 vs. relevant untreated cells ) .
adenovirus - mediated inhibition of nf-b significantly reduces il-17ra induction . p < 0.05 vs. control .
despite the intense interest provoked by the discovery of cd4 effector t - cells that secrete the signature cytokine il-17 , to date , there have been few examples in which the antigen specificity of such cells has been explored and none in the context of human autoimmune disease . here , we report that peripheral blood cd4 t - cells from patients with type 1 diabetes secrete il-17 in response to -cell autoantigens .
we show that this cytokine is actively produced in inflamed islets close to the onset of disease and provide evidence for the existence of a multistep pathway for -cell destruction in which il-17 has a key role . until the current study
, the evidence for a role for il-17 in autoimmune diabetes was largely based on animal studies , and the data are ambiguous . in the nonobese diabetic ( nod ) mouse model , it is reported that blockade of il-17 is protective ( 28 ) , whereas other studies suggest that disease actually results from the destructive actions of th1 cells that have undergone conversion from th17 cells ( 29,30 ) .
our results in human type 1 diabetes indicate a direct role for il-17secreting cells in pathways that lead to -cell damage .
moreover , we provide supportive evidence to indicate that the il-17 secretion that we detected in islets of langerhans obtained near diagnosis can be attributed to bona fide th17 cells .
we show the presence of the th17 transcription factor rorc in inflamed islets , as well as il22 , a cytokine typically secreted by th17 cells .
however , these results represent a case report , and future studies on similar cases will be required to verify these preliminary findings .
it is probable that there are multiple pathways through which -cells die during the development of type 1 diabetes .
it has been known from some years that -cells are exquisitely susceptible to the proapoptotic actions of the combination of il-1 and ifn- or tnf- and ifn- ( 22 ) , and the present and recent findings ( 9 ) highlight a prominent exacerbating role for il-17 in -cell destruction .
our results show that il-1 and ifn- condition human islets to express il-17ra and demonstrate for the first time that this is a process mediated via nf-b and stat1 , respectively . as a result
, -cells are rendered highly susceptible to the destructive actions of il-17 , which we have shown is a feature of islet inflammation and a product of cd4 t - cell autoreactivity .
stat1 is a key mediator of biological responses to ifn- in both -cells ( 31 ) and the immune system ( 32 ) , but this is the first description of stat1 affecting il-17r expression .
furthermore , recent data examining genes in type 1 diabetes pancreata report the increased expression of stat1 in the pancreas ( 13 ) , indicating that the pathway for upregulating il-17ra expression on -cells is active in diseased tissue .
il-1 exerts its effects on -cells to a large extent via the nf-b pathway ( 21,33 ) , and in our studies , blockade of nf-b resulted in a decrease in il17ra expression in relevant cell lines .
this stat1- and nf-b dependent upregulation of il17ra is a novel finding , since hitherto , the signaling cascades leading to il-17ra expression were not known .
synergy between cytokines such as il-1 , ifn- , and tnf- in the induction of apoptosis of human -cells has been well documented ( 26 ) , but the involvement of il-17 is a new finding . the requirement for il-17ra upregulation on -cells for il-17 to mediate damage lends further support to the concept that -cells are not passive bystanders in their own destruction .
rather , the -cell participates actively in its own demise via a complex cytokine transcription factor network involving ifn-stat1il-17r and il-1nf-b il-17r , whereby signaling by early inflammatory mediators secreted by th1 cells and macrophages renders -cells susceptible to subsequent il-17mediated apoptosis ( fig .
we propose that -cell death could have two phases . in the first , th1 cells and macrophages in the local milieu secrete ifn- and il-1 , respectively , which bind to specific receptors leading to the activation of stat1 and nf-b transcription factors . both stat1 and nf-b lead to the expression of il-17r through an as yet unknown mechanism . in the second phase ,
triggering through il17ra / rc exacerbates apoptosis initiated by il-1 and ifn- , leading to increased -cell death .
our results suggest that responses by il-17producing cells essentially parallel those of th1 cells near diagnosis .
there is much still to learn about the -cell specific cd4 t - cell response phenotype .
it is technically challenging to examine whether the cells we identified secrete both il-17 and ifn- , and it remains to be established whether other types of cd4 t - cell polarization exist in the disease , the time course of the responses , and whether there is a generalized tendency to generate autoreactive effector t - cells .
specific cd4 t - cells in patients with type 1 diabetes , along with additional evidence that patients have an abnormal expansion of il-17secreting cd4 t - cells after polyclonal stimulation of peripheral blood cells ( 9 ; data not shown ) , highlights a further aspect of the possible contribution of these cells to type 1 diabetes immunopathology .
these data suggest that at the time of diagnosis of type 1 diabetes , polarization toward the il-17 pathway is prominent
. however , this was not evident in the il-17 memory response to recall antigens , which we found to be normal in frequency and amplitude when tested at diagnosis ( and which would have been primed some years before via vaccination ) .
this result suggests that there is active promotion of il-17 pathway differentiation for cd4 t - cells during the peri - diagnosis period , which may be important for two reasons .
first , th17 differentiation is known to be influenced by at least one ligand - receptor system ( for aryl hydrocarbons ) that is responsive to environmental stimuli , and there is ample evidence for a role for nongenetic factors in type 1 diabetes ( 34 ) .
second , because il-17secreting cells are known to be more resistant to regulation by naturally occurring t regulatory cells , our finding could explain the consistent observation that effector cells from type 1 diabetic patients are less
regulatable ( 35 ) . taken together with the susceptibility of the human -cell to il-17
, these studies offer clear rationale for contemplating intervention in the differentiation and effector pathways of th17 cells , with several candidate biologics now available ( 36,37 ) .
| objectivecd4 t - cells secreting interleukin ( il)-17 are implicated in several human autoimmune diseases , but their role in type 1 diabetes has not been defined . to address the relevance of such cells
, we examined il-17 secretion in response to -cell autoantigens , il-17a gene expression in islets , and the potential functional consequences of il-17 release for -cells.research design and methodsperipheral blood cd4 t - cell responses to -cell autoantigens ( proinsulin , insulinoma - associated protein , and gad65 peptides ) were measured by il-17 enzyme - linked immunospot assay in patients with new - onset type 1 diabetes ( n = 50 ) .
mrna expression of il-17a and ifng pathway genes was studied by qrt - pcr using islets obtained from subjects who died 5 days and 10 years after diagnosis of disease , respectively , and from matched control subjects .
il-17 effects on the function of human islets , rat -cells , and the rat insulinoma cell line ins-1e were examined.resultsa total of 27 patients ( 54% ) showed il-17 reactivity to one or more -cell peptides versus 3 of 30 ( 10% ) control subjects ( p = 0.0001 ) . in a single case examined close to diagnosis , islet expression of il17a , rorc , and il22 was detected .
it is noteworthy that we show that il-17 mediates significant and reproducible enhancement of il-1/interferon ( ifn)-induced and tumor necrosis factor ( tnf)-/ifn-induced apoptosis in human islets , rat -cells , and ins-1e cells , in association with significant upregulation of -cell il17ra expression via activation of the transcription factors stat1 and nuclear factor ( nf)-b.conclusionscirculating il-17 + -cell specific autoreactive cd4 t - cells are a feature of type 1 diabetes diagnosis .
we disclose a novel pathway to -cell death involving il-17 and stat1 and nf-b , rendering this cytokine a novel disease biomarker and potential therapeutic target . |
fibrogenic progression of chronic liver diseases ( clds ) , eventually leading to the development of liver cirrhosis and related complications including hepatocellular carcinoma ( hcc ) , is intimately associated with pathologic angiogenesis and sinusoidal remodeling.1 , 2 , 3 , 4 , 5 , 6 this is not surprising because angiogenesis is a major feature of any wound healing response and chronic activation of wound healing is a general mechanism involved in the progression of clds.7 , 8 , 9 , 10 some researchers , including the authors of this review , go further2 , 3 , 4 , 9 , 10 , 11 , 12 , 13 , 14 , 15 in suggesting additionally that 1 ) hypoxia ( the most obvious stimulus for angiogenesis , commonly detected in progressive clds1 , 2 , 3 , 4 , 5 , 6 , 16 ) , hypoxia - inducible factors ( hifs ) , and angiogenesis may have a major role in sustaining and potentially driving liver fibrogenesis ; 2 ) hepatic myofibroblasts ( mfs ) , regardless of origin , are critical cells in governing and modulating the interactions between inflammation , angiogenesis , and fibrogenesis ; 3 ) liver angiogenesis has a role in the genesis of portal hypertension and related complications in advanced stages of clds ; and 4 ) microparticles / microvesicles released by either fat - laden hepatocytes or portal mfs have an emerging role in mediating angiogenesis and vascular remodeling .
this review offers a synthesis of the most relevant recent data and opinions regarding the close relationship between liver angiogenesis and fibrogenesis .
established concepts about mechanisms of liver angiogenesis , liver fibrogenesis , and cld progression will not be addressed . moreover , in this review , the relationship between angiogenesis and portal hypertension and related complications are not discussed ; readers interested in this specific topic should refer to a recent authoritative review .
liver angiogenesis occurs in both physiologic ( ie , liver regeneration ) and pathologic conditions , including ischemia , progressive clds , hepatocellular carcinoma , and metastatic liver cancer.1 , 2 , 3 , 4 , 5 angiogenesis in the liver is similar to angiogenesis in other tissues and organs ; however , as suggested by several groups,1 , 2 , 3 , 4 , 5 , 10 , 11 , 12 pathologic angiogenesis occurring during the progression of clds can be significantly affected by liver - specific events , interactions between different hepatic cell populations , and the involvement of atypical proangiogenic mediators . from a general point of view , the pattern of fibrosis that predominates in a specific cld is relevant to angiogenesis .
although pathologic liver angiogenesis has been described in all clds irrespective of etiology , it is much more prominent under conditions of bridging or postnecrotic fibrosis ( eg , in chronic viral infection or , to a lesser extent , in autoimmune diseases ) than in conditions characterized by pericellular or perisinusoidal fibrosis ( as in non - alcoholic fatty liver disease or alcoholic liver disease ) or by biliary fibrosis.3 , 9 , 10 this suggests an inverse correlation between angiogenesis and the potential for fibrosis reversibility , which is more evident in conditions characterized by pericellular or perisinusoidal fibrosis and biliary fibrosis than in those associated with bridging fibrosis .
this may be related to the unique tissue localization , phenotypic profile , and functional role of hepatic stellate cells ( hscs ) .
hscs , which in physiologic conditions synthesize extracellular matrix components in the space of disse , store retinoids and possibly contract in response to vasoactive mediators to modulate sinusoidal blood flux , are also liver - specific pericytes . during the progression of clds , hscs are the most relevant myofibroblast precursors and profibrogenic hepatic cells.2 , 17 , 18 , 19 , 20 , 21 hscs , particularly in their activated and mf - like phenotype ( hsc / mfs ) , modulate angiogenesis in a way that , as we will describe , relates to the role attributed to them as microcapillary pericytes .
hepatic mfs , a heterogenous population of profibrogenic , highly proliferative , and contractile cells , can also originate from portal mfs and bone marrow - derived stem cells recruited into the injured liver , and these may also play a role in angiogenesis.17 , 18 , 19 , 20 , 21 , 22 the relevance of intense cross - talk between hepatic cell populations in pathologic angiogenesis is strongly supported by the knowledge that major proangiogenic mediators such as vascular endothelial growth factor ( vegf ) and platelet - derived growth factor ( pdgf ) , are produced and released by several liver cell types involved in cld progression , including hypoxic hepatocytes and hypoxia - responsive macrophages and mfs.1 , 2 , 3 , 9 , 10 , 11 , 12 the role of liver - specific proangiogenic mediators such as angiopoietin - like-3 peptide ( angptl3 ) is controversial .
angptl3 , which belongs to a family of mediators described as playing major roles in the trafficking and metabolism of lipids , was reported to induce haptotactic endothelial cell adhesion and migration , possibly by binding to v3 integrin .
however , no further studies have been published on the role of this mediator in either physiologic or pathologic liver angiogenesis .
as we will discuss , a more interesting role is attributed to the antiangiogenic protein vasohibin , which may have a dual role in inhibiting not only angiogenesis but also fibrogenesis , likely by deactivating hscs .
angiogenesis is best defined as a dynamic , hypoxia - stimulated , and growth factor - dependent process leading to the formation of new blood vessels from preexisting ones.25 , 26 hypoxia and hifs are critical in sustaining the fibrogenic progression of clds , representing a persistent driving force able to directly affect the behavior of hepatic cell populations , including profibrogenic and proangiogenic mfs.1 , 2 , 3 , 4 , 5 , 10 , 11 , 12 , 13 , 16 detection of hypoxic areas is a common finding at any stage of cld , increasing progressively from early injury to the development of cirrhosis , with hypoxia and hifs serving throughout as proangiogenic stimuli in the overall , etiology - independent scenario of chronic wound healing .
cld progression itself is a major contributor to hypoxia due to the formation of regenerative nodules of parenchyma surrounded by evolving fibrotic septa and vascular remodeling that , along with progressive capillarization of the sinusoids , leads to an impairment of oxygen diffusion .
a vicious circle between fibrosis and pathologic angiogenesis is likely to occur10 , 12 , 27 in which parenchymal hypoxia , through the action of hifs , up - regulates expression of wound healing - related factors and mediators that should facilitate liver repair and revascularization .
however , pathologic angiogenesis can be inefficient due to the immaturity and permeability of vegf - induced neovessels and , as a result , may be unable to correct liver hypoxia .
pathologic angiogenesis and hypoxia may act synergistically in disrupting normal tissue repair , thereby promoting the development of liver fibrosis .
the connection between pathologic angiogenesis and fibrogenesis in progressive clds is strongly suggested by their parallel development in all major human forms of cld and animal models of clds , with several laboratories describing high numbers of endothelial cells and microvascular structures within fibrotic septae and in expanded portal areas.1 , 2 , 3 , 4 , 5 , 10 , 11 , 12 , 13 , 16 , 28 , 29 , 30 the response to hypoxia and vegf ( the major proangiogenic target gene ) expression can be seen in liver sinusoidal endothelial cells ( lsecs ) and in hepatocytes1 , 3 , 4 , 10 , 28 and hsc / mfs of developing septa and at the borders of more mature and larger fibrotic septa.29 , 30 studies performed in hif-1 liver conditional knockout mice provided the first definitive in vivo evidence for hypoxia - dependent induction of fibrogenesis .
however , the most convincing evidence relating angiogenesis and fibrogenesis came through in vivo studies indicating that experimental antiangiogenic therapy was highly effective in significantly reducing fibrogenic progression .
as summarized in table 1 , whatever the specific molecule or tool employed , experimental antiangiogenic therapy always resulted in a significant reduction not only of angiogenesis but also of the inflammatory infiltrate , the number of -smooth muscle actin ( -sma)-positive mfs and the extent of fibrosis.32 , 33 , 34 , 35 , 36 , 37 , 38 , 39 , 40 , 41 , 42 in some studies , experimental antiangiogenic therapy also resulted in a significant reduction of portal pressure and collateral vessel formation , as reported with sorafenib in cirrhotic animals34 , 43 and with different molecules and tools in portal hypertensive animals.9 , 24 , 44 , 45 , 46 , 47 , 48 , 49 to our knowledge , only two studies have been published that represent exceptions to this general finding .
the first one was published in 2009 by patsenker et al , who reported that pharmacologic inhibition of integrin 3 by cilengitide , although able to suppress liver angiogenesis , significantly worsened experimental liver fibrosis .
a second experimental and clinical study suggested that the development of liver fibrosis was associated with decreased expression of vegf and sinusoidal rarefication of the fibrotic scar .
these investigators reported that experimental resolution of fibrosis was accompanied by an increase in hepatic vegf levels and revascularization of fibrotic septa , events that were prevented by ablating vegf in myeloid cells or through pharmacologic inhibition of vascular endothelial growth factor receptor type 2 ( vegf - r2 ) signaling .
the question of whether available antiangiogenic drugs might be useful in treating patients with cirrhosis and portal hypertension remains unanswered , with some positive data reported in cirrhotic patients with hcc ; however , several significant concerns were raised related to the known severe side effects of these drugs ( eg , sorafenib ) as well as the increased risk of bleeding reported in cirrhotic patients with hcc . on the other hand , although vegf is unequivocally a critical profibrogenic mediator and has a well - established role in endothelial cell survival and proliferation , it may have also a role in fibrosis resolution . while blocking the interaction between vegf and vegf - r2 does not result ( as hypothesized ) in an improvement or acceleration of fibrosis resolution , the availability of vegf is critical in chemokine ( c - x - c motif ) ligand 9 ( cxcl9)- and matrix metalloprotease 13 ( mmp13)-dependent fibrosis resolution in different murine models of fibrosis .
this apparent paradox is explained by the fact that blocking vegf - r2 effectively blocks vegf - dependent recruitment of mononuclear cells and changes in vascular permeability .
blocking vegf - r2 thereby potentially reduces the recruitment of resolution macrophages , which have an important role in fibrosis reversal .
indeed , in that study , the degree of impairment of fibrosis resolution obtained by blocking vegf - r2 was nearly identical to that seen with macrophage depletion .
chronic liver inflammation is a key requirement for the progression of liver fibrosis , but there is little known about its role in promoting fibrosis - associated angiogenesis .
although inflammation - associated angiogenesis may contribute to both initiation of clds and their progression toward cirrhosis and hcc , the available data refer primarily to hcc and to macrophages in the tumor microenvironment.56 , 57 , 58 , 59 studies performed using specimens from cld patients and from mouse models have shown that chemokine - dependent accumulation of monocyte - derived macrophages represents a relevant mechanism for perpetuating hepatic inflammation and promoting fibrogenesis .
in particular , the chemokine receptor 2 ( ccr2 ) and its ligand ccl2 have a key role in promoting the accumulation of certain monocyte subsets in the liver , in particular ly6c ( gr1 ) monocytes .
macrophages derived from these monocytes release proinflammatory cytokines and can directly activate profibrogenic hepatic mfs .
accordingly , ccl2-dependent infiltrating macrophages are critical in sustaining angiogenesis in experimental models of clds .
a study has shown that these cells promote angiogenic vessel sprouting , particularly in the portal vein system , by releasing vegf - a and other potential proangiogenic factors such as matrix metalloprotease 9 ( mmp9 ) .
this study convincingly demonstrated that angiogenesis is induced even at the initial stages of liver fibrosis and that hepatic neovascularization correlates with fibrosis stage . infiltrating and ccl2-dependent inflammatory cells mediate induction of fibrosis - associated pathologic angiogenesis , and , accordingly , pharmacologic inhibition of ccl2 blocks angiogenesis in experimental fibrosis by inhibiting the infiltration of ccl2-dependent monocyte / macrophages .
hepatic mfs , particularly hsc / mfs , play a critical role in cld progression through their unique ability to act as hypoxia - sensitive and cytokine- and chemokine - modulated cellular mediators at the crossroads between chronic liver injury , inflammation , pathologic angiogenesis , and fibrogenesis.1 , 2 , 3 , 4 , 5 , 10 mfs are critical target cells , able to integrate signals from the microenvironment ( including from hypoxia , contact with altered extracellular matrix , and plasma proteins ) and from surrounding liver cells , acting as both profibrogenic and proangiogenic effector cells ( see figure 1 ) .
we propose that the overall proangiogenic role of mfs is the result of their interactions with surrounding hepatic cells and their exposure to the altered hepatic microenvironment during cld progression . in the next sections , we focus particularly on lsecs and the relevance of the interactions between lsecs , hsc , and hsc / mfs , as well as on both hypoxia - dependent and hypoxia - independent responses of activated mfs . under physiologic conditions ,
the intrahepatic vascular system is composed of different kinds of vessels , including liver sinusoids , portal venules , hepatic arterioles , central venules , and lymphatic vessels .
these vessels are composed of endothelial cells and smooth muscle cells as well as , for liver sinusoids , hscs , which act as liver specific pericytes .
all these cells , plus resident kupffer cells and other blood - derived cells , interact with each other . signaling between these cells maintains sinusoidal homeostasis and
lsecs represent the majority of liver endothelial cells and have a morphologic and functional phenotype that is strikingly different from that of endothelial cells of other vascular units in the liver and elsewhere .
fenestration and a lack of a typical basement membrane are predominant features of the lsec phenotype but are lost in clds.62 , 63 under physiologic conditions , quiescent hscs , which reside in the space of disse and interact through their multiple processes with hepatocytes and lsecs , contribute to vitamin a and retinoid metabolism as well as to deposition and remodeling of the extracellular matrix in the space of disse .
quiescent hscs , which also interact with the axonal processes of autonomic nerve fibers , are liver - specific pericytes , able to respond to a variety of vasoactive mediators and may regulate sinusoidal blood flow through their perisinusoidal processes.62 , 63 , 64 , 65 , 66 the literature suggests that human hscs contract mainly in response to endothelin-1 ( et-1 ) , thrombin , and angiotensin - ii , and relax in response to nitric oxide ( no ) and no donors , prostaglandin e2 , somatostatin , and adrenomedullin.64 , 65 there is considerable support for the paradigm that under conditions of chronic liver injury two features predominate : 1 ) overproduction of et-1 by activated hscs ; and 2 ) a significant reduction in no release by lsecs.62 , 63 , 64 , 65 hscs , which are a major source of et-1 , also represent a target for this vasoactive molecule during liver injury , with et-1 ( either from lsecs or activated hscs , acting in a paracrine or autocrine way ) exerting a prominent contractile effect on hscs and mfs , and likely contributing to portal hypertension in the cirrhotic liver67 , 68 , 69 as well as having a growth inhibitory effect on hsc / mfs . during cld progression , this results in an imbalance in vasoactive mediators and leads to abnormal sinusoids , with remodeling and constriction of the intrahepatic sinusoidal vasculature and a resulting increase in hepatic vascular resistance early in intrahepatic portal hypertension .
as highlighted by iwakiri et al , this differs from events in the mesenteric vascular bed , which are characterized by vasodilation and reduced resistance from up - regulation of vasodilators such as no ; this in turn leads to increased flow to the portal vein .
the combined result of changes in the intrahepatic and mesenteric vasculature is an increase in intrahepatic resistance and portal blood flow from the splanchnic circulation , leading to increased portal pressure and portal hypertension .
regulation of the phenotype of lsecs can be viewed as the net result of the action of different signals that include soluble factors ( mainly vegf , angiopoietins , ephrins , and fibroblast growth factors ) and mechanical forces such as those due to shear stress .
these signals are critical in modulating endothelial no synthase ( enos ) activity in lsecs , resulting in the regulation of flow and vascular tone in the liver sinusoids.62 , 71 lsecs release in a paracrine way several mediators that are involved in maintaining a physiological lsec phenotype but also affect the early response of hscs . for example
, lsecs can produce the cellular isoform of fibronectin in response to injury ; fibronectin has been reported to stimulate hsc synthesis of et-1 .
et-1 , in turn , may differentially affect proliferation of hscs , promoting proliferation in transiently activated hscs but inhibiting it in fully activated hscs.70 , 73 during cld progression , lsec phenotype changes dramatically during the so - called capillarization of sinusoids in association with a reduction in enos activity and no synthesis .
this is possibly due to extensive posttranslational dysregulation of enos as a consequence of changes in phosphorylation or interactions with proteins such as calmodulin , caveolin-1 , and protein kinase b ( akt).74 , 75 the reduction in no release by lsecs is believed to have a more direct role in sustaining fibrogenesis because no contributes to the maintenance of quiescence of hscs and may induce apoptosis or reversal of phenotype of activated hscs , with reduced levels of no facilitating hsc activation.76 , 77 the scenario may be even more complex because hscs can produce no after injury , possibly in response to up - regulation of inducible nos activity induced by proinflammatory cytokines or endotoxemia .
lsecs , through interactions between vegf and vegf - r2 , can release wnt2 and hepatocyte growth factor , which sustain proliferation of hepatocytes ; moreover , a significant amount of hepatocyte growth factor is also released by bone marrow - derived lsec progenitor cells .
lsecs from the livers of mice with biliary fibrosis have been reported to release profibrogenic mediators such as transforming growth factor , bone morphogenetic protein 2 , and pdgf - c and to decrease the release of putative antifibrotic factors such as follistatin and apelin .
increased expression of vegf in specimens from fibrotic or cirrhotic patients colocalizes with areas of hypoxia and is mostly limited to hepatocytes and mfs.28 , 29 , 30 mfs respond to hypoxic conditions through hif-1-dependent up - regulation of vegf and angiopoietin-1 ( ang-1 ) and of their respective receptors vegf - r2 and tie2.29 , 80 , 81 , 82 , 83 , 84 , 85 exposure of hsc / mf to hypoxic conditions also results in hif-1-dependent up - regulation of ccr1 , ccr5 , macrophage migration inhibitory factor , interleukin-13 receptor-1 , prolyl-4-hydroxylase-2 , and ( in part ) placental growth factor , and hif-1-independent increased expression of angiopoietin - like 4 and prolyl-4-hydroxylase-1 .
mfs can also directly respond to vegf and ang-1 , with vegf reported to elicit increased proliferation as well as increased synthesis of extracellular matrix components , and both vegf and ang-1 reported to stimulate oriented migration of hsc / mfs and mf - like cells from bone marrow - derived mesenchymal stem cells.30 , 86 , 87 hypoxia alone is able to induce oriented migration of either hsc / mfs or mf - like cells by eliciting a biphasic mechanism involving 1 ) an early phase of migration , switched on by mitochondria - released reactive oxygen species ( ros ) and requiring redox - dependent activation of extracellular - signal - regulated kinase and c - jun - nh2-terminal kinase isoforms ; and 2 ) a delayed and sustained phase of migration requiring the hif-1-mediated , ros - stabilized increased synthesis and release of vegf.29 , 30 , 86 , 87 overall these data fit well with morphologic evidence from both experimental rodent models of fibrosis and specimens from hepatitis c virus cirrhotic patients.29 , 30 positive staining for both hif-2 and heme oxygenase-1 has been reported in -sma positive mfs in developing septa and at the border of more mature and larger fibrotic septa .
this scenario is similar to the previously described finding that expression of vegf , ang-1 , and related receptors vegf - r2 and tie2 was found at the leading edge of tiny and incomplete developing septa , but not in larger bridging septa .
this suggests that pathologic angiogenesis during the fibrogenic progression of clds proceeds through two different phases : 1 ) an early phase , occurring in developing septa , in which fibrogenesis and angiogenesis are driven or modulated by hsc / mfs and by hypoxia itself ; and 2 ) a later phase , occurring in larger and more mature fibrotic septa , where chronic wound healing is likely to be less active and fibrogenic progression more established .
morphologic analyses indicate that in the late setting proangiogenic factors , particularly receptors like vegf - r2 or tie2 , are expressed only by endothelial cells , a feature that should favor stabilization of the newly formed vessels .
after exposure to hypoxia and proangiogenic mediators released by other hepatic cells , including hepatocytes , mfs may then migrate and align with the leading edge of developing fibrotic septa to drive at the same time fibrogenesis and pathologic angiogenesis , an attractive concept that fits well in the scenario of pathologic angiogenesis.88 , 89 , 90 angiogenesis is controlled by the balance between proangiogenic mediators ( eg , vegf ) and endogenous inhibitors of angiogenesis .
pathologic angiogenesis can result from up - regulation of angiogenic stimuli and concurrent down - regulation of endogenous inhibitors , with a shift of the balance in favor of proangiogenic stimuli.88 , 89 , 90 this general view has been recently integrated , particularly for angiogenesis in clds , by an elegant study investigating the role of vasohibin .
vasohibin is a natural antiangiogenic protein that is selectively induced in endothelial cells by vegf and appears to operate as an intrinsic , highly specific feedback inhibitor of activated endothelial cells engaged in the process of angiogenesis.91 , 92 expression profiling of vasohibin-1 and its relationship with vegf expression , angiogenesis , and fibrogenesis in animal models of cirrhosis and portal hypertension as well as in human cirrhotic liver specimens showed that vasohibin-1 and vegf are up - regulated in mesentery and liver in cirrhotic and precirrhotic portal hypertensive rats and cirrhotic patients .
this suggests that vasohibin-1/vegf cascades are spatially and temporally coordinated through a negative - feedback loop driving pathologic angiogenesis through an overall positive effect on the activation of hscs and fibrogenic progression.24 , 92 paradoxically , the overexpression of vasohibin-1 by adenoviral gene transfer resulted in a significant reduction in pathologic angiogenesis , portal system collateralization , splanchnic blood flow , portohepatic resistance , and portal pressure as well as an attenuation in liver fibrogenesis that was attributed , at least in part , to a vasohibin-1-mediated inhibition of hsc activation.24 , 92 the investigators suggested that ectopic overexpression of vasohibin-1 , unlike endogenous expression of the protein , is not regulated by vegf and therefore can disrupt the negative - feedback loop , resulting in constant but lower levels of vegf synthesis ; this is believed to be sufficient to maintain vascular homeostasis but not pathologic angiogenesis.24 , 92 although several questions remain unanswered , this study is extremely promising particularly because overexpression of vasohibin-1 appeared to affect pathologic angiogenesis without acting on the normal liver vasculature .
although hypoxia represents the most obvious proangiogenic environmental condition , the proangiogenic role of mfs is regulated and modulated by a number of other mediators and events typical of conditions of chronic injury and cld progression , including growth factors , oncogenes , cytokines , chemokines , metabolic or mitochondrial stress , and ros .
most if not all of these mediators , although theoretically hypoxia - independent , result in either increased transcription of hif-1 mrna levels or ( as described for any condition leading to increased intracellular levels of ros ) increased stabilization of hif-1 .
one example of a hypoxia - independent mediator is apelin , the endogenous ligand of angiotensin - like - receptor 1 ( apj ; agtrl1 ) .
apelin and apj are expressed by mfs in cirrhotic animals and have been also detected in the splanchnic vasculature of portal hypertensive rats.48 , 93 , 94 interestingly , apelin synthesis by hsc / mfs is hypoxia - independent and up - regulated by angiotensin ii and et-1 ; moreover , recombinant apelin can directly stimulate collagen - i and pdgf receptor ( pdgf - r ) expression in mf - like cells . as a proof of principle , experimental therapy based on
the use of apelin receptor antagonists resulted in a significant reduction in liver angiogenesis and fibrosis as well as splanchnic angiogenesis and portosystemic collateral vessel formation.40 , 48 , 93 , 94 , 95 two other examples of hypoxia - independent mediators are leptin and pdgf ( particularly pdgf - bb ) .
leptin is an adipocytokine that exerts a profibrogenic effect during the progression from nonalcoholic fatty liver disease ( nafld ) to nonalcoholic steatohepatitis ( nash).7 , 8 , 9 , 10 , 11 , 12 in human hsc / mfs , leptin up - regulates the expression of vegf and ang-1 as well as the proinflammatory chemokine ccl2 .
the action of leptin on hsc / mfs is mediated through its binding to its receptors ( obesity receptor , obr ) and activation of several signaling pathways , including extracellular - signal - regulated kinase , akt , and nuclear factor-b , the latter relevant to chemokine expression .
leptin also operates as a proangiogenic mediator through the recruitment and stabilization of hif-1 and the nuclear translocation of hif-1 . in vivo experiments
the proangiogenic role of leptin in nafld and nash was also confirmed by a study performed on zucker rats ( animals carrying leptin receptor mutations ) receiving the steatogenic choline - deficient and amino acid - defined diet .
the role of pdgf ( known to stimulate proliferation and chemotaxis of mfs , particularly hsc / mfs ) as a proangiogenic mediator was first described by semela et al .
phenotype in hscs by modulating hsc - driven vascular tube formation in vitro and enhanced coverage of sinusoids in vivo , with resulting effects on vascular permeability and pressure regulation .
proangiogenic signaling by pdgf required the involvement of the multifunctional ephrin - b2 receptor tyrosine kinase .
pdgf and leptin have more recently been described as playing a proangiogenic role in human hsc / mfs by sharing a common signaling pathway that leads to up - regulation of vegf expression and release of the protein into the extracellular medium .
this common signaling pathway involves activation of the mammalian target of rapamycin ( mtor ) pathway and generation of intracellular ros by nicotinamide adenine dinucleotide phosphate ( nadph)-oxidase , the latter event being relevant for increased hif-1 stabilization but not for the mammalian target of rapamycin ( mtor ) activation .
finally , another potential role for pdgf was suggested by a study performed in myeloid cell - specific hif-1 or hif-1 knockout mice subjected to bile duct ligation , which suggested that a significant amount of pdgf produced in this experimental model was dependent on the activation of hifs in macrophages .
microparticles ( mps ) are small plasma membrane vesicles surrounded by a phospholipid bilayer that are released by apoptotic or stressed cells and usually contain proteins derived from the parental cells .
mps have been detected in various biological fluids , including peripheral blood and urine . in recent years , it has become clear that mps have important biological functions , including the horizontal transfer of bioactive molecules such as receptors , integrins , growth factors , rnas , and micrornas.100 , 101 circulating mps are increased in animal models of biliary cirrhosis and human cirrhosis and nash , and at least three different studies have linked the release of mps from hepatic cells to a proangiogenic effect in the setting of cld.102 , 103 , 104 , 105 although the cells releasing these proangiogenic mps were different depending on the form of liver disease , mps released by damaged or activated cells contributed significantly to pathologic angiogenesis regardless of their cell of origin .
one study provided evidence that cholangiocytes and hsc / mfs can produce and then release ( mainly in response to pdgf ) mps containing hedgehog ( hh ) ligands .
hh ligands activate hh signaling in endothelial cells , causing significant effects in the bile duct ligation model of fibrosis .
this study found that in the normal liver the low levels of hh ligands released by immature ductular - type progenitors are efficiently counteracted by the expression of hh interacting protein ( hip ) expressed by either quiescent hscs or fenestrated lsecs . in conditions of chronic injury to biliary structures , however , hh interacting protein expression is repressed , and the activation of ductular - type progenitor cells results in pdgf - bb up - regulation and release .
pdgf - bb , in turn , induces hsc / mfs and ductular cells to produce and release hh ligands in mps ; these promote proliferation and survival of cholangiocytes and hsc / mfs as well as induce changes in lsec gene expression .
overall , this results in capillarization of the sinusoids and the release of vasoactive factors such as no , contributing to vascular remodeling in cirrhosis . a second study , focused on the role of mps in nafld and nash , showed that hepatocytes exposed to conditions mimicking the lipid accumulation and lipotoxicity that occurs in the liver during forms of steatohepatitis can also release mps .
these mps from fat - laden hepatocytes were found to act on endothelial cells and , after their internalization , to promote pathologic angiogenesis as shown in vitro and in vivo in mice exposed to a murine model of nafld / nash .
of interest , large numbers of hepatocyte - derived mps were detected in the blood of mice with diet - induced steatohepatitis , and mps levels correlated with disease severity .
genetic ablation of caspase-3 or rna interference directed against vanin-1 ( expressed on mps and mediating contact with lipid rafts of endothelial cells ) protected mice from steatohepatitis - induced pathologic angiogenesis and resulted in a loss of the proangiogenic effects of mps by reducing the extent of fibrosis . finally , a third study suggested that portal mfs release proangiogenic mps that contribute to the vascular changes leading to cirrhosis .
expression of collagen , type xv , 1 ( col15a1 ) by portal mfs was markedly increased in human and rat liver tissue at advanced stages of fibrosis caused by either biliary or hepatocellular injury . in the cirrhotic liver ,
col15a1-expressing portal mfs were detected in an unusual perivascular distribution outlining vascular capillaries proximal to reactive ductules , within large fibrotic septa .
portal mfs were found to release mps containing vegf - a ; this led to increased migration and tubulogenesis of lsecs and human umbilical vein endothelial cells and was able to trigger angiogenesis in matrigel plugs in mice .
cholangiocytes potentiated the angiogenic properties of portal mfs by increasing vegf - a expression and mp shedding .
this suggests that portal mfs have a significant role in angiogenesis and vascular remodeling , releasing vegf - a containing mp , acting as mural cells for newly formed vessels , and driving angiogenesis and scar progression from portal tracts into the parenchyma .
the literature over the last decade has provided an unequivocal mechanistic link between liver angiogenesis and fibrogenesis . in the complex scenario of a progressive cld ,
several major issues predominate in the relationship between angiogenesis and fibrogenesis : 1 ) tissue hypoxia and cellular responses mediated by hifs significantly modulate the phenotype as well as migration and functional responses of all liver cells involved in cld progression ; 2 ) the interactions ( both direct or mediated through the release of several mediators or signals ) between hepatic cells are critical to both angiogenesis and fibrogenesis , with myofibroblasts serving a critical function integrating a variety of cell - derived signals ; and 3 ) mps have an important and increasingly appreciated role in mediating angiogenesis and vascular remodeling .
future studies are needed to further unravel the molecular , cell , and tissue mechanisms linking angiogenesis and fibrogenesis ; ultimately , this has the potential to identify new targets for more selective and effective antifibrotic therapies . | pathologic angiogenesis appears to be intrinsically associated with the fibrogenic progression of chronic liver diseases , which eventually leads to the development of cirrhosis and related complications , including hepatocellular carcinoma .
several laboratories have suggested that this association is relevant for chronic liver disease progression , with angiogenesis proposed to sustain fibrogenesis .
this minireview offers a synthesis of relevant findings and opinions that have emerged in the last few years relating liver angiogenesis to fibrogenesis .
we discuss liver angiogenesis in normal and pathophysiologic conditions with a focus on the role of hypoxia and hypoxia - inducible factors and assess the evidence supporting a clear relationship between angiogenesis and fibrogenesis .
a section is dedicated to the critical interactions between liver sinusoidal endothelial cells and either quiescent hepatic stellate cells or myofibroblast - like stellate cells .
finally , we introduce the unusual , dual ( profibrogenic and proangiogenic ) role of hepatic myofibroblasts and emerging evidence supporting a role for specific mediators like vasohibin and microparticles and microvesicles . |
while it is just a small snapshot of the entire picture of the infection prevention measures needed , it is , nonetheless , an amazingly complex specialist area with its own dynamic . behind the facade of an ostensibly transparent service such as disinfection , as carried out in human medicine and other public institutions ,
lie several decades of research , carefully formulated infection control policies , intelligent marketing strategies and the experiences gleaned by users . at the core of such activities
is always public health protection , prevention of infections by means of microbiologically tested products , while taking into account their effects on humans and the environment .
however , the cost pressures arising from tight budgets in the healthcare sector mean that the need for routine disinfection measures is questioned time and again . at the same time
, increasingly more exacting demands are being addressed to the spectrum of action , human toxicological properties and environmental compatibility .
this in turn means that it is all the more important for the manufacturers and distributors of disinfectants to be able to furnish proof of the quality of their products , while using quality seals to demonstrate this to the user .
in germany , the disinfectants commission of the association for applied hygiene ( vah ) ensures that the user is given a catalog of procedures that meet the stipulated quality requirements . in this respect ,
the commission bases its endeavors on the tried and tested standard methods of the german society of hygiene and microbiology ( dghm ) as well as on european standards . acting in concert with representatives of the disinfectants industry and in agreement with the robert koch institute ,
rules of procedure were drafted for the commission which was set up in 2004 . these take account of the different requirements dictated by the various fields of application , reflecting the quality assurance system in a transparent manner . in addition to the tried and tested conformity assessment procedure , particular emphasis is placed on retesting the products already available on the market .
enormous efforts are being made at national and international level to enhance the power of these tests . in multi - center trials the inter- and intralaboratory fluctuations in the test results
are ascertained , while lending new impetus to the search for suitable standard substances ; culture conditions and detection procedures are standardized and the test procedures and potential influence factors analyzed in detail .
the aim here is to devise test procedures that will yield a reproducible and reliable result independently of the test location .
this result must then be incorporated into an evaluation procedure , reflecting the requirements applicable in everyday practice .
attention must be paid to , inter alia , the prevailing microbial spectrum in quality and quantity , the prevailing organic and inorganic load , material and surface properties and the procedures employed . often ,
these elaborate analyses and methodical approaches can not be dispensed with if one wants to attain the quality seals prescribed .
the test procedures are brought into line with the current state of scientific knowledge successively but consistently , with the aim of improving health protection .
these adaptations call for conscientiousness , tenacity and patience and continuous dialog between research , industry and practice
. they also call upon companies that wish to embrace quality assurance measures to invest time and financial resources and show commitment .
thanks to these joint efforts , we have been better able to meet the demands made by intelligent and selective prophylactic disinfection . at this juncture
i would like to thank all those persons who are helping to structure this quality assurance system at many levels .
molitor , who during my tenure of office as managing director of the disinfectants commission in the dghm and now in the vah , for the support he has given this commission .
his farsightedness , tenacity and assertiveness have set standards that have made an important contribution to the high quality of prophylactic disinfection in germany and throughout europe .
microbiologist , head of department for disinfectant testing at the institute for hygiene and public health at bonn university , secretary of the disinfectant committee of the vah ( association for applied hygiene ) jrgen gebel studied biology at the rheinische friedrich - wilhelm university , bonn and got his diploma in microbiology 1992 . in 1998 he received his degree at the hygiene institute on the subject of : standardisation of microbiological dosimetry in the area of drinking water with uv - rays and stayed at the hygiene institute as microbiologist and head of the department for disinfectant testing .
he became secretary and head of the office of the disinfectant committee of the german society for hygiene and microbiology ( dghm ) 1996 and later on of the disinfectant committee in the association for applied hygiene ( vah ) . since 1998
he is member of the task force cen tc 216 wg1 and their leader since 2004 .
jrgen gebel is assistant chairman of the taskforce chemical disinfectants and antiseptics in human medicine and joint functions in the standard committee ( named ) in din since 1999 .
microbiologist , head of department for disinfectant testing at the institute for hygiene and public health at bonn university , secretary of the disinfectant committee of the vah ( association for applied hygiene ) jrgen gebel studied biology at the rheinische friedrich - wilhelm university , bonn and got his diploma in microbiology 1992 . in 1998 he received his degree at the hygiene institute on the subject of : standardisation of microbiological dosimetry in the area of drinking water with uv - rays and stayed at the hygiene institute as microbiologist and head of the department for disinfectant testing .
he became secretary and head of the office of the disinfectant committee of the german society for hygiene and microbiology ( dghm ) 1996 and later on of the disinfectant committee in the association for applied hygiene ( vah ) . since 1998
he is member of the task force cen tc 216 wg1 and their leader since 2004 .
jrgen gebel is assistant chairman of the taskforce chemical disinfectants and antiseptics in human medicine and joint functions in the standard committee ( named ) in din since 1999 . | in germany , the disinfectants commission of the association for applied hygiene ( vah ) ensures that the user can avail of procedures that meet the stipulated quality requirements .
these requirements are based on the tried and tested standard methods of the german society of hygiene and microbiology ( dghm ) as well as on european standards .
they take account of the different requirements dictated by the various fields of application , reflecting the quality assurance system in a transparent manner .
special emphasis is increasingly placed on retesting the products already available on the market . in multi - center trials the inter- and intralaboratory fluctuations in the test results
are ascertained , the quest for suitable standard substances intensified , culture conditions and detection procedures are standardized and the test procedures and potential influence factors analyzed in detail .
the aim here is to devise test procedures that will yield a reproducible and reliable result independently of the test location , and meet the requirements of everyday practice .
attention must be paid to , inter alia , the prevailing microbial spectrum in quality and quantity , the prevailing organic and inorganic load as well as material and surface properties .
the test procedures are gradually brought into line with the current stock of scientific knowledge , because such a task calls for conscientiousness , tenacity and patience as well as continuous dialog between research , industry and practice .
thanks to these joint efforts , we are increasingly better able to meet the demands made by intelligent and selective prophylactic disinfection . |
a prevailing model explaining cell fate specification during central nervous system ( cns ) development is the combinatorial code , in which cell identity is determined by the expression of specific combinations of transcription factors ( tfs ) .
tf codes controlling cell fate during neurogenesis are indeed well established in many cns regions , such as the drosophila ventral nerve cord or vertebrate spinal cord . in these systems ,
neural progenitors interpret morphogen gradients to acquire distinct tf signatures based on their position in space .
these tf combinations act as a code to endow progenitors with the potential to give rise to the appropriate neuronal subtypes .
particularly prominent are homeodomain tfs from the hox and extended hox families , as well as bhlh tfs of the proneural he s and olig families . while tf combinations represent a powerful and elegant framework for explaining cell fate decisions during cns development , it has proven to be difficult to
decode the most complicated neural lineages . in regions such as the vertebrate neocortex and retina , multipotent progenitors of a given spatial domain
alter their output over time to generate sequences of different types of neuronal and glial cells . in the retina ,
this sequence begins with the production of retinal ganglion cells , followed closely by the overlapping generation of cone photoreceptors , horizontal , and amacrine neurons . as the generation of these cell types peaks , rod photoreceptors begin to be produced . at birth , production of most of the early - born neuronal subtypes
ceases , rod production continues , and bipolar neurons and latest - born mller glia begin to be generated in an overlapping fashion . in the neocortex and hindbrain , evidence suggests that both extrinsic factors , and cell - intrinsic processes are important to control the temporal identity of progenitors . in the developing retina ,
however , even though extrinsic signaling has been shown to alter progenitor output , these factors are thought to mostly control proliferation and act as negative feedback inhibition signals to refine the size of a specific neuronal population , rather than regulate temporal identity in retinal progenitors .
instead , cell - intrinsic processes appear to play a dominant role to control progenitor fate output , both when retinal progenitors were cultured in heterochronic transplants or cell pellets , or when cultured individually .
retinal progenitors can not maintain the same combinatorial tf code during retinogenesis , or their output would be static . yet ,
identification of the key tfs that define the competence state of retinal progenitors at specific phases of development has remained largely elusive . while a great deal of progress has been made in identifying tfs that contribute to the generation of particular cell fates during retinal development , many of these factors , such as lhx2 , pax6 , rx , and vsx2
are expressed in retinal progenitors throughout development and therefore can not explain on their own the sequential order of cell birth .
conversely , fate determinants such as ascl1 , atoh7 , olig2 , onecut1/2 , otx2 , prdm1 ( blimp-1 ) , and sox4/11 exhibit dynamic patterns temporally , but in each case , data suggests that these factors act at the last or penultimate cell division rather than in bona fide multipotent progenitors .
thus , although the collective efforts of a large number of laboratories have identified many tfs that contribute to the combinatorial coding of each retinal cell type , we have little understanding of how multipotent vertebrate retinal progenitors select between them to generate lineages of the appropriate size and complexity .
in contrast , in drosophila , the mechanism controlling temporal competence during development has been well characterized . in neuroblasts of the ventral nerve cord or medulla ,
additional tf cascades operate in time , acting to diversify the tf code established spatially by the actions of morphogens . in the fly ventral nerve cord ,
landmark work had shown that most neuroblasts express a sequence of tfs as development proceeds : hunchback , krppel , nub / pdm2 ( collectively pdm ) , and castor ( fig .
these tfs are necessary and sufficient to confer temporal identity to neuroblasts , such that they produce daughter cell types in the correct sequence .
( a ) roles of the temporal tfs hunchback ( hb ) and castor ( cas ) in the sequential generation of cell fates during development of the drosophila neuroblast nb71 motoneuron lineage .
( b ) analogous roles of the murine hb and cas orthologues ikzf1 and casz1 in regulating the temporal competence of retinal progenitors .
casz1 might function by suppressing the competence of genes to be expressed rather than by directly controlling target gene levels .
elucidation of the co - factor complexes utilized by casz1 should allow this hypothesis to be addressed .
this model is not meant to be exclusive , and alternative factors not shown here could be involved .
( a ) roles of the temporal tfs hunchback ( hb ) and castor ( cas ) in the sequential generation of cell fates during development of the drosophila neuroblast nb71 motoneuron lineage .
( b ) analogous roles of the murine hb and cas orthologues ikzf1 and casz1 in regulating the temporal competence of retinal progenitors .
casz1 might function by suppressing the competence of genes to be expressed rather than by directly controlling target gene levels .
elucidation of the co - factor complexes utilized by casz1 should allow this hypothesis to be addressed .
this model is not meant to be exclusive , and alternative factors not shown here could be involved .
previously , we had shown that the zinc finger tf ikzf1 ( ikaros ) , an ortholog of drosophila hunchback , was sufficient to impart late retinal progenitors with the competence to generate early - born neuronal subtypes , as well as being required for the generation of normal numbers of early - born fates during development . because ikzf1 had no effect on retinal progenitor proliferation
, these results indicated that ikzf1 does not control the timing of cell cycle exit , but instead directly confers early temporal identity in retinal progenitors , which are biased to give rise to early - born neurons when they express ikzf1 ( fig .
interestingly , recent work showed that ikzf1 misexpression in cortical progenitors resulted in prolonged period of production of early - born neurons well into the late temporal identity window .
although ikzf1 was not absolutely required for early - born neuronal cell type production in the developing cortex , these results suggest that ikzf1 could be a general factor regulating early temporal identity in vertebrates , much like hunchback is essential to control early temporal identity in multiple fly neuroblast lineages .
while ikzf1 and hunchback appear to function analogously , it remained unclear how ikzf1 achieved these effects in the retina . whereas drosophila hunchback operates within the context of a tf cascade that changes over time ,
indeed , a single homolog of the drosophila zinc finger tf castor , called casz1 , was previously shown to be expressed in later phases of retinogenesis , as well as in differentiated photoreceptors .
we therefore hypothesized that an ikzf1 casz1 sequence might represent conservation of the drosophila temporal code . to test this hypothesis , we recently used mouse genetics and retroviral lineage tracing to study casz1 function during retinogenesis .
we showed that casz1 is not expressed at significant levels at early stages , but is upregulated in retinal progenitors during mid / late stages of retinal development , suggesting a role in controlling temporal identity at these later stages ( fig .
consistently , we found that precocious misexpression of casz1 in retinal progenitors was sufficient to increase the production of mid / late fates like bipolar cells and rod photoreceptors , at the expense of early fates such as cone photoreceptors , horizontal cells , and amacrine cells .
conversely , analysis of retinal progenitor cell lineages in conditional casz1 knockout retinas showed increased production of early - born fates at the expense of rod photoreceptors within a lineage .
importantly , similar to what was observed with ikzf1 , the cell fate changes observed following casz1 manipulations were independent of an effect on proliferation or cell death , indicating that casz1 directly controls temporal identity in retinal progenitors and not cell cycle exit .
interestingly , we also found that ikzf1 normally represses casz1 expression in retinal progenitors , and identified the cis - regulatory modules in casz1 that can mediate the transcriptional effects of ikzf1 .
together these results suggest that , much like in drosophila neuroblasts , a cross - regulatory mechanism of tf expression operate to control temporal identity progression in mouse retinal progenitor cells .
although the biochemical activities underlying casz1 function have been studied to some extent in the developing heart , muscle and vascular systems , where casz1 is thought to control progenitor proliferation and differentiation , the mechanism by which casz1 confers temporal identity in neural progenitors remains unknown . in drosophila neuroblasts , cas was initially suggested to function as a transcriptional repressor , but a mechanistic understanding of its activities remains incomplete . in an effort to provide some clues about casz1 mechanism of action , we recently carried out a gene expression profiling experiment using rna - seq on casz1 knockout retinal progenitors .
however , this approach failed to identify strong effects on cell fate determinants that could explain casz1 function ( mattar and cayouette , unpublished data ) . although changes were observed in the levels of transcripts for genes that are known to control the production of cell types affected by casz1 conditional deletion , the changes observed were no larger than 5-fold for any of them .
these results suggest that casz1 might not directly modify the combinatorial tf code , but instead could function by mediating more subtle epigenetic changes .
indeed , castor proteins have recently been shown to associate with a variety of transcriptional regulators , including the mi-2/nurd and polycomb repressor complexes .
intriguingly , previous work has shown that mi-2/nurd is required for ikzf1-mediated fate determination functions in lymphocytic development .
similarly , in fly neuroblasts and mouse cortical or retinal progenitors , polycomb repressive complexes have been shown to regulate temporal identity , suggesting that temporal tfs might regulate competence via common epigenetic pathways .
in such a model , casz1 could act by altering the probability and/or competence of specific fate determination genes to be transcribed , rather than acting as a straightforward modifier of tf expression levels ( fig .
consistent with this hypothesis , changes in the temporal competence of drosophila neuroblasts are controlled by alterations in the competence of fate determinant genes to be expressed . whether casz1 could function together with mi-2/nurd and/or polycomb to provide
target gene specificity for chromatin modifications remains unknown , but this is certainly an interesting possibility .
other potential explanations for the effects of ikzf1/casz1 on progenitor competence are of course possible , based on the identification of factors that have also been shown to regulate progenitor temporal identity or progression . in xenopus
laevis , dicer , a key determinant of microrna biosynthesis and function , was shown to control the correct onset of markers of late - born cell types , and several micrornas were implicated in this effect .
subsequently , a role for dicer and the micrornas let7 , mir9 , and mir125 in the progression of retinal progenitor temporal identity from early- to late - phase was uncovered .
these effects may be mediated in part by protogenin and lin28 , which were shown to be targets of the micrornas .
moreover , the requirement for dicer / micrornas in the progression of temporal competence states is very similar to that of lhx2 , which when mutated , stalls retinal progenitors in the earliest phase of neurogenesis .
whether there are mechanistic connections between ikzf1/casz1 , dicer / micrornas , and lhx2 remains unclear , but in c. elegans , the hunchback ortholog hbl-1 and the let-7 microrna cross - regulate each other , with hbl-1 repressing let-7 expression , and let-7 binding and inhibiting the hbl-1 3 ' utr . if conservation of this negative feedback loop is maintained in the retina , this might suggest a possible point of convergence for these pathways . alternatively , these pathways might act independently , providing redundancy and robustness to the system . elucidating
how temporal identity factors function in vertebrate neural progenitors to control the production of specific cell types associated with a given developmental window will be important , as this knowledge could provide novel ways of manipulating stem cell differentiation for cell replacement therapies . a recent study ,
for example , reported that tgf functions as a regulator of temporal identity in hindbrain progenitors and provided proof of concept that manipulating tgf can be applied to control temporal specification of specific neuronal types from stem cells .
this work was supported by research grants from the foundation for fighting blindness canada , and the canadian institutes of health research ( mop-77570 ) .
pm was supported by a cihr postdoctoral fellowship , and mc is a senior fellow of the fond de la recherche du qubec | while much progress has been made in recent years toward elucidating the transcription factor codes controlling how neural progenitor cells generate the various glial and neuronal cell types in a particular spatial domain , much less is known about how these progenitors alter their output over time . in the past years ,
work in the developing mouse retina has provided evidence that a transcriptional cascade similar to the one used in drosophila neuroblasts might control progenitor temporal identity in vertebrates .
the zinc finger transcription factor ikzf1 ( ikaros ) , an ortholog of drosophila hunchback , was reported to confer early temporal identity in retinal progenitors and , more recently , the ortholog of drosophila castor , casz1 , was found to function as a mid / late temporal identity factor that is negatively regulated by ikzf1 .
the molecular mechanisms by which these temporal identity factors function in retinal progenitors , however , remain unknown . here
we briefly review previous work on the vertebrate temporal identity factors in the retina , and propose a model by which they might operate . |
current rates of species extinction have been inferred to be orders of magnitude above background levels , based on well known groups with a fossil record , but whether these taxa are representative for biodiversity in general is not known .
there is concern that we are currently entering a mass extinction event comparable to the previous five greatest mass extinctions in the history of life .
currently , most human - induced extinctions result from habitat destruction and overexploitation of species .
climate change caused by greenhouse gas emissions may become a similarly severe threat , and will add to present challenges for global biodiversity . the ability to assess the potential species loss
the number of species on earth is not known even to the order of magnitude , and the geographic distributions and ecology is poorly known for most taxa described by science .
extinctions of species where climate change is thought to be the ultimate cause may have already occurred , but pinpointing the ultimate cause of an extinction event is difficult . to assess the magnitude of the problem in the future , modelling is required . despite the challenge of incomplete knowledge ,
rough predictions of the number of species threatened with extinction by climate change have been made [ 8 - 10 ] .
the general method in such predictions has been to project the degree of habitat loss expected as a result of geographic range adjustments in response to climate change , and then use species - area equations to assess potential species loss as a result of range contractions .
depending on the climate change scenario used and dispersal ability of species , thomas et al
. projected that 18 - 35% of species are at risk of extinction in their sample of regions across the earth .
the paper and similar efforts are controversial , however , and both the methods and the way the results have been presented in the media have been criticized .
nevertheless , realistic predictions of climate - driven extinction risk are needed for climate mitigation and species conservation efforts .
the accuracy of extinction risk predictions depends on the ability to project how species will adjust to climate change . to survive climate change that is beyond its tolerance
, a species may either adapt to the new climate locally , or shift its geographical distribution to climatically suitable areas .
a critical question is whether species will be able to evolve tolerance to new climatic conditions at the pace of climate change .
species distribution models utilize species distribution records and correlations with climate to project where species will occur in the future under the assumption of no evolutionary change .
these models are not only used to predict extinction risk , but by providing projections of where species may occur in a future climate , they form the basis of adaptation strategies to conserve biodiversity .
when the ability of species distribution models to predict future distributions has been checked , however , their performance has been less than satisfying , although the methods are rapidly improving .
reasons for poor model performance and inconsistencies among models include variation among climate change scenarios , poor species distribution data , variation in modelling methods , and violation of model assumptions .
one way of gaining insight into the constraints and opportunities offered by extinction risk assessments is to examine how species have responded to similar climatic challenges in the past , and to examine how biodiversity is distributed at large spatial scales and the underlying causes of this distribution , sometimes referred to as
below i highlight recent advances in the knowledge of how species adjust their ranges in response to climate change now and in the past , which may help assess the risk for climate - induced species extinctions .
if species can evolve rapidly enough in response to climate change , projections of extinction risk based on species distribution modelling may be overestimated .
recent empirical evidence suggests that evolution is responsive to climate variation and may occur at rates that make evolutionary change a plausible alternative to geographical range shifts .
adaptive evolution in local populations may , however , be counteracted by competition from immigrating organisms pre - adapted to the new climate . the dominant response to quaternary climate changes comprised geographical range shifts rather than morphological adaptation in situ .
both in situ persistence and range shifts may involve evolutionary processes that enhance adaptations to new climatic conditions - for example , by selection for enhanced tolerance in migrating populations .
an increasing body of data documents that species and assemblages respond to warming as predicted by models , adjusting their ranges polewards and towards higher elevations .
this lends support to bioclimatic modelling approaches and projections of species extinction . the most common data record latitudinally expanding range limits of temperate taxa colonizing habitats that have become climatically suitable [ 21 - 23 ]
. records of latitudinal range contractions are mostly anecdotal , but a few recent studies document elevational range shifts [ 25 - 27 ]
. range shifts on tropical mountains in response to warming raise double concerns ; many high - elevational species are at risk of becoming extinct if mountains are too low to offer them habitat after the climate zones have shifted upwards .
in addition , tropical lowlands might be depleted of species as more and more species shift their ranges towards higher latitudes and elevations .
data on range shifts from tropical mountains have been mostly missing , but recently chen et al . documented 102 insect species shifting their distributions an average of 62 m upwards on mount kinabalu , borneo , between 1965 and 2007 .
raxworthy et al . found that 30 species of reptiles and amphibians had shifted their elevational midpoints upslope by 19 - 51 m on madagascar , in agreement with recent regional warming .
some climate types are predicted to completely disappear , threatening species assemblages living in these vanishing environments with extirpation . in the majority of cases , however , the environments of species will shift geographically to various degrees , necessitating modelling of range adjustments and extinction risk .
beale et al . found that null models of species - climate associations established using bioclimatic models were no better than chance for 68 of 100 european bird species .
many factors other than climate , such as species interactions and human impact , set the limit of species ranges , all contributing to weaken species - climate associations . if species do not occur everywhere their climatic requirements are met ( global dispersal ) due to migration barriers , this will also reduce the ability of projecting future geographic distributions . as a result ,
modelled climatic tolerances will be more narrow than real ones , overstating extinction risk . on the other hand
, such a situation also implies that many species are likely to have problems colonizing new areas that become climatically suitable .
svenning and skov found that european tree species occupy only 38% of their calculated potential climatic ranges .
frequent successful naturalizations of tree species outside their native ranges confirm the climatic suitability of modelled potential ranges . if dispersal limitation is common , species or species diversity patterns can not be expected to be in steady - state with contemporary climate .
the pattern of diversity for european tree species with restricted ranges was best explained by climatic conditions occurring at the last glacial maximum about 21,000 years ago , whereas the diversity of widely distributed species correlates with present day climate .
this suggests that some species were unable to expand their ranges following deglaciation and holocene warming to occupy their potential niches .
this interpretation is supported by the demonstration that species richness of tree species in northern europe can be explained by differential rates of dispersal out of glacial refugia . in line with these results ,
arajo et al . demonstrated that species richness of narrow - ranging amphibians and reptiles in europe was better explained by climatic stability between the last glacial maximum and the present day than by the present climate .
a question related to dispersal limitation in explaining present distributions is whether species will be able to keep up with future climate change .
models of species ' future distributions usually assume either full adjustment of species ranges - that is , that species can colonize any suitable habitat - or no range adjustment at all , implying only range contraction in response to climate change .
the finding that species richness of butterflies in britain increases with climate change , but that species richness change lags behind climate change sheds some light on this question .
generalist species responded quicker than specialist ones , suggesting that species with high specialization and low vagility will have the greatest difficulties in tracking their habitat .
lag times should be longer and dispersal barriers more difficult to overcome in taxa less vagile than butterflies . when using information on how species responded to past climatic shifts , we are faced with a conundrum .
climate shifts during the quaternary were thought to be as rapid and large as those expected during the 21st century . despite that
filtered by a previous challenge or catastrophe usually experience little extinction when faced by similar subsequent challenges . for example , the first continental glaciation in europe in the plio - pleistocene caused a wave of extinction in , for example , tree species , but the following glacial periods resulted in few extinctions , despite an increase in the amplitude of climate shifts .
the first cooling event pruned the european tree species pool of cold - intolerant taxa , whereas the remaining taxa could persist . secondly
, present global climate is close to the peak average temperature attained during the quaternary .
many species have never experienced such a situation , potentially resulting in extinction of intolerant taxa .
thirdly , during previous climatic shifts , species were able to shift their distributions without interference from humans .
humans now dominate most ecosystems , resulting in barriers to dispersal and combined threats of climate change , habitat destruction and overexploitation of species . the combined effect of human overexploitation and climate change has been implicated in the extinction of the megafauna in eurasia and america at the beginning of the holocene .
bioclimatic models show that the ranges of many species , such as the woolly mammoth , drastically declined during deglaciation , but megafaunal species had survived many similar climate shifts in the past , and the coup de grce was probably caused by human hunting . where are species most at risk from climate change ? given that observed and projected climate change is larger towards the poles , most attention has been directed towards high - latitude environments , with polar bears as iconic species in the public mind . depending on the vulnerability of species , geographic patterns of extinction risk may , however , deviate from geographic patterns of warming .
deutsch et al . showed that tropical insects are more sensitive to warming than high - latitude ones , because they already live close to their limit of climatic tolerance , whereas the fitness of many high - latitude species may increase as a result of warming .
moreover , tropical assemblages contain more species with poor dispersal ability , high specialization and small geographic ranges , all characteristics that make them vulnerable to climate change .
mccain recently found support for janzen 's hypothesis that the elevational range sizes of organisms are smaller on tropical than high - latitude mountains , making them more vulnerable to warming and upslope range shifts .
narrow range - sizes also mean that for many tropical mountain species there will be no overlap between present and projected future distributions , increasing the risk for dispersal failure and extinction . in an assessment of global extinction risk in birds , jetz et al .
projected many extinctions of tropical narrow - ranged species , although land conversion is presently a greater threat than climate change . as a result of narrow climatic tolerances
, relatively small temperature changes may have large effects on species distributions in the tropics .
in a record of forest turnover during 48,000 years in the amazon basin , forest cover was found to be continuous , but a temperature change of only 1c during the transition from glacial to holocene resulted in a large turnover in species composition .
if species can evolve rapidly enough in response to climate change , projections of extinction risk based on species distribution modelling may be overestimated .
recent empirical evidence suggests that evolution is responsive to climate variation and may occur at rates that make evolutionary change a plausible alternative to geographical range shifts .
adaptive evolution in local populations may , however , be counteracted by competition from immigrating organisms pre - adapted to the new climate . the dominant response to quaternary climate changes comprised geographical range shifts rather than morphological adaptation in situ .
both in situ persistence and range shifts may involve evolutionary processes that enhance adaptations to new climatic conditions - for example , by selection for enhanced tolerance in migrating populations .
an increasing body of data documents that species and assemblages respond to warming as predicted by models , adjusting their ranges polewards and towards higher elevations .
this lends support to bioclimatic modelling approaches and projections of species extinction . the most common data record latitudinally expanding range limits of temperate taxa colonizing habitats that have become climatically suitable [ 21 - 23 ] .
records of latitudinal range contractions are mostly anecdotal , but a few recent studies document elevational range shifts [ 25 - 27 ] .
range shifts on tropical mountains in response to warming raise double concerns ; many high - elevational species are at risk of becoming extinct if mountains are too low to offer them habitat after
in addition , tropical lowlands might be depleted of species as more and more species shift their ranges towards higher latitudes and elevations .
data on range shifts from tropical mountains have been mostly missing , but recently chen et al . documented 102 insect species shifting their distributions an average of 62 m upwards on mount kinabalu , borneo , between 1965 and 2007 .
raxworthy et al . found that 30 species of reptiles and amphibians had shifted their elevational midpoints upslope by 19 - 51 m on madagascar , in agreement with recent regional warming .
some climate types are predicted to completely disappear , threatening species assemblages living in these vanishing environments with extirpation . in the majority of cases , however , the environments of species will shift geographically to various degrees , necessitating modelling of range adjustments and extinction risk .
beale et al . found that null models of species - climate associations established using bioclimatic models were no better than chance for 68 of 100 european bird species .
many factors other than climate , such as species interactions and human impact , set the limit of species ranges , all contributing to weaken species - climate associations .
if species do not occur everywhere their climatic requirements are met ( global dispersal ) due to migration barriers , this will also reduce the ability of projecting future geographic distributions . as a result , modelled climatic tolerances will be more narrow than real ones , overstating extinction risk .
on the other hand , such a situation also implies that many species are likely to have problems colonizing new areas that become climatically suitable .
svenning and skov found that european tree species occupy only 38% of their calculated potential climatic ranges .
frequent successful naturalizations of tree species outside their native ranges confirm the climatic suitability of modelled potential ranges . if dispersal limitation is common , species or species diversity patterns can not be expected to be in steady - state with contemporary climate .
the pattern of diversity for european tree species with restricted ranges was best explained by climatic conditions occurring at the last glacial maximum about 21,000 years ago , whereas the diversity of widely distributed species correlates with present day climate .
this suggests that some species were unable to expand their ranges following deglaciation and holocene warming to occupy their potential niches .
this interpretation is supported by the demonstration that species richness of tree species in northern europe can be explained by differential rates of dispersal out of glacial refugia . in line with these results ,
arajo et al . demonstrated that species richness of narrow - ranging amphibians and reptiles in europe was better explained by climatic stability between the last glacial maximum and the present day than by the present climate .
a question related to dispersal limitation in explaining present distributions is whether species will be able to keep up with future climate change
. models of species ' future distributions usually assume either full adjustment of species ranges - that is , that species can colonize any suitable habitat - or no range adjustment at all , implying only range contraction in response to climate change .
the finding that species richness of butterflies in britain increases with climate change , but that species richness change lags behind climate change sheds some light on this question .
generalist species responded quicker than specialist ones , suggesting that species with high specialization and low vagility will have the greatest difficulties in tracking their habitat .
lag times should be longer and dispersal barriers more difficult to overcome in taxa less vagile than butterflies . when using information on how species responded to past climatic shifts , we are faced with a conundrum .
climate shifts during the quaternary were thought to be as rapid and large as those expected during the 21st century . despite that
first , assemblages that have been filtered by a previous challenge or catastrophe usually experience little extinction when faced by similar subsequent challenges .
for example , the first continental glaciation in europe in the plio - pleistocene caused a wave of extinction in , for example , tree species , but the following glacial periods resulted in few extinctions , despite an increase in the amplitude of climate shifts .
the first cooling event pruned the european tree species pool of cold - intolerant taxa , whereas the remaining taxa could persist . secondly
, present global climate is close to the peak average temperature attained during the quaternary .
many species have never experienced such a situation , potentially resulting in extinction of intolerant taxa .
thirdly , during previous climatic shifts , species were able to shift their distributions without interference from humans .
humans now dominate most ecosystems , resulting in barriers to dispersal and combined threats of climate change , habitat destruction and overexploitation of species . the combined effect of human overexploitation and climate change has been implicated in the extinction of the megafauna in eurasia and america at the beginning of the holocene .
bioclimatic models show that the ranges of many species , such as the woolly mammoth , drastically declined during deglaciation , but megafaunal species had survived many similar climate shifts in the past , and the coup de grce was probably caused by human hunting .
where are species most at risk from climate change ? given that observed and projected climate change is larger towards the poles , most attention has been directed towards high - latitude environments , with polar bears as iconic species in the public mind . depending on the vulnerability of species , geographic patterns of extinction risk may , however , deviate from geographic patterns of warming .
deutsch et al . showed that tropical insects are more sensitive to warming than high - latitude ones , because they already live close to their limit of climatic tolerance , whereas the fitness of many high - latitude species may increase as a result of warming .
moreover , tropical assemblages contain more species with poor dispersal ability , high specialization and small geographic ranges , all characteristics that make them vulnerable to climate change .
mccain recently found support for janzen 's hypothesis that the elevational range sizes of organisms are smaller on tropical than high - latitude mountains , making them more vulnerable to warming and upslope range shifts .
narrow range - sizes also mean that for many tropical mountain species there will be no overlap between present and projected future distributions , increasing the risk for dispersal failure and extinction . in an assessment of global extinction risk in birds , jetz et al .
projected many extinctions of tropical narrow - ranged species , although land conversion is presently a greater threat than climate change .
as a result of narrow climatic tolerances , relatively small temperature changes may have large effects on species distributions in the tropics . in a record of forest turnover during 48,000 years in the amazon basin ,
forest cover was found to be continuous , but a temperature change of only 1c during the transition from glacial to holocene resulted in a large turnover in species composition .
even if the assumptions in bioclimatic models are frequently violated , they remain a primary strategy to project the future of biodiversity .
the methods are also developing rapidly , with prospects of accounting for variability among models and relaxing some of the critical assumptions .
information on how species responded to climate change in the past serves as a reality check of model projections .
further advances could be made by taking advantage of new knowledge about past climate change .
for example , studies of species responses to pleistocene climate oscillations have focused on the role of refugia , with recent interest in microrefugia ( survival of taxa in small habitat pockets ) , to explain the speed of post - glacial recolonization .
the concept of microrefugia should also be relevant in projecting the effects of climate change .
survival in small patches may be incorporated into models , and management and creation of microrefugia could be used as an active adaptation strategy . an area in need of more
. some species may be more tolerant than indicated by present species - environment relationships , whereas others live close to their tolerance limits .
much of our present knowledge is derived from studies of europe and north america , which may not be relevant for the rest of the world , such as the hotspots of species richness and endemism where most of earth 's biodiversity reside .
rapid progress in this field is urgently needed to avoid the prospect of a wave of climate change - induced extinction becoming inevitable .
| human - induced climate change may threaten a large proportion of earth 's biota , but the uncertainties involved in projecting the future geographical distributions of species make quantitative predictions of extinction risk difficult to make .
i discuss how insight from recent advances in macroecology and knowledge about species responses to past climate change can help predict extinction risks more accurately . |
tp53 ( tumor protein p53 ) is the paradigm of tumor suppressors and the most frequently mutated gene in cancer ( present in about 50% of cancers ) .
alteration of the p53 gene is often associated with environmental mutagens such as ultraviolet ( uv ) light or smoking . as a result of sun exposure ,
the incidence of p53 mutations is strikingly high in skin squamous carcinomas ( 8090% ) . in spite of these numbers ,
the role of p53 in human tissue renewal , including epidermal homeostasis , remains intriguing .
since experimenting in humans in vivo is not possible for obvious reasons , mouse models have become a strong reference for skin research .
however , as observed for other key genes , p53 knockout ( ko ) mice develop normally .
in addition , mouse and human skin displays different physiological features . to address the role of p53 in human skin using new approaches ,
we have adapted the shrna technology for the inactivation of endogenous proteins in human primary keratinocytes .
we describe the consequences of silencing p53 in these epidermal cells in a recent issue of cell reports .
we can now achieve efficiencies of about 90% for cell transduction with lentiviral vectors , which is the closest we can get to human ko epidermis . in steady - state healthy epidermis , p53 is thought to play a role in cell differentiation .
however , contrary to our expectations , we found that knockdown of p53 in human keratinocytes favored squamous differentiation .
we have proposed a novel model of the keratinocyte epidermal cell cycle in which differentiating cells do not undergo quiescence but instead continue post - mitotic dna replication in the absence of cell division ( endoreplication ) .
mitotic slippage. this term is somewhat controversial but in our opinion it expresses well the following sequence of events that occur during endoreplication : ( 1 ) in the case of difficulty or danger , mitotic checkpoints block cell division ; ( 2 ) these cells are unable to properly stay in mitosis ( g2/m ) ; ( 3 ) as a result , they slip through the g2/m checkpoints and restart dna replication ; ( 4 ) however , as the cells can not divide , they become polyploid .
endoreplication is controversial within the field of skin biology because it is still a poorly understood concept despite some very good reviews that highlight its biological importance .
defining endoreplication and explaining its potential functions is often a challenge . nonetheless , endoreplication might be more widespread in human tissues than we think .
we have shown that in keratinocytes mitotic slippage occurs in response to cell cycle deregulation that causes dna replication errors ( replication stress ) and triggers mitotic checkpoints .
our current paper in cell reports we believe that we answer 2 questions regarding epidermal homeostasis : ( 1 ) the function of p53 in normal keratinocytes ; and ( 2 ) the importance of endoreplication against precancerous mutations .
p53 is upregulated by uv irradiation and is thought to trigger apoptosis of sunburn cells but , as mentioned above ,
we also overexpressed a temperature - sensitive form of p53 that behaves as an inactive mutation at 39c or as the active wild - type protein at 32c .
similar to our observations for the knockdown , overexpression of the inactive conformation triggered terminal differentiation .
p53 is a critical keeper of cell cycle pace , thus its absence caused cell cycle deregulation leading to replication stress , mitotic slippage , and endoreplication .
this in turn triggered squamous differentiation , therefore forcing mutant cells to detach ( fig . 1 ) .
interestingly , at 32c the wild - type conformation attenuated differentiation , indicating that p53 protects the proliferative compartment and putatively the stem cells .
p53 surveys for correct execution of the cell cycle in epidermal homeostasis ( a ) .
loss of p53 function as a single mutation causes mitotic slippage , squamous differentiation , and cell shedding , thus maintaining homeostasis ( b ) , or contributes to malignancy when additional mutations ( m2 ) affect cell division control and allow p53-deficient cells to divide ( c ) .
p53 surveys for correct execution of the cell cycle in epidermal homeostasis ( a ) .
loss of p53 function as a single mutation causes mitotic slippage , squamous differentiation , and cell shedding , thus maintaining homeostasis ( b ) , or contributes to malignancy when additional mutations ( m2 ) affect cell division control and allow p53-deficient cells to divide ( c ) .
mitotic slippage , replication stress , and endoreplication are all fairly new concepts that we will probably read increasingly more about in the near future as they most likely have a role in cancer .
we propose that these events represent a mitosis - differentiation checkpoint ( mdc ) , a tool used by normal skin to remove precancerous cells bearing irreparable damage .
even though skin carcinomas are the most common malignancies , their incidence seems low considering that cells in the epidermis are continuously dividing and are chronically exposed to the mutagenic power of uv light .
therefore , the skin must have powerful protective mechanisms in play when the level of pigment ( melanin ) is not sufficient and the dna repair machinery can not repair the damage produced .
mutations of p53 are highly frequent in skin carcinomas yet we propose the existence of a protective mechanism in normal skin . how can both concepts be reconciled ?
inactivation of p53 in the whole mouse did not provoke early skin carcinomas although the animals died by 4 months of age from other types of cancer .
however , skin - specific p53 ko mice did develop squamous cell carcinomas from 4 months onwards .
it therefore seems likely that additional mutations are required for the loss of p53 to be tumorigenic .
our model proposes a second guardian of the genome through the cell division block imposed by the mdc .
if this checkpoint is mutated , the additional absence of p53 , the original guardian of the genome , makes those cells more malignant ( fig . 1 ) .
our data point to a homeostatic maintenance role of endoreplication that might be common to other endoreplicating tissues .
the list of these tissues in the human body has increased to include megakaryocytes , hepathocytes , endometrium , keratinocytes , and cardiomyocytes and may expand further in the future .
finally , we interpret our data to mean that skin carcinoma cells are malignant not simply because their cell cycle is deregulated but because , in addition , they are able to divide .
we apologize that we could not cite every relevant publication because of length limitations ; many missing articles are cited within the papers we do list .
we thank the instituto de salud carlos iii for their support , grant fis pi11/02070 . | the role of p53 , the original guardian of the genome , in skin has remained elusive .
we have explored p53 function in human epidermal cells and demonstrated the importance of a mitosis - differentiation checkpoint to suppress potentially precancerous cells . this model places epidermal endoreplication as an antioncogenic mechanism in the face of irreparable genetic alterations . |
premenstrual syndrome ( pms ) is a set of somatic and psychological symptoms which occur during the luteal phase of the menstrual cycle ( 1 ) . up to 85% of women of reproductive age report experiencing one or more of the symptoms of pms ( 2 ) and approximately 5%
suffer from a severe form of pms called premenstrual dysphoric disorder ( pmdd ) ( 3 ) .
the exact cause of pms still remains unclear and many factors are supposed to contribute to the condition ; therefore , it has been proposed that pms is a multicausal problem ( 4 ) . according to the neurobiological data on the subject
, the activities of the neurotransmitter system are mainly affected by gonadal steroids . through this process
, estrogens , progestins , and androgens may be indirectly implicit in the development of depression ( 5 , 6 ) .
although several studies have focused on the hormone and lipid profiles of women with pms , they report findings that are controversial . for example , several studies that have assessed the connection between pms and testosterone levels , present findings that conflict ( 7 , 8) .
similarly , there is also disagreement over the association between prolactin ( prl ) levels and pms ; while benedek - jaszmann and hearn - sturtevant reported higher levels of prl in women with pms ( 9 ) , backstrom and aakvaag failed to find any association ( 10 ) .
there are also conflicting data on the mean concentration of cholesterol among women with and without pms ( 11 ) . by conducting epidemiological and clinical studies ,
many researchers have uncovered conflicting results regarding the association between estradiol ( e2 ) , follicle - stimulating hormone ( fsh ) , testosterone ( t ) , dehydroepiandrosterone ( dhea ) , and dehydroepiandrosterone sulfate ( dhea - s ) levels and depression ( 12 - 15 ) . to date , few studies have evaluated the association between hormonal and metabolic factors and pms .
most of the results were restricted by their small sample size , inappropriate inclusion criteria for women with pms , and by their comparison of a limited number of variables between the two groups of women , both with and without pms ( 10 , 16 ) .
as a result , in the present study we aimed to investigate the association of hormonal and metabolic factors with pms among iranian women of reproductive age .
the subjects of this study were selected from among the participants in the iranian pcos prevalence study which was a community based cross - sectional study of 1026 women , aged 18 - 45 years , conducted between 2009 - 2010 ( 17 ) .
the eligible women were invited to participate in a comprehensive interview and their blood pressure , anthropometric , hormonal , and metabolic measurements were documented .
data were completed for all but 97 women who did not come to the clinics and 19 participants whose hormonal and metabolic profiles were unavailable .
we also excluded women who were using antidepressants ( n = 34 ) , oral contraceptive pills ( n = 151 ) , or taking hormonal medication for irregular menses . furthermore , those women who were pregnant at the time of the study ( n = 43 ) and menopausal women ( n = 37 ) were also excluded .
finally , after the exclusion of the women who did not meet our inclusion criteria , 656 women were enrolled in the study . the american college of obstetricians and gynecologists ( acog ) criteria were used to diagnose pms .
according to these criteria , the women needed to experience at least one of each of the following affective symptoms ( depression , angry outbursts , irritability , anxiety , confusion , social withdrawal ) and one somatic symptom ( breast tenderness , abdominal bloating , headache , swelling of extremities ) during the five days before menses in order to be diagnosed as having pms .
each symptom must appear in three consecutive menstrual cycles and be scored on a scale of 1 ( low intensity ) , 2 ( moderate intensity ) and 3 ( severe intensity ) .
the sum of the pms score ranges from 2 to 30 . using these criteria ,
our study participants were categorized into two groups : women with pms ( n = 354 ) and those without pms ( n = 302 ) .
all the participants underwent clinical examinations , where their body weight , height , waist and hip circumferences , and blood pressure were measured by trained staff .
height and weight were also measured with the subjects wearing light clothes but without shoes , using standard apparatus .
height and waist circumferences ( wc ) were measured to the nearest 0.5 cm using a measuring tape .
the waist was measured midway between the lower rib margin and the iliac crest , after a gentle expiration .
body mass index ( bmi ) was calculated by dividing a participant s weight in kilograms by their height in meters squared ( kg / m ) . a blood sample that was used to determine biochemical measurements was taken from each subject on the second or third day of their menstrual cycle , after 12 hours of overnight fasting .
blood samples were collected in edta treated test tubes . written informed consent was obtained from all participants before study entry .
17-hydroxyprogesterone ( 17oh - p ) , total testosterone ( tt ) and androstenedione ( a4 ) were measured by enzyme immunoassay ( eia ) , ( diagnostics biochem canada inc . ,
ontario , canada ) . sex hormone - binding globulin ( shbg ) was measured by immunoenzymometric assay ( iema ) , ( mercodia , uppsala , sweden ) .
all elisa tests were performed using a sunrise elisa reader ( tecan co. , salzburg , austria ) .
luteinizing hormone ( lh ) , follicle stimulating hormone ( fsh ) , prolactin ( prl ) , and thyroid stimulating hormone ( tsh ) were measured by immunoradiometric assay ( irma ) ( izotop , budapest , hungary ) using a gamma counter ( wallac wizard , turku , finland ) .
it has been shown that in women , the free androgen index ( fai ) has a good correlation with free testosterone measured by a physical separation method ( 18 ) ; therefore , in this study the fai was calculated by using the formula : the intra- and inter - assay coefficients of variation for tt were 5.6% and 6.6% ; for shbg , 1.2% and 5.7% ; for a4 , 2.2% and 3.5% ; for lh , 3% and 5.8% ; for fsh , 3.5% and 4% ; for tsh , 1.7% and 3.4% , and for prl , they were 2.1% and 4.1% , respectively . according to the acog practice guidelines for the diagnosis of pms ,
one or more of the disturbing affective or somatic symptoms must have occurred in the five days before menses in each of three previous menstrual cycles .
metabolic syndrome , based on the joint interim statement ( jis ) definition , was considered to be the presence of any three of the following five risk factors ( 19 ) : wc 95 ( country - specific cutoff point for iranians ( 20 ) , hdl < 50 , sbp 130 or dbp 85 , tg 150 and fbs 100 . according to the sixth report of the joint national committee
( jnc - vi ) criteria , hypertension or high bp was defined as mean sbp _ 140 mmhg , mean dbp _ 90 mmhg , or applied to a person undergoing current treatment for hypertension with prescription medication ( 21 ) .
based on the american diabetes association s ( ada ) definition of diabetes , participants who met the following criteria were considered to be diabetic : 1 ) using anti - diabetic drugs or with fasting blood sugar ( fbs ) of _ 7 mmol / l or 2-h plasma glucose ( 2hpg ) _ 11.1 mmol / l ; 2 ) those with 2hpg between 7.77 and 11.1
mmol / l were defined as igt , and ; 3 ) fbs between 5.6 - 6.9
mmol / l was defined as impaired fasting plasma glucose ( ifg ) ( 10 , 22 ) . based on atp ii ,
dyslipidemia was defined as tc _ 240 mg / dl or ldl _ 160 mg / dl or tg _ 200 mg / dl or hdl < 35 mg / dl ( 23 ) .
biochemical hyperandrogenism was detected by fai and/or a4 levels above the upper 95 percentile for the 362 women studied who were not on any hormonal medication and had no clinical evidence of hyperandrogenism , anovu , or pco . specifically , the upper normal limits for total t were = 0.88 ng / ml , a4 = 2.3 ng / ml , and fai = 5.47 .
continuous variables were checked for normality using the one - sample kolmogorov - smirnov test , and expressed as mean , standard deviation , and/or median ( iq : 25 - 75 ) , as appropriate .
linear regression ( forward method ) was used to identify the association between pms scores and metabolic disorders ( dependent variable ) after adjustment for age and bmi .
the data analysis was performed using the spss 15.0 pc package ( spss inc . ,
the subjects of this study were selected from among the participants in the iranian pcos prevalence study which was a community based cross - sectional study of 1026 women , aged 18 - 45 years , conducted between 2009 - 2010 ( 17 ) .
the eligible women were invited to participate in a comprehensive interview and their blood pressure , anthropometric , hormonal , and metabolic measurements were documented .
data were completed for all but 97 women who did not come to the clinics and 19 participants whose hormonal and metabolic profiles were unavailable .
we also excluded women who were using antidepressants ( n = 34 ) , oral contraceptive pills ( n = 151 ) , or taking hormonal medication for irregular menses . furthermore , those women who were pregnant at the time of the study ( n = 43 ) and menopausal women ( n = 37 ) were also excluded .
finally , after the exclusion of the women who did not meet our inclusion criteria , 656 women were enrolled in the study . the american college of obstetricians and gynecologists ( acog ) criteria were used to diagnose pms .
according to these criteria , the women needed to experience at least one of each of the following affective symptoms ( depression , angry outbursts , irritability , anxiety , confusion , social withdrawal ) and one somatic symptom ( breast tenderness , abdominal bloating , headache , swelling of extremities ) during the five days before menses in order to be diagnosed as having pms .
each symptom must appear in three consecutive menstrual cycles and be scored on a scale of 1 ( low intensity ) , 2 ( moderate intensity ) and 3 ( severe intensity ) .
the sum of the pms score ranges from 2 to 30 . using these criteria ,
our study participants were categorized into two groups : women with pms ( n = 354 ) and those without pms ( n = 302 ) .
all the participants underwent clinical examinations , where their body weight , height , waist and hip circumferences , and blood pressure were measured by trained staff .
height and weight were also measured with the subjects wearing light clothes but without shoes , using standard apparatus .
height and waist circumferences ( wc ) were measured to the nearest 0.5 cm using a measuring tape .
the waist was measured midway between the lower rib margin and the iliac crest , after a gentle expiration .
body mass index ( bmi ) was calculated by dividing a participant s weight in kilograms by their height in meters squared ( kg / m ) . a blood sample that was used to determine biochemical measurements was taken from each subject on the second or third day of their menstrual cycle , after 12 hours of overnight fasting .
blood samples were collected in edta treated test tubes . written informed consent was obtained from all participants before study entry .
17-hydroxyprogesterone ( 17oh - p ) , total testosterone ( tt ) and androstenedione ( a4 ) were measured by enzyme immunoassay ( eia ) , ( diagnostics biochem canada inc . , ontario , canada ) .
sex hormone - binding globulin ( shbg ) was measured by immunoenzymometric assay ( iema ) , ( mercodia , uppsala , sweden ) .
all elisa tests were performed using a sunrise elisa reader ( tecan co. , salzburg , austria ) .
luteinizing hormone ( lh ) , follicle stimulating hormone ( fsh ) , prolactin ( prl ) , and thyroid stimulating hormone ( tsh ) were measured by immunoradiometric assay ( irma ) ( izotop , budapest , hungary ) using a gamma counter ( wallac wizard , turku , finland ) .
it has been shown that in women , the free androgen index ( fai ) has a good correlation with free testosterone measured by a physical separation method ( 18 ) ; therefore , in this study the fai was calculated by using the formula : the intra- and inter - assay coefficients of variation for tt were 5.6% and 6.6% ; for shbg , 1.2% and 5.7% ; for a4 , 2.2% and 3.5% ; for lh , 3% and 5.8% ; for fsh , 3.5% and 4% ; for tsh , 1.7% and 3.4% , and for prl , they were 2.1% and 4.1% , respectively .
according to the acog practice guidelines for the diagnosis of pms , one or more of the disturbing affective or somatic symptoms must have occurred in the five days before menses in each of three previous menstrual cycles .
metabolic syndrome , based on the joint interim statement ( jis ) definition , was considered to be the presence of any three of the following five risk factors ( 19 ) : wc 95 ( country - specific cutoff point for iranians ( 20 ) , hdl < 50 , sbp 130 or dbp 85 , tg 150 and fbs 100 . according to the sixth report of the joint national committee
( jnc - vi ) criteria , hypertension or high bp was defined as mean sbp _ 140 mmhg , mean dbp _ 90 mmhg , or applied to a person undergoing current treatment for hypertension with prescription medication ( 21 ) .
based on the american diabetes association s ( ada ) definition of diabetes , participants who met the following criteria were considered to be diabetic : 1 ) using anti - diabetic drugs or with fasting blood sugar ( fbs ) of _
7 mmol / l or 2-h plasma glucose ( 2hpg ) _ 11.1 mmol / l ; 2 ) those with 2hpg between 7.77 and 11.1
mmol / l were defined as igt , and ; 3 ) fbs between 5.6 - 6.9
mmol / l was defined as impaired fasting plasma glucose ( ifg ) ( 10 , 22 ) . based on atp ii
, dyslipidemia was defined as tc _ 240 mg / dl or ldl _ 160 mg / dl or tg _ 200 mg / dl or hdl < 35 mg / dl ( 23 ) .
biochemical hyperandrogenism was detected by fai and/or a4 levels above the upper 95 percentile for the 362 women studied who were not on any hormonal medication and had no clinical evidence of hyperandrogenism , anovu , or pco .
specifically , the upper normal limits for total t were = 0.88 ng / ml , a4 = 2.3 ng / ml , and fai = 5.47 .
continuous variables were checked for normality using the one - sample kolmogorov - smirnov test , and expressed as mean , standard deviation , and/or median ( iq : 25 - 75 ) , as appropriate .
correlations between hormone concentrations and premenstrual syndrome scores were checked using the pearson correlation . linear regression ( forward method )
was used to identify the association between pms scores and metabolic disorders ( dependent variable ) after adjustment for age and bmi .
the data analysis was performed using the spss 15.0 pc package ( spss inc . ,
the demographic characteristics of the women who did not complete the questionnaire or those without hormonal measurements available did not significantly differ from those who completed the study procedure ( data has not been presented ) .
according to our findings , the education levels in women with pms were significantly higher than those without the condition .
table 1 demonstrates some of the demographic , reproductive , and anthropometric characteristics of the studied women , as divided into two groups , i.e. women with pms and those without it .
the values are expressed as mean sd except income , marital and job statuses that are presented as % .
comparison carried out using t - tests or tests for continuous and categorical variables , respectively .
table 2 shows a comparison of the hormonal , metabolic , and lipid profiles of women with and without pms .
our results suggest that there was a significant increase in prl and tg among women with pms , whereas tes , hdl and 17oh - p were significantly decreased when compared to women without pms .
mean prl and tes for women with pms were 18.5 and 0.58 , respectively , whereas for the controls these values were 16.3 and 0.67 , respectively ( p < 0.05 ) .
the mean serum levels of tg , hdl and 17oh - p for women with and without pms were 147.3 , 44.2 , and 1.8 versus 128.2 , 46.3 , and 2 , respectively . using ancova , we compared all the above variables between the two groups in order to adjust for age and bmi ; the results of the t - test analysis were not significantly different .
the results of the pearson correlation analysis showed that t4 , prl , tes , and 17oh - p had significant correlations with pms scores among the affected women ( p < 0.05 ) .
other variants , including serum levels of tsh , lh , shbg , hdl , tg , insulin , fbs , ldl , chl , systolic bp , diastolic bp , lh / fsh , fai , and a4 were not significantly correlated with pms . after adjusting for age and bmi , using partial correlation
after adjusting for age and bmi , a linear regression analysis demonstrated a significant association between pms scores and the prevalence of metabolic syndrome ( p = 0.033 ) . according to our findings ,
for every one unit increase in pms score there was a 12% increase in the probability of having metabolic syndrome . using a linear regression analysis we found a non - significant association between pms scores and other metabolic disorders , such as diabetes , hyperandrogenism , dyslipidemia , hypertension , and hypothyroidism ( table 3 ) .
abbreviations : bmi , body mass index ; ci , confidence interval ; pms , premenstrual syndrome .
in the present study , we assessed a wide range of hormones and metabolites in order to establish their associations with pms .
the association found between lower tt levels and pms in the present study contradicts the findings of bloch et al.s study ( 7 ) which indicated significantly lower tt and free t levels in women with pms .
however , eriksson et al . ( 16 ) and backstrom and aakvaag ( 10 ) discussed the contrary .
it has been found that low testosterone in women can cause a number of physical and emotional symptoms , including depression , loss of sexual desire , and declining libido ( 24 - 26 ) .
despite there being a limited number of studies concerning the effects of testosterone on mood , the results show that testosterone treatment per se , or with estrogen , improves mood in women ( 27 ) .
we observed significantly lower 17-ohp levels among women with pms which are findings that contradict those of eriksson et al .
( 16 ) who assessed women with pms during the luteal phase and reported higher 17-ohp levels when compared to age - matched controls ; these differences may be explained by the difference in the age of the subjects and the effects of age on hormone levels .
the present study demonstrated that the serum prl level is significantly higher in women with pms , which is a finding that is in line with previous studies ( 9 , 28 ) .
by contrast , another study ( 10 ) reported that the mean plasma prolactin level among 15 women affected with pms was not significantly different from that of the 17 women in the control group ; it appears that because of the small sample size , the researchers found no association between prl and pms .
prolactin plays an indirect role in premenstrual syndrome and may cause renal retention of water , sodium , and potassium , and it interacts with lithium .
prolactin can also interact with ovarian hormones to cause symptoms of depression , anxiety , or irritable hostility ( 29 ) .
the data report higher levels of total cholesterol in women with pms with no significant alteration in tg and hdl ( 11 ) ; conversely , our data showed higher tg and lower hdl levels .
to our knowledge , this is the first study examining the association of metabolic disorders in iranian women with pms .
several studies have demonstrated that depression is significantly associated with metabolic syndrome ; for example , raikkonen et al .
( 30 ) also suggest that there is an association between metabolic syndrome and higher rates of depression .
since depression is one of the main criteria of diagnosing pms , it can be speculated that pms and metabolic syndrome are associated . according to the data produced by this study
, there was no significant association between insulin levels and pms , which is a consonant result with that of zarei et al .
eriksson et al . ( 16 ) found no significant difference between pms cases and controls in shbg levels , which was also consistent with our findings .
one of the strengths of our study was the large cross - sectional community based sample that it used .
since most similar studies have focused on the small number of variants that contribute to pms disorders , we were able to investigate a broad range of anthropometric parameters and hormone levels in order to determine the independent association between endogenous hormones and metabolic parameters in multivariate analyses .
however , a potential limitation that must be recognized is that we used homa - ir as a surrogate marker for assessing insulin resistance ( ir ) ; in spite of the good correlation between homa - ir and gold standard clamp methods , it might be inaccurate in women with pcos .
moreover , we did not measure free testosterone due to our inability to access a proper method for its measurement .
however , we calculated the fai using equation 1 it has been shown that fai correlates with free testosterone as measured by the physical separation method in women , which is the same as calculated free testosterone ( 17 ) . in conclusion
, we found a significant association between pms scores and the prevalence of metabolic syndrome .
moreover , we observed higher levels of prl and lower levels of tes , ohp-17 , tg , and hdl among women with pms , when compared to women without the syndrome .
further studies are needed to confirm and validate the relationships between lipid profile abnormalities and metabolic disorders with pms . | backgroundpremenstrual syndrome ( pms ) is reported by up to 85% of women of reproductive age .
although several studies have focused on the hormone and lipid profiles of females with pms , the results are controversial.objectivesthis study was designed to investigate the association of hormonal and metabolic factors with pms among iranian women of reproductive age.materials and methodsthis study was a community based cross - sectional study .
anthropometric measurements , biochemical parameters , and metabolic disorders were compared between 354 women with pms and 302 healthy controls selected from among 1126 women of reproductive age who participated in the iranian pcos prevalence study .
p values < 0.05 were considered significant.resultsprolactin ( prl ) and triglycerides ( tg ) were significantly elevated in women with pms , whereas their testosterone ( tes ) , high density lipoprotein ( hdl ) and 17-hydroxyprogesterone ( 17-ohp ) levels were significantly less than they were in women without the syndrome ( p < 0.05 ) . after adjusting for age and body mass index ( bmi ) , linear regression analysis demonstrated that for every one unit increase in pms score there was 12% rise in the probability of having metabolic syndrome ( p = 0.033).conclusionsthere was a significant association between pms scores and the prevalence of metabolic syndrome .
further studies are needed to confirm and validate the relationships between lipid profile abnormalities and metabolic disorders with pms . |
these changes are in response to the rapidly increasing number of available ribosomal rna gene sequences ( rrna sequences ) and the trend toward high - throughput rrna sequencing with the concomitant need for high volume rrna analysis tools .
details about the data and analysis services can be found at the rdp - ii website ( http://rdp.cme.msu.edu/ ) .
the rdp obtains bacterial rrna sequences from the international nucleotide sequence databases ( insd : genbank / embl / ddbj ) on a monthly basis .
these sequences are aligned against a general bacterial rrna model using a modified version of rnacad ( 2 ) , a stochastic context free grammar ( scfg)-based rrna aligner that directly incorporates rrna secondary structure information into its internal model .
this aligner is trained on a set of high - quality hand - aligned sequences and incorporates the conserved bacterial secondary structure model of gutell and co - workers ( 3 ) . as of release 9.21 ( august 2004 ) , the database contained 101 632 total small subunit bacterial rrna sequences . of these ,
39 772 were near full - length ( 1200 bases ) , 54 316 came from uncultured organisms and 4431 were from type strains of validly named bacterial species .
all release 9 tools use a new hierarchical framework ( rdp hierarchy ) differing significantly from the hierarchy provided with previous rdp releases .
the rdp hierarchy is based on the new phylogenetically consistent higher - order bacterial taxonomy proposed by garrity et al .
it provides a phylogenetic framework into which to place results of the rdp analysis functions , and it provides an entry point for users looking for sequences from specific groups of organisms .
new sequences are placed into the rdp hierarchy using the rdp classifier ( see below ) .
the rdp analysis services have been completely revised to support the emerging trend toward high - throughput rrna sequence analysis in microbial ecology and related disciplines .
the user can choose to apply up to three data filters on the view or analysis . by applying the three filters ,
the user can ( i ) include only environmental clone or only isolate sequences ; ( ii ) include only sequences 1200 bases in length ( near full - length ) or only shorter sequences ; and ( iii ) include only sequences from type strains or only non - type strain sequences .
the latter filter is of special importance since type strains act as a link between rrna - based phylogeny and taxonomy .
a more detailed description of each analysis service can be found at the rdp website .
the browser presents views of the rdp sequences placed either in the rdp hierarchy , or optionally in the ncbi taxonomy hierarchy ( 5 ) .
while navigating , the browser automatically expands an appropriate number of hierarchical levels to fit the display . at any time
, the user can select for later download of both individual sequences and those of entire taxa .
data filters can be applied at any time to limit the display to specific data subsets .
in addition , the user can quickly search for words or phrases in the sequence definition line .
this includes the organism name and strain designation ( if available ) , culture collection identifiers and insd nucleic acid accession identifiers .
optimized for large query sets , it can be used to give an initial taxonomic placement for a single sequence or hundreds of sequences .
the first result page summarizes the assignments on an interactive display similar to that of the hierarchy browser .
each node in the hierarchy lists the number of user queries assigned to that taxonomic rank .
a confidence estimate is generated for each assignment , and the assignments are displayed only when the estimate is above a user - specified confidence threshold .
at any time , the user can switch to a detail view showing the detailed taxonomic assignments and confidence scores for any subset of query sequences .
these assignment details can also be downloaded in a file suitable for import into popular spreadsheet programs .
sequence match is a complete re - implementation of the original sequence match method ( 1 ) .
sequence match finds sequences similar to a user 's query sequences using a word matching strategy not requiring prior alignment .
sequence match is more accurate than blast ( 6 ) at finding closely related rrna sequences ( table 1 ) .
the related sequences returned by sequence match serve as a good starting point for more detailed examination of relatedness by classical phylogenetic or other methods .
the initial result page presents a k - nearest neighbor ( k - nn ) classifier assignment of the query sequences . a query is assigned to the lowest taxonomic rank that includes the k highest scoring database sequences . the value of k , as well as the three data filters can be changed at will in this view .
the user can switch from the summary k - nn view to a detailed results view for any query sequence . in this view
, the top k database matches to the query are displayed in the rdp hierarchy . in this mode
, any subset of the matches can be selected for transfer to the hierarchy browser and later download .
probe match is a complete re - implementation of our previous probe match program ( 1 ) .
it uses a more efficient algorithm that is better suited to the amount of rrna data available today and in the foreseeable future .
the new probe match accepts a candidate primer / probe , optionally with ambiguity codons , of up to 64 bases in length .
while our previous version searched for hits within a specified number of mismatches ( hamming distance ) , the new version finds hits with a combination of mismatches and insertion / deletions ( edit distance ) .
since some single insertion / deletion may be no more deleterious than a single mismatch , this new capability offers a significant improvement in the detection of potential cross - hybridization . in our previous implementation ,
the high percentage of partial sequences in the database limited the program 's utility ; it was difficult to determine if database entries failed to match simply because the sequence was incomplete in the target region . in this new version
, the users can restrict analysis to database entries containing sequence data for the candidate probe target region of the rrna molecule .
( however , the search is not limited to this region of the molecule . )
similar to the other new programs , the results are displayed in an interactive version of the rdp hierarchy .
each taxonomic rank lists the total number of sequences searched and the number matching within a user - specified edit distance .
the rdp analysis services have been completely revised to support the emerging trend toward high - throughput rrna sequence analysis in microbial ecology and related disciplines .
the user can choose to apply up to three data filters on the view or analysis . by applying the three filters ,
the user can ( i ) include only environmental clone or only isolate sequences ; ( ii ) include only sequences 1200 bases in length ( near full - length ) or only shorter sequences ; and ( iii ) include only sequences from type strains or only non - type strain sequences .
the latter filter is of special importance since type strains act as a link between rrna - based phylogeny and taxonomy .
a more detailed description of each analysis service can be found at the rdp website .
the browser presents views of the rdp sequences placed either in the rdp hierarchy , or optionally in the ncbi taxonomy hierarchy ( 5 ) .
while navigating , the browser automatically expands an appropriate number of hierarchical levels to fit the display . at any time
, the user can select for later download of both individual sequences and those of entire taxa .
data filters can be applied at any time to limit the display to specific data subsets .
in addition , the user can quickly search for words or phrases in the sequence definition line .
this includes the organism name and strain designation ( if available ) , culture collection identifiers and insd nucleic acid accession identifiers .
optimized for large query sets , it can be used to give an initial taxonomic placement for a single sequence or hundreds of sequences .
the first result page summarizes the assignments on an interactive display similar to that of the hierarchy browser .
each node in the hierarchy lists the number of user queries assigned to that taxonomic rank .
a confidence estimate is generated for each assignment , and the assignments are displayed only when the estimate is above a user - specified confidence threshold . at any time , the user can switch to a detail view showing the detailed taxonomic assignments and confidence scores for any subset of query sequences .
these assignment details can also be downloaded in a file suitable for import into popular spreadsheet programs .
sequence match is a complete re - implementation of the original sequence match method ( 1 ) .
sequence match finds sequences similar to a user 's query sequences using a word matching strategy not requiring prior alignment .
sequence match is more accurate than blast ( 6 ) at finding closely related rrna sequences ( table 1 ) .
the related sequences returned by sequence match serve as a good starting point for more detailed examination of relatedness by classical phylogenetic or other methods .
the initial result page presents a k - nearest neighbor ( k - nn ) classifier assignment of the query sequences . a query is assigned to the lowest taxonomic rank that includes the k highest scoring database sequences . the value of k , as well as the three data filters can be changed at will in this view .
the user can switch from the summary k - nn view to a detailed results view for any query sequence . in this view , the top k database matches to the query are displayed in the rdp hierarchy . in this mode
, any subset of the matches can be selected for transfer to the hierarchy browser and later download .
probe match is a complete re - implementation of our previous probe match program ( 1 ) .
it uses a more efficient algorithm that is better suited to the amount of rrna data available today and in the foreseeable future .
the new probe match accepts a candidate primer / probe , optionally with ambiguity codons , of up to 64 bases in length . while our previous version searched for hits within a specified number of mismatches ( hamming distance ) , the new version finds hits with a combination of mismatches and insertion / deletions ( edit distance ) .
since some single insertion / deletion may be no more deleterious than a single mismatch , this new capability offers a significant improvement in the detection of potential cross - hybridization . in our previous implementation ,
the high percentage of partial sequences in the database limited the program 's utility ; it was difficult to determine if database entries failed to match simply because the sequence was incomplete in the target region . in this new version
, the users can restrict analysis to database entries containing sequence data for the candidate probe target region of the rrna molecule .
( however , the search is not limited to this region of the molecule . )
similar to the other new programs , the results are displayed in an interactive version of the rdp hierarchy .
each taxonomic rank lists the total number of sequences searched and the number matching within a user - specified edit distance .
the rdp - ii data and analysis services can be found at http://rdp.cme.msu.edu/. the rdp 's mission includes user support .
the rdp - ii staff may also be contacted via fax ( + 1 517 353 8957 attn : rdp ) or regular mail .
we thank several individuals for their past contributions : robin gutell ( and his colleagues ) , chuck parker , paul saxman , bonnie maidak , tim lilburn , niels larsen , tom macke , michael j. mccaughey , ross overbeek , sakti pramanik , scott dawson , mitch l. sogin , gary olsen and carl woese .
the rdp is supported through us doe ober / nih grant de - fg02 - 99er62848 and nsf grant dbi-0328255 . | the ribosomal database project ( rdp - ii ) provides the research community with aligned and annotated rrna gene sequences , along with analysis services and a phylogenetically consistent taxonomic framework for these data .
updated monthly , these services are made available through the rdp - ii website ( http://rdp.cme.msu.edu/ ) .
rdp - ii release 9.21 ( august 2004 ) contains 101 632 bacterial small subunit rrna gene sequences in aligned and annotated format .
high - throughput tools for initial taxonomic placement , identification of related sequences , probe and primer testing , data navigation and subalignment download are provided .
the rdp - ii email address for questions or comments is rdpstaff@msu.edu . |
neuromyelitis optica ( nmo ) is a chronic inflammatory demyelinating disease of putative autoimmune etiology .
it mainly affects the optic nerves and spinal cord with the majority of cases having a relapsing disease course . in 2004 , a serum autoantibody nmo ig g , which binds selectively to central nervous system microvessels , pia , subpia , and virchow - robin spaces , was identified .
the binding sites of this autoantibody were found to co - localize with aquaporin-4 . the anti - aquaporin-4 ig g antibody ( anti - aqp4 ig g ) or nmo ig g has been found to be highly specific and sensitive for nmo . it has been incorporated into the revised wingerchuk criteria of 2006 for the diagnosis of nmo [ 1 , 2 ] . with the aid of this antibody , the spectrum of nmo is now known to be wider than previously recognized . in view of this heterogeneity , a new term , that is , neuromyelitis optica spectrum disorders ( nmosd ) , was introduced which encompasses patients with recurrent or isolated , longitudinally extensive myelitis or optic neuritis , longitudinally extensive myelitis or optic neuritis associated with systemic autoimmune disease , brain lesions typical for nmo , and some cases of asian opticospinal multiple sclerosis ( osms ) [ 2 , 3 ] . based on previous literature ,
neuromyelitis optica had been recognized as relatively uncommon in malaysia . in the past , it was defined as a monophasic disease [ 4 , 5 ] .
literature on nmo in malaysia is lacking especially after the revision of the wingerchuk 2006 criteria [ 2 , 3 ] .
hence , it was decided to pursue the testing of this antibody to find out the frequency of anti - aqp4 ig g amongst malaysian patients presenting with a phenotype suggestive of nmo and other idiopathic inflammatory demyelinating diseases at high risk for neuromyelitis optica spectrum disorder ( nmosd ) .
there have been a number of reports suggesting that treatment response in patients with nmo / nmosd differs from that used for multiple sclerosis and thus identifying these patients has important therapeutic implications [ 69 ] .
kuala lumpur hospital is a major tertiary referral center with a neurology service that caters for patients throughout the country .
the patient population attending the neurology service is representative of the demographics of the country . to our knowledge
this is the first study assessing the frequency of the antibody in patients with iidds suggestive of nmo / nmosd and high risk syndromes for this condition from a single tertiary referral center in malaysia .
the main objective of this study was to evaluate the frequency of anti - aqp4 ig g in patients with idiopathic inflammatory demyelinating diseases such as nmo and high risk syndromes for neuromyelitis optica spectrum disorder ( nmosd ) such as monophasic or multiphasic recurrent transverse myelitis and optic neuritis as well as brain lesions at onset of disease either monophasic / multiphasic , monofocal , or multifocal .
a secondary objective was to look at the differences between the seropositive and seronegative groups .
this was a hospital based retrospective time series with longitudinal followup looking at consecutive patients who had presented to the department of neurology , kuala lumpur hospital , between january 2009 and january 2014 with idiopathic inflammatory demyelinating disease .
kuala lumpur hospital is a tertiary referral center with a neurology service that caters for patients from all the states within malaysia .
patients were stratified into the following groups barring the presence of anti - aqp4 ig g antibody ( anti - aqp4 ) testing based on the criteria highlighted below : all patients with idiopathic inflammatory demyelinating disease who fulfilled wingerchuk criteria of 2006 for nmo excluding anti - aqp4 testing , patients at high risk for nmosd , that is , those with single episode , monophasic or recurrent relapsing optic neuritis ( ron / mon ) and single ( monophasic ) episode or recurrent myelitis ( mtm / rtm ) , as outlined by wingerchuk et al .
[ 2 , 3],patients with brain symptoms and signs at onset of disease with magnetic resonance imaging ( mri ) of the brain showing demyelinating lesions atypical for multiple sclerosis but typical for nmosd as described by pittock et al . , patients with monophasic or multiphasic , monofocal or multifocal demyelinating disease involving the brain but did not fulfil the criteria for brain involvement in nmosd , acute demyelinating encephalomyelitis , or ms .
all patients with idiopathic inflammatory demyelinating disease who fulfilled wingerchuk criteria of 2006 for nmo excluding anti - aqp4 testing , patients at high risk for nmosd , that is , those with single episode , monophasic or recurrent relapsing optic neuritis ( ron / mon ) and single ( monophasic ) episode or recurrent myelitis ( mtm / rtm ) , as outlined by wingerchuk et al . [ 2 , 3 ] , patients with brain symptoms and signs at onset of disease with magnetic resonance imaging ( mri ) of the brain showing demyelinating lesions atypical for multiple sclerosis but typical for nmosd as described by pittock et al . , patients with monophasic or multiphasic , monofocal or multifocal demyelinating disease involving the brain but did not fulfil the criteria for brain involvement in nmosd , acute demyelinating encephalomyelitis , or ms .
those excluded from the study were patients who refused or were unable to consent for inclusion of their data in the study and/or for the purpose of testing for anti - aqp4 antibody . also excluded were patients with nondemyelinating idiopathic inflammatory diseases involving the brain.patients diagnosed with symptoms and signs suggestive of multiple sclerosis based on mcdonalds 's 2005 and 2010 diagnostic criteria [ 11 , 12 ] were excluded from the study.patients diagnosed with acute demyelinating encephalomyelitis were also excluded .
those excluded from the study were patients who refused or were unable to consent for inclusion of their data in the study and/or for the purpose of testing for anti - aqp4 antibody . also excluded were patients with nondemyelinating idiopathic inflammatory diseases involving the brain .
patients diagnosed with symptoms and signs suggestive of multiple sclerosis based on mcdonalds 's 2005 and 2010 diagnostic criteria [ 11 , 12 ] were excluded from the study .
institutional ethics committee approval was sought from the national medical research register under the auspices of the ministry of health of malaysia and approved for the establishment of a demyelinating diseases registry , for review of available data , and for longitudinal followup .
data was collected with regard to demographics , clinical features , neuroimaging , and laboratory parameters . in order to maintain accuracy of interpretation of brain and cord mri 's , neuroimaging at disease onset or
statistical analysis was done with spss version 16 looking at absolute medians , means , and descriptive data was expressed as total numbers and percentages .
parametric data was looked at with student 's t - test and nonparametric data using the chi square test .
the anti - aqp4 test was conducted at the autoimmune unit , allergy & immunology research centre , institute for medical research , kuala lumpur .
anti - aquaporin-4 antibodies ( aqp4 ) were determined in the serum of the patients by indirect immunofluorescence ( euroimmun iift , germany ) on a cell line which had been molecular - biologically modified ( aqp4 transfected cells ) to produce large quantities of aqp4 .
biochip slides containing aqp4 transfected cells and nontransfected cells ( eu-90 ) were incubated with diluted patient samples . in the case of positive reactions , specific antibodies of the classes iga , ig g , and
the attached antibodies are stained with fluorescein - labelled anti - human antibodies and made visible with the fluorescence microscope .
these patients had visited the neurology department at kuala lumpur hospital between january 2009 and january 2014 .
out of the total number of patients in the database , there were 58 patients with neuromyelitis optica .
in addition , there were 22 subjects with brain involvement at onset of disease suggestive of nmosd .
twenty of whom went on to develop optic neuritis or transverse myelitis or both on longitudinal followup .
nineteen patients had transverse myelitis alone of which 14 had relapsing transverse myelitis ( rtm ) and 5 had monophasic transverse myelitis ( mtm ) .
one hundred and thirty - two patients had multiple sclerosis and twenty were diagnosed to have acute demyelinating encephalomyelitis and both groups were excluded from the study .
five patients out of the 266 patients in the database had monofocal monophasic disease affecting the brain stem not typical of multiple sclerosis or nmo of which 3 patients had incomplete data and were excluded .
these patients were still considered at high risk for nmosd as all other causes had been excluded .
testing for anti - aquaporin-4 antibody was done on 48 patients with nmo , 21 with nmosd ( brain involvement at onset ) , 15 with transverse myelitis , 7 with relapsing optic neuritis , 3 with monophasic optic neuritis , and 2 in patients with monophasic , monofocal brain disease at onset ( whose brain mri lesions did not look like those described by pittock et al . but still were possibly at high risk for nmosd ) .
10 patients with nmo and one with brain nmosd did not have anti - aquaporin-4 testing done as they were lost to followup or had incomplete data .
patient demographics are shown in ( table 1 ) . overall , the majority of patients were females ( 88.5% versus 11.5% ) .
the malays were the predominant racial group affected , 47/96 ( 49.0% ) , followed by the chinese , 41/96 ( 42.7% ) , indians , 6/96 ( 6.2% ) , and other indigenous groups such as ibans ( 1.1% ) and bajaus ( 1.1% ) .
relapsing remitting disease ( 90/96 , 93.8% ) was the commonest disease course with progressive course ( 1% ) and monophasic disease ( 5.2% ) being rare .
median age at onset was 30 11.5 years , median duration of disease was 2.0 0.99 years ( range 1 to 6 years ) , and median edss on the last followup was 3.0 2.48 , ( range 0 to 9 ) .
median duration between first and second attack was 0.5 1.1 years ( range 17 years ) .
median annualized relapse rate was 1.0 0.73 ( 0 to 3.4 ) . clinically , transverse myelitis was the commonest initial presentation followed by optic neuritis in this mixed group ( table 1 ) .
overall , 65 subjects or 67.7% tested positive out of 96 patients with idiopathic inflammatory demyelinating disease at high risk for nmo / nmosd . in the nmo group , 38/48 ( 79.1% ) subjects tested positive for anti - aqp4 antibody .
out of the 21 subjects with brain involvement at onset suggestive of nmosd , 12 ( 57.1% ) were positive for the antibody ( table 2 ) . in the transverse myelitis group , 12 ( 80% )
patients were antibody positive of which 11 patients had relapsing myelitis ( table 2 ) .
out of the 11 with relapsing myelitis who were positive , the majority had either longitudinally contiguous lesions ( 6/11 ) , linear lesions ( 3/11 ) , and finally a combination of longitudinally linear and contiguous lesions ( 1/12 ) ( figure 1 ) .
one patient with short segment relapsing myelitis was also positive for anti - aqp4 antibody .
she initially presented with paroxysmal tonic spasms , weakness of left upper limb , and a short cord cervical lesion of 2 vertebral segments ( cervical 1 to 2 ) in length and was seronegative .
at first she was started on beta - interferon i - b but after one and a half years she experienced another cord relapse with a longitudinally extensive cervical cord lesion from cervical vertebrae 2 to 5 and subsequently after the second relapse a thoracic vertebrae 6 ( t6 ) to 10 ( t10 ) lesion and became anti - aqp4 antibody positive .
another patient with monophasic longitudinally extensive contiguous cord lesion involving the entire spinal cord was also seropositive ; she was started on immunosuppressants and maintenance steroids and remained relapse free till today .
however , at year 5 on immunosuppressants she developed neutropenia necessitating an interruption in azathioprine .
only three patients ( 42.8% ) with relapsing optic neuritis were positive for anti - aqp4 antibody out of 7 patients .
three patients with monophasic optic neuritis were seronegative . out of the three seropositive patients ,
one went on to develop an asymptomatic short cord lesion on surveillance magnetic resonance imaging ( mri ) after 2 years .
none of the patients with monophasic or monofocal demyelinating disease of the brain were anti - aqp4 antibody positive .
we also looked at the association between the presence of the anti - aqp4 antibody and certain clinical and neuroimaging parameters .
seropositivity was significantly associated with a higher current mean edss ( 4.5 2.4 versus 2.4 2.6 , p < 0.001 ) compared to seronegatives .
it was also significantly associated with an increased mean number of relapses , that is , 5.15 ( 4.42 ) versus 2.10 ( 1.68 ) , in the seronegative group , p = 0.001 .
furthermore , seropositive nmo / nmosd was significantly associated with paroxysmal tonic spasms , p = 0.004 , and longer length in spinal cord lesions , that is , 6.66 ( 4.9 ) versus 2.90 ( 2.5 ) vertebral segments , p < 0.001 .
blindness in one or both eyes and poor visual acuity were significantly seen in seropositive patients rather than seronegative patients ( tables 3 and 4 ) .
nmo / nmosd patients with longitudinally extensive cord lesions of contiguous or linear nature with or without fragmentation / interrupted lesions were significantly associated with being anti - aqp4 antibody positive rather than being negative , p < 0.001 .
seropositive patients had significantly more lesions in the cervical , thoracic , and cervicothoracic cord regions , p < 0.001 .
more seropositive patients had cord atrophy ; however , this did not achieve statistical significance , p = 0.056 .
holocord or central gray matter lesions were significantly associated with seropositivity ( p < 0.001 ) .
seronegative patients had significantly lesser number of cord lesions as compared to seropositive patients ( 0.96 versus 1.17 ) , p = 0.025 ( table 3 ) .
on the other hand , seropositivity and seronegativity did not discriminate between the different ethnic races or the type of clinical presentation at onset probably due to the small sample sizes .
the analysis also showed seropositivity was not significantly associated with time to conversion to nmo / nmosd , age at onset and duration of disease ( table 4 ) .
for those patients who were seronegative in our study we plan to closely follow their clinical phenotype and repeat their antibody testing for anti - aqp4 in the future .
this study attempted to determine the frequency of anti - aqp4 ig g amongst malaysian patients presenting with idiopathic inflammatory demyelinating disease ( iidds ) at high risk for nmo / nmosd . in our study , 65 out of 96 patients , that is , 67.7% , were positive .
historically , studies conducted in malaysia , from the 1980s , found very few patients presenting with neuromyelitis optica [ 4 , 5 ] .
this could be due to the fact that nmo , according to the original definition used by devic , was considered to be a monophasic disease . at that time , relapsing forms of severe optic nerve and cord disease were characterized as opticospinal multiple sclerosis regardless of cord length and most were of chinese origin [ 4 , 5 ] . however with the revised wingerchuk criteria of 2006 , relapsing forms have been recognised and the spectrum of nmo has been found to be wider with the discovery of the anti - aqp4 antibody for nmo [ 13 ] . in our study , the majority of patients with an opticospinal presentation had neuromyelitis optica with a longitudinally extensive cord lesion and were seropositive .
the diagnosis of opticospinal multiple sclerosis had been made initially in some cases but was revised with the recognition of the significance of the longitudinally extensive cord lesions . in some cases the diagnosis had to be reviewed after obtaining the initial mri spine at presentation showing such a lesion .
furthermore in other patients , the persistently normal looking brain mri in the absence of typical lesions for multiple sclerosis on longitudinal followup and development in some cases of typical lesions for nmo / nmosd aided by seropositivity led to better characterization and diagnosis .
we also noted that cord lesions tend to change with time , disease activity , and treatment .
so if patients with opticospinal presentation were reviewed without the initial mri of the spine at onset , there was a possibility of classifying them as multiple sclerosis rather than neuromyelitis optica . in a study from japan ,
out of 19 japanese patients with opticospinal multiple sclerosis ( osms ) and 13 with classical multiple sclerosis tested for nmo - ig g , 14 patients with osms and two patients with classical ms were positive .
the two ms patients who were positive had features suggestive of nmo / nmosd with longitudinally extensive cord lesions and brain involvement atypical for ms , respectively . in their study ,
nmo seropositive patients frequently had longitudinally extensive transverse myelitis ( 93% versus 57% ) and more severe visual deficits in at least one eye ( 50% versus 0% ) .
furthermore , this similarity can be seen in our own analysis . in our study despite the ethnic differences , seropositivity was associated with poorer visual outcome and longer length of cord lesions .
this disease in our country is not so dissimilar to nmo / nmosd in other asian /african ethnic cohorts .
we noted that , among the patients who fulfilled the wingerchuk 2006 criteria for nmo , 79.2% were positive . in the other iidd groups at high risk for nmo / nmosd , 11/15 ( 73.3% ) patients with relapsing transverse myelitis and 1/15 with monophasic disease were positive as were 3/7 ( 42.8% ) with relapsing optic neuritis .
none of the patients with poorly characterized idiopathic isolated monofocal monophasic demyelinating disease of the brain ( brainstem ) were positive .
the majority of the patients in our study , 69/94 , had longitudinally extensive cord lesions at onset of disease either longitudinally contiguous ( 44 ) , linear ( 11 ) , both ( 5 ) , or longitudinally linear and contiguous with patchy interrupted and fragmented lesions ( 9 ) ( table 1 ) .
seropositive nmo / nmosds in our study were found to be significantly associated with a mean length of the spinal cord lesions of more than 6 vertebral segments ( 6.6 versus 2.9 , p < 0.001 ) , holocord and central gray matter involvement , and a tendency for the cervical , thoracic , and cervicothoracic cord to be involved as compared to seronegative patients .
we found seropositivity to be significantly associated with the presence of a longitudinally extensive cord lesion ( lescl ) whether linear or contiguous in nature .
seronegative patients had significantly lesser number of cord lesions compared to seropositive patients , that is , 0.96 versus 1.16 , p = 0.025 . in the seronegative group ,
a number of patients had short cord segments of between 2 to 3 vertebral segments and hence the mean cord length in the seronegative group was borderline at 2.9 vertebral segments .
a nearly equal number of patients with opticospinal or brain involvement at onset of disease who were seropositive and seronegative were found in our study . in the past ,
some of these patients with opticospinal disease might have been characterized as opticospinal multiple sclerosis .
regionally , a study by lu et al . compared 29 patients with nmo to 22 patients with ms and found that nmo patients have significantly more linear lesions than ms patients , that is , 48.3% versus 0% , p < 0.001 with these lesions distributed in the spinal cord , medullospinal region , and medulla . in their study , longitudinally extensive cord lesions were also seen more frequently in nmo than ms , that is , 72.4% versus 22.7% , p < 0.001 .
studies from thailand and taiwan have demonstrated similar observations in their cohorts with lescls and seropositivity [ 16 , 17 ] .
in fact , wingerchuk et al . found the presence of a longitudinally extensive cord lesion extending more than 3 vertebral segments to be the most reliable feature in the diagnosis of nmo [ 1 , 2 ] . in a review of literature by jarius and wildemann , the frequency of anti - aqp4 antibody in patients with longitudinally extensive transverse myelitis ( letm ) ranged between 0% and 100% with a median of 53.3% .
though not all of the studies reviewed differentiated between monophasic and recurrent letm , a higher frequency of antibody positivity was seen in patients with letm .
these results are very comparable to the rates that we got in our relapsing transverse myelitis cohort , that is , 80% .
one patient with monophasic transverse myelitis was also seropositive and had a longitudinally extensive contiguous cord lesion affecting the entire spinal cord .
we found an equal number of patients with nmo / nmsod with short segment myelitis who were seropositive and seronegative .
this occurred usually at the onset of the disease . therefore , in patients with opticospinal or short segment spinal lesions we still feel it may be worthwhile to do the anti - aqp4 antibody test .
this is especially so if the brain mri is not in keeping with ms and is persistently so even when repeated and has some atypicality resembling nmo / nmosd such as the presence of paroxysmal tonic spasms .
later on , their lesions lengthened to become longitudinally extensive , an observation of ours not reported in the outcome . in the overall cohort ,
similarly , if we compare our results in patients with ron to a study from jarius and wildemann , in a large cohort , the antibody was found in three out of 89 patients with a single attack of optic neuritis and in five out of 50 with relapsing on ( 10% ) using cell based assays .
nmo antibody seropositivity has been tested within a diverse population in the west and in asia .
regionally , a study from thailand found seropositivity in 18 out of 23 patients ( 78% ) who fulfilled wingerchuk 's criteria for nmo which is very similar to the rate in our study and all the more interesting since thailand is geographically to the north of malaysia [ 1 , 2 , 16 ] .
reported 80.9% of nmo patients and 54.5% of patients with longitudinally extensive cord lesions to be seropositive .
y. long et al . from china reported 88.6% of nmo patients , 4.3% with ms , 30.8% with optic neuritis , and 51.7% with longitudinally extensive transverse myelitis to be seropositive .
in western literature , though ms is far more common than nmo / nmosd , jarius et al . found a seropositivity of 61.1% ( 22 of 26 ) among nmo patients as defined by the 1999 wingerchuk criteria , with 35 of the 36 having long cord lesions .
more recently , the same review by jarius and wildemann highlighted the heterogeneity in sensitivities for anti - aqp4 antibody existing amongst different studies when different assays were used .
this occurred not only due to technical difficulties with the assays but also due to the smaller sample sizes . in their review , when adequate sample sized studies were included , that is , > 40 nmo patients , the sensitivities were in the realm of 73.58% , similar to what we see in our study for patients with nmo .
this brings home the fact that rates seem to be fairly comparable amongst different ethnic groups in different regions provided there is heightened awareness , the diagnosis is made carefully ( i.e. , to exclude patients with multiple sclerosis ) , samples have good effect sizes , and more importantly if a sensitive assay is used taking into account the treatment effect and assessment coinciding with disease remission .
other salient differences were that seropositive patients were more likely to go blind , experience tonic spasms , have more relapses , and have a higher edss .
therefore , when faced with the seropositive nmo / nmosd patient , therapy needs to be more aggressive as patients have a higher chance of disease worsening due to tendency to relapse more ( table 4 ) . a large multicentre study from germany and a single center study from thailand described similar findings . in their cohort of patients ,
those who were seropositive were more likely to have poorer visual acuity , longer cord lesions , and in the former study to relapse more .
our ratio for female preponderance in the seropositive group was 12 : 1 .
a similar observation was also noted by lin et al . in their sample of 57 chinese patients where the ratio was noted to be 8.5 to 1 .
this high female predisposition has already been well described in caucasian patients where nmo / nmosd is nine times more prevalent in women than in men .
this study showed that patients with the nmo / nmosd phenotype do exist in our country in larger numbers than was previously estimated and are not dissimilar from descriptions elsewhere .
this is the first study to characterize the frequency of this group of patients in multiethnic malaysia . when diagnostic criteria is applied appropriately with sensitive and specific assays for anti - aquaporin 4 antibody , it is evident across different ethnic groups around the world that similarities in clinical features , presentation , and neuroimaging exist
. the majority of the patients with nmo / nmosd are anti - aqp4 antibody positive with rates comparable to those around the region .
however , we acknowledge the limitations of the study , that is , retrospective nature , descriptive study with small sample size especially within the optic neuritis , transverse myelitis , and demyelinating brain disease at onset groups , referral bias being a tertiary referral centre , and lack of comparison between the nmo / nmosd and ms groups , as well as the need to use more sensitive and specific assays . we are hoping to address these issues in future studies . with better diagnostic ability to characterize these patients | background . in the past
the occurrence of neuromyelitis optica in malaysia was thought to be uncommon and the frequency of anti - aquaporin-4 ig g antibody was unknown . objective . to evaluate the frequency of anti - aquaporin-4 ig g antibody ( anti - aqp4 antibody ) amongst patients with neuromyelitis optica ( nmo ) and its spectrum disorders ( nmosd ) and the differences between the seropositive and seronegative groups
. methods .
retrospectively , 96 patients with nmo / high risk syndromes for nmosd ( hrs - nmosd ) were identified out of 266 patients with idiopathic inflammatory demyelinating disease from a single center hospital based registry .
anti - aqp4 seropositivity was found in 38/48 ( 79.2% ) with nmo , 12/21 ( 57.1% ) with brain involvement at high risk for nmosd , 12/15 ( 80% ) with transverse myelitis ( i.e. , 11/15 with relapsing transverse myelitis and one with monophasic transverse myelitis ) , and 3/7 ( 42.8% ) with relapsing optic neuritis .
sixty - five out of 96 patients , that is , 67.7% , with nmo / hrs for nmosd were seropositive .
seropositivity was significantly associated with female gender , a higher number of mean relapses , that is , 5.15 4.42 versus 2.10 1.68 , longer length of spinal cord lesions , that is , 6.6 4.9 versus 2.9 2.5 , vertebral bodies , higher edss , 4.5 2.4 versus 2.4 2.6 , presence of paroxysmal tonic spasms , and blindness ( unilateral / bilateral ) ; p < 0.001 .
longitudinally extensive cord lesions ( contiguous or linear ) , presence of lesions in the cervical and thoracic regions , and involvement of the central gray matter or holocord regions on axial scans , were also significantly associated with seropositivity ; p < 0.001 . conclusion .
nmo and hrs for nmosd are present in larger numbers than previously thought in malaysia .
more than 2/3rds are seropositive .
seropositive and seronegative nmo / nmosd have differences that are useful in clinical practice . |
tissue was collected from postnatal - day-21 albino wistar rats ( rattus norvegivus ) that were euthanized by asphyxiation and subsequent cervical dislocation .
all procedures were approved by the animal ethical review committee of the university of sydney , australia and were carried out in accordance with the national health medical research council ( australia ) guidelines and the association for research in vision and ophthalmology statement for the use of animals in ophthalmic and vision research ( united states ) .
lens epithelial explants were prepared as described previously and cultured in medium 199 with earle 's salts ( life technologies , waltham , ma , usa ) , supplemented with 50 g / ml l - glutamine , 50 iu / ml penicillin/50 g / ml streptomycin ( thermo fisher scientific , waltham , ma , usa ) , 2.5 g / ml amphostat b ( thermo fisher scientific ) , and 0.1% bsa ( sigma - aldrich corp . ,
recombinant tgf-2 was added to media in each culture dish at a working concentration ranging from 200 pg / ml up to 1ng / ml ( r&d systems , minneapolis , mn , usa ) . before the addition of tgf-2
, some explants were pretreated with the pan - nadph oxidase inhibitor vas2870 at a working concentration of 5 m .
the progression of emt in live cells was observed and captured using phase - contrast microscopy ( olympus ck2 , tokyo , japan ) and a digital camera ( leica dfc-280 ; leica camera , wetzlar , germany ) .
percentage cell loss quantification was performed using the thresholding function of imagej ( national institutes of health [ nih ] , bethesda , md , usa ) , such that cells could be distinguished from bare lens capsule . at the end of the culture period , explants were fixed in 100% methanol for 45 seconds , followed by 3 consecutive 15-second rinses in pbs .
explants were blocked in 10% normal goat serum ( ngs ) for 1 hour at room temperature .
excess normal goat serum was removed and primary antibody was applied , diluted in 0.15% ngs / pbs supplemented with 1% bsa .
-smooth muscle actin ( sma ) was labeled with a specific monoclonal mouse antibody ( sigma - aldrich corp . ) , diluted 1:100 .
nox4 was labeled with a specific polyclonal rabbit antibody ( santa cruz biotechnology , dallas , tx , usa ) , diluted 1:50 .
e - cadherin was labeled with a specific polyclonal mouse antibody ( bd biosciences , north ryde , nsw , australia ) , diluted at 1:100 . the following day , dishes were equilibrated to room temperature and subject to 3 5 minute washes in pbs / bsa .
an appropriate secondary antibody was diluted in pbs / bsa and applied to each explant for 2 hours in dark conditions .
-smooth muscle actin and e - cadherin were detected using goat anti - mouse alexa - fluor 488 ( cell signaling , danvers , ma , usa ) .
nox4 was detected using an anti - rabbit whole igg conjugated to cy3 ( sigma - aldrich corp . ) .
dishes subsequently were rinsed in pbs / bsa and a 1:2000 solution of bisbenzimide ( hoechst dye ) diluted in pbs / bsa was applied for 3 minutes to visualize cell nuclei .
immunofluorescent labeling was viewed and captured using epifluorescence microscopy ( leica - dmlb ; leica camera ) , and a digital camera ( micropublisher 3.3 rtv ; q - imaging , surrey , bc canada ) . where appropriate , fluorescence also was visualized and images were collected using a zeiss lsm-5pa confocal microscope ( carl zeiss ag , jena , germany ) .
after appropriate treatment , lens epithelial explants were placed into a 1.5 ml centrifuge tube .
total rna extraction was performed using a quick - rna miniprep kit ( zymo research , irvine , ca , usa ) according to manufacturer 's instructions .
first - strand cdna synthesis was done using 150 ng of rna with a reverse transcription system ( bioline , eveleig , nsw , australia ) according to the manufacturer 's instructions .
control reactions omitting reverse transcriptase were set up and later tested , confirming that genomic dna was absent from the purified rna template ( data not shown ) . for pcr amplification , the mytaq red dna polymerase ( bioline ) kit
primers used for the detection of nox14 and gapdh mrna were based on those previously published . for detection of nox14 mrna ,
sequence - specific primers were used : nox1 : fw : 5-cttcctcactggctgggata-3 , rev : 5-tgacagcatttgcgcaggc-3 ; nox2 : 5-ccagtgaagatgtgttcagct-3 , 5-gcacagccagtagaagtagat-3 ; nox3 : 5-gtgtccttcgtggttgctct-3 , 5-tctacagcacacgctcgttc-3 ;
nox4 : 5-agtcaaacagatggga-3 , 5-tgtcccatatgagttgtt-3 ; gapdh : 5-tgaacgggaagctcactgg-3 , 5-tccaccaccctgttgctgta-3. cdna was amplified for 35 cycles : nox1 and nox2 : 95c for 45 seconds , 51c for 45 seconds , and 72c for 45 seconds ; nox1 , nox3 , and gapdh : 95c for 30 seconds , 62c for 30 seconds , and 72c for 30 seconds .
amplified dna was visualized with sybr safe dna gel stain ( diluted at 1:10,000 ; invitrogen , carlsbad , ca , usa ) in a 2% agarose tae gel , run at 100 v for 20 minutes .
positive control tissues for each nox homologue also were examined ( nox1 , rat colon ; nox2 and nox4 , rat kidney ) .
dihydroethidium ( life technologies ) was added to explants in each dish containing 1 ml of medium to yield a working concentration of 30 m .
explants then were rinsed in cold pbs ( 3 15 seconds ) before being mounted in 10% glycerol / pbs . in
its reduced form , dhe fluoresces blue , but when exposed to ros , including the superoxide anion radical and hydrogen peroxide , it readily reacts to form the oxidized red fluorescent dhe form that can be viewed readily using epifluorescence microscopy .
fluorescence microscopy gives an indication of the level of total ros produced by tgf-2dependent nox4 ; however , absolute quantification of specific types of ros can not be determined using this method .
after appropriate treatment , lens epithelial explants were placed into a 1.5 ml tube with cold lysis buffer containing 2.5 mm edta , 25 mm tris , 0.375 m nacl , 250 mm sodium orthovandate , 10 mm sodium deoxycholate , and a protease inhibitor cocktail ( complete , mini ; roche , basel , switzerland ) .
tubes were rotated for 2 hours and protein lysates were extracted by centrifuging for 15 minutes ( 15,500 g , 4c ) .
the direct detect spectrophotometer system ( merck millipore , bayswater , vic , australia ) was used to determine the protein concentration for each lysate , according to manufacturer 's instructions .
equal amounts of protein per sample were loaded onto 10% sds - page gels for electrophoresis at 200 v for 1.5 hours and transferred onto a polyvinylidene fluoride membrane ( merck millipore ) at 100 v for 1 hour .
membranes then were incubated with a blocking solution containing 5% skim milk diluted in 0.1% tween-20 in tris - buffered saline ( tbs - t ) for 1 hour at room temperature and probed overnight at 4c with specific antibodies , appropriately diluted in blocking buffer .
rabbit monoclonal anti - nox4 antibody ( santa - cruz biotechnology ) was diluted at 1:200 , mouse monoclonal anti--sma ( sigma - aldrich corp . )
was diluted at 1:2000 and mouse monoclonal anti - gapdh ( sigma - aldrich corp . )
membranes were then rinsed ( 3 5 minutes ) in tbs - t and anti - rabbit or anti - mouse secondary antibodies conjugated to horseradish peroxidase ( diluted at 1:5000 in tbs - t ) were applied to each membrane and incubated at room temperature for 2 hours with gentle rocking .
membranes were rinsed ( 3 5 minutes ) with tbs - t and an equal volume of peroxidase and luminol reagent was applied immediately before imaging using the chemidoc mp imaging system ( bio - rad laboratory , hercules , ca , usa ) .
tissue was collected from postnatal - day-21 albino wistar rats ( rattus norvegivus ) that were euthanized by asphyxiation and subsequent cervical dislocation .
all procedures were approved by the animal ethical review committee of the university of sydney , australia and were carried out in accordance with the national health medical research council ( australia ) guidelines and the association for research in vision and ophthalmology statement for the use of animals in ophthalmic and vision research ( united states ) .
lens epithelial explants were prepared as described previously and cultured in medium 199 with earle 's salts ( life technologies , waltham , ma , usa ) , supplemented with 50 g / ml l - glutamine , 50 iu / ml penicillin/50 g / ml streptomycin ( thermo fisher scientific , waltham , ma , usa ) , 2.5 g / ml amphostat b ( thermo fisher scientific ) , and 0.1% bsa ( sigma - aldrich corp . , st .
recombinant tgf-2 was added to media in each culture dish at a working concentration ranging from 200 pg / ml up to 1ng / ml ( r&d systems , minneapolis , mn , usa ) . before the addition of tgf-2
, some explants were pretreated with the pan - nadph oxidase inhibitor vas2870 at a working concentration of 5 m .
the progression of emt in live cells was observed and captured using phase - contrast microscopy ( olympus ck2 , tokyo , japan ) and a digital camera ( leica dfc-280 ; leica camera , wetzlar , germany ) .
percentage cell loss quantification was performed using the thresholding function of imagej ( national institutes of health [ nih ] , bethesda , md , usa ) , such that cells could be distinguished from bare lens capsule .
at the end of the culture period , explants were fixed in 100% methanol for 45 seconds , followed by 3 consecutive 15-second rinses in pbs .
explants were blocked in 10% normal goat serum ( ngs ) for 1 hour at room temperature .
excess normal goat serum was removed and primary antibody was applied , diluted in 0.15% ngs / pbs supplemented with 1% bsa .
-smooth muscle actin ( sma ) was labeled with a specific monoclonal mouse antibody ( sigma - aldrich corp . ) , diluted 1:100 .
nox4 was labeled with a specific polyclonal rabbit antibody ( santa cruz biotechnology , dallas , tx , usa ) , diluted 1:50 .
e - cadherin was labeled with a specific polyclonal mouse antibody ( bd biosciences , north ryde , nsw , australia ) , diluted at 1:100 .
the following day , dishes were equilibrated to room temperature and subject to 3 5 minute washes in pbs / bsa .
an appropriate secondary antibody was diluted in pbs / bsa and applied to each explant for 2 hours in dark conditions .
-smooth muscle actin and e - cadherin were detected using goat anti - mouse alexa - fluor 488 ( cell signaling , danvers , ma , usa ) .
nox4 was detected using an anti - rabbit whole igg conjugated to cy3 ( sigma - aldrich corp . ) .
dishes subsequently were rinsed in pbs / bsa and a 1:2000 solution of bisbenzimide ( hoechst dye ) diluted in pbs / bsa was applied for 3 minutes to visualize cell nuclei .
immunofluorescent labeling was viewed and captured using epifluorescence microscopy ( leica - dmlb ; leica camera ) , and a digital camera ( micropublisher 3.3 rtv ; q - imaging , surrey , bc canada ) . where appropriate , fluorescence also was visualized and images were collected using a zeiss lsm-5pa confocal microscope ( carl zeiss ag , jena , germany ) .
after appropriate treatment , lens epithelial explants were placed into a 1.5 ml centrifuge tube .
total rna extraction was performed using a quick - rna miniprep kit ( zymo research , irvine , ca , usa ) according to manufacturer 's instructions .
first - strand cdna synthesis was done using 150 ng of rna with a reverse transcription system ( bioline , eveleig , nsw , australia ) according to the manufacturer 's instructions .
control reactions omitting reverse transcriptase were set up and later tested , confirming that genomic dna was absent from the purified rna template ( data not shown ) . for pcr amplification , the mytaq red dna polymerase ( bioline ) kit
primers used for the detection of nox14 and gapdh mrna were based on those previously published . for detection of nox14 mrna ,
sequence - specific primers were used : nox1 : fw : 5-cttcctcactggctgggata-3 , rev : 5-tgacagcatttgcgcaggc-3 ; nox2 : 5-ccagtgaagatgtgttcagct-3 , 5-gcacagccagtagaagtagat-3 ; nox3 : 5-gtgtccttcgtggttgctct-3 , 5-tctacagcacacgctcgttc-3 ; nox4 : 5-agtcaaacagatggga-3 , 5-tgtcccatatgagttgtt-3 ; gapdh : 5-tgaacgggaagctcactgg-3 , 5-tccaccaccctgttgctgta-3. cdna was amplified for 35 cycles : nox1 and nox2 : 95c for 45 seconds , 51c for 45 seconds , and 72c for 45 seconds ; nox1 , nox3 , and gapdh : 95c for 30 seconds , 62c for 30 seconds , and 72c for 30 seconds .
amplified dna was visualized with sybr safe dna gel stain ( diluted at 1:10,000 ; invitrogen , carlsbad , ca , usa ) in a 2% agarose tae gel , run at 100 v for 20 minutes .
positive control tissues for each nox homologue also were examined ( nox1 , rat colon ; nox2 and nox4 , rat kidney ) .
dihydroethidium ( life technologies ) was added to explants in each dish containing 1 ml of medium to yield a working concentration of 30 m .
explants then were rinsed in cold pbs ( 3 15 seconds ) before being mounted in 10% glycerol / pbs . in its reduced form , dhe fluoresces blue , but when exposed to ros , including the superoxide anion radical and hydrogen peroxide , it readily reacts to form the oxidized red fluorescent dhe form that can be viewed readily using epifluorescence microscopy .
fluorescence microscopy gives an indication of the level of total ros produced by tgf-2dependent nox4 ; however , absolute quantification of specific types of ros can not be determined using this method .
after appropriate treatment , lens epithelial explants were placed into a 1.5 ml tube with cold lysis buffer containing 2.5 mm edta , 25 mm tris , 0.375 m nacl , 250 mm sodium orthovandate , 10 mm sodium deoxycholate , and a protease inhibitor cocktail ( complete , mini ; roche , basel , switzerland ) .
tubes were rotated for 2 hours and protein lysates were extracted by centrifuging for 15 minutes ( 15,500 g , 4c ) .
the direct detect spectrophotometer system ( merck millipore , bayswater , vic , australia ) was used to determine the protein concentration for each lysate , according to manufacturer 's instructions .
equal amounts of protein per sample were loaded onto 10% sds - page gels for electrophoresis at 200 v for 1.5 hours and transferred onto a polyvinylidene fluoride membrane ( merck millipore ) at 100 v for 1 hour .
membranes then were incubated with a blocking solution containing 5% skim milk diluted in 0.1% tween-20 in tris - buffered saline ( tbs - t ) for 1 hour at room temperature and probed overnight at 4c with specific antibodies , appropriately diluted in blocking buffer .
rabbit monoclonal anti - nox4 antibody ( santa - cruz biotechnology ) was diluted at 1:200 , mouse monoclonal anti--sma ( sigma - aldrich corp . )
was diluted at 1:2000 and mouse monoclonal anti - gapdh ( sigma - aldrich corp . )
membranes were then rinsed ( 3 5 minutes ) in tbs - t and anti - rabbit or anti - mouse secondary antibodies conjugated to horseradish peroxidase ( diluted at 1:5000 in tbs - t ) were applied to each membrane and incubated at room temperature for 2 hours with gentle rocking .
membranes were rinsed ( 3 5 minutes ) with tbs - t and an equal volume of peroxidase and luminol reagent was applied immediately before imaging using the chemidoc mp imaging system ( bio - rad laboratory , hercules , ca , usa ) .
nox4 expression in tgf-driven lens emt was assayed using immunolabeling of lens epithelial explant lysates . specifically , we determined nox4 expression using western blotting at different time points ( 0 , 2 , 4 , 6 , and 8 hours ) .
1 ) , and this level was significantly different to that of the untreated control at the same time period ( fig .
expression of nox4 also was significantly more pronounced at 8 hours to that observed at 6 hours ( fig .
8 hours after tgf-2 treatment , the level of nox4 protein expression was approximately 2.5-fold higher to that of the matching control without tgf-2 ( fig .
this increase in nox4 protein expression also was accompanied by an upregulation in nox4 mrna transcription in tgf-2treated explants compared to untreated explants at 8 hours ( fig .
transforming growth factor-2 induces an increase in nox4 expression in lens epithelial explants : western blot .
( a ) representative western blot depicting the time - course ( 4 , 6 , and 8 hours ) of nox4 expression following tgf-2 treatment ( t4 , t6 , and t8 ) , compared to control explants ( c4 , c6 , and c8 ) .
was performed to normalize the expression of nox4 protein to gapdh expression . upon tgf-2 treatment ,
nox4 expression was significantly upregulated at 6 hours ( t6 ) , and these levels significantly increased by 8 hours ( t8 ) .
one - way anova , tukey 's post hoc test ( 2-tailed t - test ) * p < 0.05 and * * p < 0.01 .
( b ) a representative rt - pcr of nox14 mrna transcripts after 8 hours , in the absence ( c8 ) and presence ( t8 ) of tgf-2 .
for each lane , the pcr products shown correspond to the expected base pair length ( nox1 , 201 base pairs [ bp ] ; nox2 , 149 bp ; nox3 , 234 bp ; nox4 , 191 bp , and gapdh , 287 bp ) .
given that the expression level of nox4 was more pronounced at 8 hours after tgf-2 treatment , this time - point was used to examine the spatial localization of nox4 in rat lens epithelial cells using immunofluorescence and confocal microscopy ( fig .
some punctate labeling was observable , and appeared to localize throughout the cytosol ( fig .
this was in contrast to the two distinct expression patterns of nox4 seen at 8 hours following tgf-2 treatment ( fig .
2b ) . specifically , increased nox4 expression was seen throughout all tgf-2treated epithelial cells at 8 hours , with more pronounced punctate labeling in the cell nuclei and perinuclear areas .
interestingly , nox4 localization in lens epithelial cells treated for longer periods with tgf-2 , at 48 hours , was predominantly cytosolic ( fig .
2e ) , which was first detectable by 24 hours after tgf-2 treatment ( data not shown ) . at 48 hours treatment , nox4 appeared to colocalize with particular areas of the -sma reactive stress fibers ( see fig .
this colocalization of nox4 and -sma has been observed in other models of tgf-driven emt .
interestingly , not all cells with a strong nox4 label colabeled with -sma ( see fig .
similar to untreated cells at 8 hours , untreated cells at 48 hours yielded negligible nox4 expression and -sma positive stress fiber formation . transforming growth factor-2 induces variable nox4 localization patterns in lens epithelial cells with the progression of tgf- induced emt .
nox4 ( red ) was merged with the cell nuclear hoechst dye ( blue ) .
untreated explants at 8 ( a ) and 48 ( c ) hours exhibited little nox4 expression .
explants treated with tgf-2 ( + tgf-2 ) exhibited a stronger distinct punctate label by 8 hours ( b ) that primarily localized to the perinuclear regions of the cells . at 48 hours ( d ) , this label became more pronounced throughout the cytosol . by 48 hours compared to control explants ( e ) , epithelial cells treated with tgf-2 also labeled for -sma ( green ; [ f ] , with -sma labeled - stress fibres colocalizing with nox4 ( shown at higher magnification , inset 1 ) .
punctate cytosolic distribution of nox4 , independent of stress fibers , also was apparent in some cells ( [ f ] , shown at higher magnification , inset 2 ) .
scale bar : 40 and 20 m for the inserts . to validate the specificity of the nox4 label seen in response to tgf-2 treatment
, we repeated our immunolabeling experiments with the pan - nadph oxidase inhibitor vas2870 ( fig .
3 ) . inhibition of nadph oxidase blocked nox4 protein expression in lens epithelial cells in response to tgf-2 treatment ( fig .
3i , inset ) when compared to tgf-2treated cells without the inhibitor ( see fig .
we next determined whether this tgf-2dependent expression of nox4 was accompanied with nadph oxidase activity , using dhe fluorescence microscopy to assay for ros production in lens epithelial cells . as nox4 was first detected between 6 and 8 hours after tgf-2 treatment , dhe reactivity was assayed before and at these time points .
reassuringly , nox4 expression was accompanied by a significant increase in nadph oxidase specific activity ( dhe oxidation ) in tgf-2treated lens explants ( figs
. 3 g , 3h ) compared to the control explants not treated with tgf-2 ( figs .
the ros probe , dhe , was used to assess increases in ros following tgf-2 treatment . in untreated explants , basal levels of ros were observable ( a d ) .
importantly , tgf-2 treated explants yielded noticeable increases in ros by 6 hours , that increased up to 8 hours ( h ) .
treatment with vas2870 blocked tgf-2induced ros ( i ) as shown by diminished fluorescence intensity , with less ros produced .
this was in conjunction to vas2870 also blocking nox4 protein expression ( see [ i ] inset ) .
representative images from 3 independent experiments , consisting of at least 2 replicates for each treatment group .
scale bar : 100 m ( a i ) and 20m for ( inset [ i ] ) .
total fluorescence intensity also was calculated using imagej ( j ) , with significant increases after 6 and 8 hours , effectively blocked by vas2870 .
one - way anova , tukey 's post hoc test ( 2-tailed t - test ) * *
further earlier time points that did not exhibit nox4 expression ( 2 and 4 hours ) did not produce a dhe label , either in the presence or absence of tgf-2 ( fig .
3 ) . in addition , when tgf-driven nox4 expression was inhibited with vas2870 ( see also fig .
3i ) . given that we have shown nox4 expression and activity is induced in lens epithelial cells and that we could inhibit nox4 expression and concomitant activity with vas2870 , we next investigated the role nox4 had in the development of tgf-dependent lens emt .
the development of tgf-2induced lens emt can be followed morphologically in lens explants using phase - contrast microscopy . in our experimental model ,
4b ) , and from day 3 exhibit progressive cell loss indicated by acellular patches ( fig . 4c , asterisk ) , cellular blebbing ( fig .
4d ) . interestingly , in explants treated with tgf-2 in the presence of vas2870 , cellular loss was significantly delayed by day 3 and most pronounced at 5 days after tgf-2 treatment ( fig .
4i ; day 3 , + tgf vs. + tgf/vas , p = 0.0001 and day 5 + tgf vs. + tgf/vas , p = 0.005 ) .
moreover , cells in these explants did not appear to undergo tgf-2dependent cellular elongation , with cells remaining on day 5 of culture , exhibiting a typical uniformly packed lens epithelial phenotype ( fig .
vas2870-alone treatment had no effect on e - cadherin labeling ( 5a ) . moreover ,
consistent with the absence of cell elongation earlier in the culture period , indicating the absence of myofibroblastic cells that are thought to promote lens capsular wrinkling , under these conditions there was no apparent capsular wrinkling ( fig .
phase contrast images of explants at days 2 ( b , f ) , 3 ( c , g ) , and 5 ( a , e , d , h ) .
explants treated with tgf-2 alone ( b d ) underwent morphologic changes associated with emt compared to untreated cells ( a ) .
explants treated with vas2870 and tgf-2 ( f h ) showed a delayed emt response , with less cell elongation apparent by day 2 ( f ) , reduced cell loss by day 3 ( g ) and the retention of epithelial - like cells by day 5 ( h ) .
note that , vas2870 alone ( e ) had little effect on cells treated up to 5 days of culture .
( i ) percentage cell loss was calculated using the threshold feature of imagej in relation to areas of bare lens capsule , with significant cell loss in tgf-2treated explants from day 3 , which was abrogated by application of vas2870 .
two - way anova , tukey 's post hoc test ( 2-tailed t - test ) * * p < 0.01 and * * * * p < 0.0001 .
explants were treated with vas2870 ( a ) , or cotreated with tgf-2 and vas2870 ( b ) and fixed at 5 days of culture .
immunolabeling for the membrane marker e - cadherin ( green ) was performed and nuclei were counterstained with hoechst ( blue ) . in cells treated only with vas2870 ( a ) , e - cadherin localized to the cell membrane and there was no apparent cell loss .
cotreatment with tgf-2 and vas2870 promoted increased cell survival and the maintenance of a membranous e - cadherin label ( b ) .
explants cultured for 24 hours in the presence of tgf-2 ( a c ) , or tgf-2 and vas2870 ( d f ) were immunolabeled for nox4 ( [ b , e ] , red ) , -sma ( green ; [ c , f ] ) and counterstained with hoechst dye ( a , d ) .
compared to explants treated with tgf alone ( a c ) , vas2870 was shown to block tgf-2-induced nox4 ( e ) and -sma ( f ) expression .
( g ) protein lysates also were collected from explants treated with and without tgf-2 and vas2870 and western blotting demonstrated similar reductions in nox4 and -sma expression in the presence of vas2870 at 24 and 48 hours .
2d ) to localize with some -sma reactive stress fibers , it was proposed that nox4 may have a role in -sma accumulation .
cells treated with both vas2870 and tgf-2 yielded undetectable nox4 expression ( figs . 6b , 6 g ) , compared to explants treated with tgf-2 alone ( figs .
6e , 6 g ) , and in the process halted the progression of emt , with little to no detectable labeling for -sma expression in cells ( fig .
6 g ) that retained their epithelial - like appearance ( see hoechst staining , figs .
nox4 expression in tgf-driven lens emt was assayed using immunolabeling of lens epithelial explant lysates . specifically , we determined nox4 expression using western blotting at different time points ( 0 , 2 , 4 , 6 , and 8 hours ) .
1 ) , and this level was significantly different to that of the untreated control at the same time period ( fig .
expression of nox4 also was significantly more pronounced at 8 hours to that observed at 6 hours ( fig .
8 hours after tgf-2 treatment , the level of nox4 protein expression was approximately 2.5-fold higher to that of the matching control without tgf-2 ( fig .
this increase in nox4 protein expression also was accompanied by an upregulation in nox4 mrna transcription in tgf-2treated explants compared to untreated explants at 8 hours ( fig .
transforming growth factor-2 induces an increase in nox4 expression in lens epithelial explants : western blot .
( a ) representative western blot depicting the time - course ( 4 , 6 , and 8 hours ) of nox4 expression following tgf-2 treatment ( t4 , t6 , and t8 ) , compared to control explants ( c4 , c6 , and c8 ) .
was performed to normalize the expression of nox4 protein to gapdh expression . upon tgf-2 treatment ,
nox4 expression was significantly upregulated at 6 hours ( t6 ) , and these levels significantly increased by 8 hours ( t8 ) .
one - way anova , tukey 's post hoc test ( 2-tailed t - test ) * p < 0.05 and * * p < 0.01 .
( b ) a representative rt - pcr of nox14 mrna transcripts after 8 hours , in the absence ( c8 ) and presence ( t8 ) of tgf-2 .
for each lane , the pcr products shown correspond to the expected base pair length ( nox1 , 201 base pairs [ bp ] ; nox2 , 149 bp ; nox3 , 234 bp ; nox4 , 191 bp , and gapdh , 287 bp ) .
given that the expression level of nox4 was more pronounced at 8 hours after tgf-2 treatment , this time - point was used to examine the spatial localization of nox4 in rat lens epithelial cells using immunofluorescence and confocal microscopy ( fig .
some punctate labeling was observable , and appeared to localize throughout the cytosol ( fig .
this was in contrast to the two distinct expression patterns of nox4 seen at 8 hours following tgf-2 treatment ( fig .
specifically , increased nox4 expression was seen throughout all tgf-2treated epithelial cells at 8 hours , with more pronounced punctate labeling in the cell nuclei and perinuclear areas .
interestingly , nox4 localization in lens epithelial cells treated for longer periods with tgf-2 , at 48 hours , was predominantly cytosolic ( fig .
2e ) , which was first detectable by 24 hours after tgf-2 treatment ( data not shown ) . at 48 hours treatment , nox4 appeared to colocalize with particular areas of the -sma reactive stress fibers ( see fig .
this colocalization of nox4 and -sma has been observed in other models of tgf-driven emt .
interestingly , not all cells with a strong nox4 label colabeled with -sma ( see fig .
2 , inset 2 ) . similar to untreated cells at 8 hours , untreated cells at 48 hours yielded negligible nox4 expression and -sma positive stress fiber formation .
transforming growth factor-2 induces variable nox4 localization patterns in lens epithelial cells with the progression of tgf- induced emt .
nox4 ( red ) was merged with the cell nuclear hoechst dye ( blue ) .
untreated explants at 8 ( a ) and 48 ( c ) hours exhibited little nox4 expression .
explants treated with tgf-2 ( + tgf-2 ) exhibited a stronger distinct punctate label by 8 hours ( b ) that primarily localized to the perinuclear regions of the cells . at 48 hours ( d ) , this label became more pronounced throughout the cytosol . by 48 hours compared to control explants ( e ) , epithelial cells treated with tgf-2 also labeled for -sma ( green ; [ f ] , with -sma labeled - stress fibres colocalizing with nox4 ( shown at higher magnification , inset 1 ) .
punctate cytosolic distribution of nox4 , independent of stress fibers , also was apparent in some cells ( [ f ] , shown at higher magnification , inset 2 ) .
to validate the specificity of the nox4 label seen in response to tgf-2 treatment , we repeated our immunolabeling experiments with the pan - nadph oxidase inhibitor vas2870 ( fig .
3 ) . inhibition of nadph oxidase blocked nox4 protein expression in lens epithelial cells in response to tgf-2 treatment ( fig .
3i , inset ) when compared to tgf-2treated cells without the inhibitor ( see fig .
we next determined whether this tgf-2dependent expression of nox4 was accompanied with nadph oxidase activity , using dhe fluorescence microscopy to assay for ros production in lens epithelial cells . as nox4 was first detected between 6 and 8 hours after tgf-2 treatment , dhe reactivity was assayed before and at these time points .
reassuringly , nox4 expression was accompanied by a significant increase in nadph oxidase specific activity ( dhe oxidation ) in tgf-2treated lens explants ( figs
. 3 g , 3h ) compared to the control explants not treated with tgf-2 ( figs .
the ros probe , dhe , was used to assess increases in ros following tgf-2 treatment . in untreated explants , basal levels of ros were observable ( a d ) .
importantly , tgf-2 treated explants yielded noticeable increases in ros by 6 hours , that increased up to 8 hours ( h ) . treatment with
vas2870 blocked tgf-2induced ros ( i ) as shown by diminished fluorescence intensity , with less ros produced .
this was in conjunction to vas2870 also blocking nox4 protein expression ( see [ i ] inset ) .
representative images from 3 independent experiments , consisting of at least 2 replicates for each treatment group .
scale bar : 100 m ( a i ) and 20m for ( inset [ i ] ) .
total fluorescence intensity also was calculated using imagej ( j ) , with significant increases after 6 and 8 hours , effectively blocked by vas2870 .
one - way anova , tukey 's post hoc test ( 2-tailed t - test ) * * * p < 0.001 and * * * * p <
0.0001 . further earlier time points that did not exhibit nox4 expression ( 2 and 4 hours ) did not produce a dhe label , either in the presence or absence of tgf-2 ( fig .
3 ) . in addition , when tgf-driven nox4 expression was inhibited with vas2870 ( see also fig .
given that we have shown nox4 expression and activity is induced in lens epithelial cells and that we could inhibit nox4 expression and concomitant activity with vas2870 , we next investigated the role nox4 had in the development of tgf-dependent lens emt .
the development of tgf-2induced lens emt can be followed morphologically in lens explants using phase - contrast microscopy . in our experimental model ,
4b ) , and from day 3 exhibit progressive cell loss indicated by acellular patches ( fig .
4d ) . interestingly , in explants treated with tgf-2 in the presence of vas2870 , cellular loss was significantly delayed by day 3 and most pronounced at 5 days after tgf-2 treatment ( fig . 4i ; day 3 , + tgf vs. + tgf/vas , p = 0.0001 and day 5 + tgf vs. + tgf/vas , p = 0.005 ) .
moreover , cells in these explants did not appear to undergo tgf-2dependent cellular elongation , with cells remaining on day 5 of culture , exhibiting a typical uniformly packed lens epithelial phenotype ( fig .
moreover , consistent with the absence of cell elongation earlier in the culture period , indicating the absence of myofibroblastic cells that are thought to promote lens capsular wrinkling , under these conditions there was no apparent capsular wrinkling ( fig .
phase contrast images of explants at days 2 ( b , f ) , 3 ( c , g ) , and 5 ( a , e , d , h ) . explants treated with tgf-2 alone ( b d ) underwent morphologic changes associated with emt compared to untreated cells ( a ) .
explants treated with vas2870 and tgf-2 ( f h ) showed a delayed emt response , with less cell elongation apparent by day 2 ( f ) , reduced cell loss by day 3 ( g ) and the retention of epithelial - like cells by day 5 ( h ) . note that , vas2870 alone ( e ) had little effect on cells treated up to 5 days of culture .
( i ) percentage cell loss was calculated using the threshold feature of imagej in relation to areas of bare lens capsule , with significant cell loss in tgf-2treated explants from day 3 , which was abrogated by application of vas2870 .
two - way anova , tukey 's post hoc test ( 2-tailed t - test ) * * p < 0.01 and * * * * p < 0.0001 .
explants were treated with vas2870 ( a ) , or cotreated with tgf-2 and vas2870 ( b ) and fixed at 5 days of culture .
immunolabeling for the membrane marker e - cadherin ( green ) was performed and nuclei were counterstained with hoechst ( blue ) . in cells treated only with vas2870 ( a ) ,
e - cadherin localized to the cell membrane and there was no apparent cell loss .
cotreatment with tgf-2 and vas2870 promoted increased cell survival and the maintenance of a membranous e - cadherin label ( b ) .
explants cultured for 24 hours in the presence of tgf-2 ( a c ) , or tgf-2 and vas2870 ( d f ) were immunolabeled for nox4 ( [ b , e ] , red ) , -sma ( green ; [ c , f ] ) and counterstained with hoechst dye ( a , d ) .
compared to explants treated with tgf alone ( a c ) , vas2870 was shown to block tgf-2-induced nox4 ( e ) and -sma ( f ) expression .
( g ) protein lysates also were collected from explants treated with and without tgf-2 and vas2870 and western blotting demonstrated similar reductions in nox4 and -sma expression in the presence of vas2870 at 24 and 48 hours .
2d ) to localize with some -sma reactive stress fibers , it was proposed that nox4 may have a role in -sma accumulation .
cells treated with both vas2870 and tgf-2 yielded undetectable nox4 expression ( figs . 6b , 6 g ) , compared to explants treated with tgf-2 alone ( figs .
6e , 6 g ) , and in the process halted the progression of emt , with little to no detectable labeling for -sma expression in cells ( fig .
6 g ) that retained their epithelial - like appearance ( see hoechst staining , figs .
in the present study , for the first time , we describe the novel temporal and spatial expression of nox4 and characterize its activity in the rat lens explant system following tgf- treatment .
we demonstrated that nox4 is the only nox homologue expressed in rat lens epithelial explants , and is responsive to tgf , consistent with other studies .
nox4 inhibition using a pan - nadph oxidase inhibitor slowed the progression of emt through the promotion of cell survival .
furthermore , we also impeded the expression of the known myofibroblastic emt marker , -sma , and subsequent development of capsular wrinkling .
the remaining / surviving cells on the lens capsule following the nox4 blockade retain an epithelial - like phenotype and do not progress down the emt pathway , highlighting an important role for nox4 in tgf-dependent emt in the lens .
reactive oxygen species are known to have a key role in the development of cataract .
these antioxidants include the glutathione synthesis machinery , the detoxification enzyme peroxiredoxin , and glutaredoxins , that are responsible for the maintenance of the thiol redox status of lens proteins , including the crystallins .
previous studies have shown the indirect role of ros in the development of tgf-dependent cataract in rats . in these studies ,
addition of the antioxidant gsh was shown to strongly suppress tgf-induced opacification and subcapsular plaque formation in whole rat lens cultures .
furthermore , microarray studies using tgf-treated human lens cell lines highlighted that nox4 was not only induced in response to tgf- , but was one of the highest induced transcripts in these cultured human lens epithelial cells compared to controls .
while we demonstrate nox4 to be involved in tgf-dependent emt in lens , it has previously been reported to be involved in a number of other different models of emt .
for example , in breast cancer cells , tgf- has been shown to induce nox4 expression and activity ( by measuring the production of ros in the presence of a nadph oxidase inhibitor ) .
furthermore , in this earlier study , tgf-dependent nox4 expression and activity was dependent on the smad signaling pathway , given inhibition of this pathway ( via sirna or pharmacological inhibition ) inhibited nox4 labeling / activity and abolished the development of an emt .
additionally , blocking nadph oxidase activity alone ( either pharmacologically or by sirna ) , also prevented emt in breast cancer cells , highlighting that nox4 expression and activity was responsible for the development of emt in breast cancer cells .
it is likely that tgf- signals through the smad pathway to induce nox4 expression and activity in lens . in future studies
, it would be of great interest to determine whether direct or indirect tgf-smad signaling contributes to the differential nuclear ( at 8 hours ) and cytosolic ( 48 hours ) nox4 labeling we observed here .
furthermore , nox4 expressed at later time points in the emt process may directly influence the actin cytoskeleton with which it is colocalized .
novel studies in the kidney have highlighted the role of nox4 in actin cytoskeleton rearrangement , leading to regulation of focal adhesion turnover and vsmc migration .
members of the rho gtpase family are strongly associated with the formation of the actin cytoskeleton .
ascertained that rhoa / rock activation is upstream of poldip2 and nox4 in tgf- signaling in the kidney , and controls myofibroblast activation . indeed , in the rat lens explant
cellular blebbing is an indirect indication of apoptosis , where cells are physically dismantled from within by modulation of the actin cytoskeleton .
this process is mediated by members of the proapoptotic caspase family , where the plasma membrane is weakened allowing the cytosol to protrude from the cell .
caspases have been shown to cleave and activate rho kinase ( rock i ) , a downstream effector of rhoa signaling .
activation of rock i leads to the formation of actin stress fibers , cell contraction and dynamic membrane blebbing induced by active rock i. therefore , it is feasible that the changes seen in the tgf-driven emt in the lens may signal at least in part through the rhoa / rock pathway via nox4 .
reflective of its multiple roles in proinflammatory responses , migration , differentiation and apoptosis , nox4-derived ros appear enigmatic in their downstream targets . using tgf-stimulated human aortic muscle cells , martin - garrido et al
. found that silencing of nox4 abolished the formation of stress fibers and -sma accumulation in a mapk / p38-dependent manner .
this is in contrast to research using fibroblastic cells that report the c - jun n - terminal kinase ( jnk pathway ) as capable of regulating -sma accumulation in a nox4-dependent fashion .
together , these findings from other fibrotic models and the lens suggest that the progressive accumulation of -sma is the result of a heterogeneous process that converges on the -sma promoter , and that nox4 may act as a common mediator of these pathways .
more broadly , this is indirect evidence to the notion that redox signaling is capable of serving as a nexus between parallel signaling pathways , such as smads and mapks .
the present study shows for the first time to our knowledge that nox4 has a role in tgf-driven lens emt .
in particular , nox4 appears to have a crucial role in the regulation of -sma , potentially apoptosis , cell elongation and capsular wrinkling .
the findings of this study provide new avenues for the potential treatment of cataract and the prevention of secondary cataract , given tgf-driven emt is integral to the development of some cataracts in humans .
future studies will concentrate on expanding these findings in genetic models of tgf-dependent cataract , ascertaining the upstream events crucial to nox4 expression and delineating the specific targets of ros during lens emt . | purposetransforming growth factor- induces an epithelial to mesenchymal transition ( emt ) in the lens , presented as an aberrant growth and differentiation of lens epithelial cells .
studies in other models of emt have shown that tgf-driven emt is dependent on the expression of the reactive oxygen species ( ros)producing enzyme nicotinamide adenine dinucleotide phosphate ( nadph)oxidase-4 ( nox4 ) .
we investigate the role of this enzyme in tgf-induced lens emt and determine whether it is required for this pathologic process.methodsrat lens epithelial explants were used to investigate the role of nox4 in tgf-driven lens emt .
nox14 expression and localization was determined by immunolabeling and/or rt - pcr .
nadph
oxidase produced ros were visualized microscopically using the fluorescent probe , dihydroethidium ( dhe ) .
vas2870 , a pan - nadph oxidase inhibitor , was used to determine the specificity of nox4 expression and its role in ros production , and subsequently tgf-driven emt.resultswe demonstrate , for the first time to our knowledge , in rat lens epithelial explants that tgf- treatment induces nox4 ( but not nox13 ) expression and activity . increased nox4 expression was first detected at 6 to 8 hours following tgf- treatment and was maintained in explants up to 48 hours . at 8 hours after tgf- treatment , nox4 was observed in cell nuclei , while at later stages in the emt process ( at 48 hours ) , nox4 was predominately colocalized with -smooth muscle actin .
the inhibition of nox4 expression and activity using vas2870 inhibited emt progression.conclusionstransforming growth factor- drives the expression of the ros - producing enzyme nox4 in rat lens epithelial cells and nox4 inhibition can impede the emt process . |
hypoglycaemia is a major side effect of some glucoselowering therapies , in particular , insulin and the insulin secretagogues ( sulphonylureas and glinides ) .
it is a frequent occurrence in people treated with insulin and is more common in type 1 than in type 2 diabetes 1 , 2 , 3 .
hypoglycaemia increases in frequency and severity with the duration of insulin treatment 3 . impaired awareness of hypoglycaemia
is common in people treated with insulin , particularly those with type 1 diabetes 4 .
hypoglycaemia has a significant burden as it can engender fear and anxiety , disrupt sleep and adversely affect domestic and social life 5 , 6 .
interestingly , rates of severe hypoglycaemia have not generally diminished over the years , despite the introduction of insulin analogues and advances in glucose monitoring 7 , 8 .
this may be a consequence of the drive to tighter glucose targets , which have diminished the benefits of technological advances . attempting to reduce this burden
can encourage the maintenance of a suboptimal glycaemic control 5 , 9 , 10 , and both the severity and frequency of hypoglycaemia unequivocally reduce healthrelated quality of life 11 , 12 , 13 , 14 .
hypoglycaemia is also a burden on healthcare resources and on society as a consequence of the direct costs of its treatment and the indirect costs associated with lost productivity 9 , 15 , 16 .
these and other factors can negatively influence physicians and people with diabetes , promoting reluctance to initiate or intensify therapy insulin in particular because of the perceived burden of hypoglycaemia 10 , 17 .
the benefits of stricter glycaemic control are counterbalanced by the enhanced risk of concomitant hypoglycaemia .
the most expensive aspect of treating severe hypoglycaemia is hospital admission and inpatient care 15 , 16 .
severe hypoglycaemia is a common cause of hospitalization in elderly people with diabetes 18 , 19 , 20 , 21 . to our knowledge
, no largescale studies of the resource use associated with severe hypoglycaemia have been performed that have examined this resource use according to type of diabetes and insulin regimen .
the aim of this analysis was to estimate the resource use attributable to severe hypoglycaemia events in a large cohort of people with insulintreated diabetes , using data from a largescale clinical trial programme .
this analysis used data from the insulin degludec ( ideg ) and insulin degludec / insulin aspart ( idegasp ) phase 3a clinical trial programme , including 15 phase 3a therapeutic confirmatory trials , which involved more than 8000 participants .
all trials were registered on clinicaltrials.gov , the trial protocols were approved by independent ethics committees or institutional review boards , and written informed consent was obtained from participants before enrolment .
trials were undertaken in accordance with the declaration of helsinki and good clinical practice guidelines 22 , 23 .
the trials were categorized into three groups , depending on the patient population and the type of insulin regimen : type 1 diabetes receiving basal bolus therapy , type 2 diabetes receiving multiple daily injections and type 2 diabetes receiving basal oral therapy .
severe hypoglycaemia events were identified using information relating to adverseevent case reports in the clinical trial safety database .
only clearly stated resource use was included in the analysis , and all events were analysed independently .
the data were analysed descriptively , taking into consideration three variables ( that are not necessarily mutually exclusive ) :
nonmedical assistance only , with the response categories : yes , no.ambulance or ( onsite ) emergency team , with the response categories : yes , no.hospital or emergency room visit , with the response categories : no , yes 24 h ,
yes > 24 h.
nonmedical assistance only , with the response categories : yes , no . ambulance or ( onsite ) emergency team , with the response categories : yes , no . hospital or emergency room visit , with the response categories : no , yes 24 h , yes > 24 h. given the multiplicity of countries in the trial programme , it was decided not to attempt to distinguish between hospital inpatient treatment and emergency room treatment ( with subsequent discharge ) , because the local procedures varied .
hospital visits were therefore distinguished solely on duration ( 24 h or > 24 h ) .
details of the trials and characteristics of the patient populations are summarized in table 1
24 , 25 , 26 , 27 , 28 , 29 , 30 , 31 , 32 , 33 , 34 , 35 , 36 . in general , people with type 1 diabetes had a lower mean age , longer disease duration and lower bmi compared with those with type 2 diabetes . across the 15 openlabel ,
randomized , treattotarget clinical trials , severe hypoglycaemia was an infrequent occurrence : 536 severe hypoglycaemia events were recorded and analysed from a total of 8364 patient records .
most occurred in people with type 1 diabetes receiving basal bolus therapy , fewer occurred in those with type 2 diabetes on multiple daily injections , and a small number occurred in people with type 2 diabetes on basal oral therapy ( table 1 ) .
all but one event occurred in the iglar arm in the trials 3590 and 3593 . as a proportion of the number of randomized patients included in the safety analysis set in each trial .
biasp 30 , biphasic insulin aspart ; bid , twice daily ; bmi , body mass index ; ideg , insulin degludec ; idegasp , insulin degludec / insulin aspart ; idet , insulin detemir ; iglar , insulin glargine .
overall , 157/536 ( 29.3% ) of severe hypoglycaemia events required an ambulance / emergency team ( mainly involving paramedics , but also including physicians , although the analysis did not distinguish between them ) ( table 2 ) . additionally , following 100/536 ( 18.7% ) of the severe hypoglycaemia events , hospital or emergency room treatment
was required , with 36/536 ( 6.7% points ) of these requiring a hospital stay of > 24 h ( table 2 ) .
proportion of severe hypoglycaemia events leading to ambulance , emergency room or hospital visits although fewer events were recorded in people with type 2 compared with type 1 diabetes , once an event occurred , the proportion resulting in hospital or emergency room treatment was greatest in the basal oral therapy treatment group .
resources were used in relation to 57.1% , 45.3% and 37.9% of severe hypoglycaemia events in the type 2 diabetes basal oral therapy , type 2 diabetes multiple daily injections and type 1 diabetes basal
the greatest difference was in the proportion of events requiring a hospital stay of > 24 h : 47.6% in the type 2 diabetes basal
oral therapy , 5.3% for type 2 diabetes multiple daily injections , and 5.0% for the type 1 diabetes basal bolus group ( table 2 ) . the proportion of severe hypoglycaemia events utilizing medical resources . medical resource use includes use of ambulance / emergency team , hospital visit < 24 h and > 24 h. the variables measured were not necessarily mutually exclusive .
poor glycaemic control in diabetes is associated with serious complications such as sightthreatening retinopathy and renal failure , and optimizing glycaemic control is fundamental to minimize this morbidity 37 , 38 , 39 . however
, severe hypoglycaemia , a serious adverse effect of some glucoselowering therapies particularly insulin can also cause significant morbidity and have quality of life and economic consequences .
this analysis utilized data from a largescale clinical trial programme and subdivided records by type of diabetes and also by insulin regimen .
this has shown that in the controlled clinical trial setting , severe hypoglycaemia seldom occurs , but when it does , it leads to direct healthcare resource use in ~ 40% of cases .
a large difference was observed between the different regimens in the proportion of people experiencing severe hypoglycaemia who required hospitalization for > 24 h. the proportion of participants using resources following a severe hypoglycaemia event was highest in the people with type 2 diabetes on basal oral therapy , despite the overall lower number of events compared with the patients on multiple daily injections .
this may be because many of the participants receiving basal oral therapy ( and their family / carers ) were much less experienced in treating severe hypoglycaemia .
they may be more inclined to seek medical assistance , in contrast to people with type 1 diabetes and those with insulintreated type 2 diabetes taking multiple daily injections who probably have greater experience of exposure to severe hypoglycaemia .
people with diabetes and their family members in these groups are less likely to seek assistance because they have learned to cope effectively with this emergency and the immediate requirements of treating an episode of severe hypoglycaemia .
furthermore , the people with type 2 diabetes included in the trials had a higher mean age than those with type 1 diabetes , and this may mean that more of them had more comorbidities , were frailer and were more vulnerable to morbidity associated with severe hypoglycaemia , so contributing to the longer duration of hospital admissions .
several studies have shown that hypoglycaemia requiring hospital treatment is more frequent in the elderly compared with younger people with diabetes 19 , 20 , 21 .
it should also be noted that , in some trials , people with type 2 diabetes were taking concomitant oral antidiabetes agents , including sulphonylureas , and it is not clear whether these contributed to any of the severe hypoglycaemia events .
some specialists believe that hospital admission is necessary for people who experience severe hypoglycaemia as a consequence of sulphonylurea therapy , which might account for some of the hospital attendances by the participants receiving basal oral therapy 40 , 41 .
a previous study observed that the proportion of people for whom emergency medical assistance was sought was higher in those with type 2 diabetes than with type 1 diabetes ( 33% vs. 10% ) 2 . a populationbased study analysing resource use associated with severe hypoglycaemia treated by emergency medical services in tayside , scotland , recorded 260 episodes over a 12month period .
of these , 34% involved the ambulance service alone , 7% were treated by emergency / primary care services alone and 52% used both .
some 28% of cases required hospital admission , resulting in hospital occupancy of 230 bed days 16 .
although this is higher than the 18.6% who visited hospital in our study , it should be noted that this scottish study analysed only those events that had required emergency medical treatment , thus representing a more severe end of the spectrum .
the use of medical resources following severe hypoglycaemia is costly , considering the large number of people with insulintreated diabetes and the extensive use of sulphonylureas .
for example , an estimate of this resource utilization and associated financial burden based on the 20132014 uk national health service tariffs for admitted patient care & outpatient procedures diabetes with hypoglycaemic disorders equates to 1269 for people aged 69 years and 2187 for people aged 70 years , in addition to 235 for an ambulance transfer .
this yields an average cost per event across treatment regimens of 305 [ ( 11.9% + 6.7% ) * 1269 + 29.3% * 235 ] for people aged 69 years and 476 [ ( 11.9% + 6.7% ) * 2187 + 29.3% * 235 ] for people aged 70 years 42 .
hypoglycaemia is more costly in elderly populations with diabetes , who may be much more susceptible to severe hypoglycaemia , perhaps in relation to coexisting comorbidities 18 , 19 , 20 , 21 .
several other studies using different methodologies have provided results that are in broad agreement with our study .
hospitalization is the major cost associated with treating hypoglycaemia , but costs vary depending on the countries involved and local practices and procedures .
for example , the costs associated with treating hypoglycaemia in germany are higher than elsewhere because people with diabetes are often admitted to hospital for several days to receive additional education 43 .
the study based in tayside estimated the direct costs of treating severe hypoglycaemia based on 127 for an ambulance , 89 for emergency room treatment and 218 for each patient admitted to a ward to be over 13 million per year for the uk in 2003 ( equivalent to 17 million in 2014 prices ) 16 .
based on these values , the cost of a single severe hypoglycaemia event , which required some medical assistance , has been estimated at 335 44 .
data obtained using a questionnaire regarding a participant 's most recent severe hypoglycaemia event performed in germany , spain and the uk revealed that the cost of treating a person with type 2 diabetes ( germany , 533 ; spain , 691 ; uk , 537 ) was higher than for a person with type 1 diabetes ( 441 , 577 and 236 , respectively ) 15 . a usbased study investigating the cost of hypoglycaemia in people with insulintreated type 2 diabetes estimated the direct costs of severe events requiring assistance from a healthcare professional as $ 1729 , whereas those events requiring nonmedical assistance were $ 242 per event 45 .
most previous studies have been retrospective assessments of all people with severe hypoglycaemia , and often exclusively those who had utilized additional healthcare resources .
although some of these studies included cost analyses , they did not include all severe hypoglycaemia events , so it is difficult to determine what proportion of events required ambulance and hospital treatment .
this study benefits from its size and the more accurate and reliable recording associated with controlled clinical trials , by contrast with data that have been collected in realworld observational or retrospective studies .
this study has the limitation that the rates of hypoglycaemia may be lower than in a realworld setting , because people at high risk of severe hypoglycaemia or who were judged by the investigator to have impaired hypoglycaemia awareness were excluded from the clinical trials .
however , because the examination was not primarily aimed at assessing the risk of events , but rather exploring the consequences of such an event , it is unclear to what extent such a limitation would influence the results .
additionally , because of the global nature of the clinical trials , a huge variation in healthcare utilization and local procedures are to be expected , making estimates of resource use difficult to determine with accuracy .
healthcare utilization in trials is often protocol driven , but this has not been assumed in this analysis because of the acute nature of severe hypoglycaemia .
potential exists for the underreporting of ambulance use , or inconsistent reporting of resource use .
although it is known when people had attended hospital , information about how they arrived there was not consistently recorded , so only clearly stated resource use was included in the analysis .
this study did not attempt to capture length of stay or what medication was received because this would be healthcaresector specific and could be confounded by other comorbidities .
the study has reported only the direct resource use relating to the involvement of emergency services and hospital treatment and does not include additional blood glucose measurements , costs of glucagon or intravenous dextrose , or any subsequent medical consultations that were necessary following a severe hypoglycaemia episode .
this analysis suggests that severe hypoglycaemia events often result in emergency / ambulance calls and treatment in hospital , thereby incurring a substantial economic burden .
a high rate of resource utilization was observed with all insulin regimens , and although the incidence of severe hypoglycaemia events was low , the greatest level of resource use following an event occurred in people with type 2 diabetes on a regimen of basal insulin combined with oral antidiabetes drugs .
novo nordisk contributed to the study design and conduct , data collection , analysis and interpretation of the trials .
sh has served on advisory panels and speaker bureaus for eli lilly , novo nordisk and takeda , and received research support from medtronic .
bmf has served on advisory panels and speaker bureaus for eli lilly , novo nordisk , msd , sanofi , boehringer ingelheim and janssen .
sg has served on advisory boards for novo nordisk , sanofi , takeda and eli lilly and has received research support from novo nordisk , sanofi and takeda .
all authors contributed to the design , interpretation , writing , evaluation and approval of this manuscript . | abstractaimsto assess resource utilization associated with severe hypoglycaemia across three insulin regimens in a large phase 3a clinical programme involving people with type 1 diabetes treated with basal bolus insulin , people with type 2 diabetes treated with multiple daily injections and people with type 2 diabetes treated with basal
oral therapy.methodsdata relating to severe hypoglycaemia events ( defined as episodes requiring external assistance ) from the insulin degludec and insulin degludec / insulin aspart programme ( 15 trials ) were analysed using descriptive statistics .
comparators included insulin glargine , biphasic insulin aspart , insulin detemir and sitagliptin .
mealtime insulin aspart was used in some regimens .
this analysis used the serious adverse events records , which documented the use of ambulance / emergency teams , a hospital / emergency room visit 24 h , or a hospital visit > 24 h.resultsin total , 536 severe hypoglycaemia events were analysed , of which 157 ( 29.3% ) involved an ambulance / emergency team , 64 ( 11.9% ) led to hospital / emergency room attendance of 24 h and 36 ( 6.7% ) required hospital admission ( > 24 h ) .
although there were fewer events in people with type 2 diabetes compared with type 1 diabetes , once a severe episode occurred , the tendency to utilize healthcare resources was higher in type 2 diabetes vs. type 1 diabetes . a higher proportion ( 47.6% ) in the basal
oral therapy group required hospital treatment for > 24 h versus the type 1 diabetes ( 5.0% ) and type 2 diabetes multiple daily injections ( 5.3% ) groups.conclusionthis analysis suggests that severe hypoglycaemia events often result in emergency / ambulance calls and hospital treatment , incurring a substantial health economic burden , and were associated with all insulin regimens . |
olfactory dysfunction has been reported in several neurodegenerative conditions such as alzheimer 's disease ( ad ) and parkinson 's disease ( pd ) and may be used as a diagnostic tool in these conditions . however , whilst olfactory dysfunction has been reported in korsakoff 's syndrome ( ks ) , the possibility of using olfactory dysfunction to aid in a differential diagnosis in patients with working memory dysfunction has not been fully explored .
ks patients usually first present with wernicke 's encephalopathy ( we ) , characterized by opthalmoplegia , gait abnormalities , confusion , and some signs of short - term memory loss , before developing ks .
we patients typically respond well to thiamine ( vitamin b1 ) , but unfortunately , the signs of we are often overlooked , and without proper thiamine treatment , the we patient may then develop ks .
acute ks may also benefit from early thiamine treatment ; however ks symptoms may be ascribed to other alcohol - related problems , or the memory impairments ( especially working memory ) may be confused with frontal lobe ( fl ) dysfunction .
given the potential of thiamine treatment , improving the diagnostic pick - up rate for early ks would be a useful clinical goal . in this study
, we examine the relationship between olfaction and memory in ks patients , fl patients , and healthy volunteers to explore the implications of olfactory testing in the diagnosis of ks .
we predicted that both patient groups would perform poorly on the memory test and that ks patients would show a more severe olfactory deficit than fl patients .
twelve ks patients , four age - matched fl patients ( one left frontoparietal , two right frontotemporal , and one bilateral frontal lesions , due to stroke ) and nine healthy age - matched controls were tested .
ethical approval was obtained from the school of psychology at bangor university and the north west wales research ethics committee before this research began .
twelve individuals with a clinical diagnosis of alcoholic korsakoff 's syndrome and a history of wernicke 's encephalopathy were recruited from 4 nursing and private care facilities in the uk . written and verbal informed consent was obtained from individual patients and their primary caregiver or family member .
the diagnostic guidelines used to verify wernicke - korsakoff syndrome were based on consensus guidelines used for inclusion in an ongoing biomedical study investigating the genetic susceptibility of alcohol - related brain damage .
the specific guidelines used were wernicke 's encephalopathy confirmed by medical history of hospitalization with a presentation of confusion and either ataxia or nystagmus ; korsakoff 's syndrome confirmed by a medical history of memory impairment and a past presentation of either confabulation or disorientation , as well as a present indication of short - term memory impairment .
only adults under the age of 65 were included . only individuals with an onset date of 6 months prior to recruitment were included to ensure that the patient group represented those in a chronic stage of the illness .
the onset date of korsakoff 's syndrome recorded in their medical or care facility admission notes varied from 6 months to 10 years prior to recruitment .
only individuals who were currently abstinent from alcohol , and had been for at least 6 months , were included .
there was no indication of spontaneous confabulation in any individual included in the korsakoff 's group .
all korsakoff 's patients lived in a care environment where proper nutrition and medical attention were offered , although there was variation in the administration of prescription medication and vitamin supplementation , including thiamine . the benton visual retention test - fifth edition ( bvrt - v ) and the brief smell identification test ( b - sit ) .
the benton visual retention test - fifth edition ( bvrt - v ) was administered .
performance was quantified by the numerical error score calculated relative to the participants iq and age .
performance was assessed by the number error score calculated relative to the participants ' iq and age .
the 12-item b - sit [ 5 , 6 ] was used to evaluate olfactory function .
the b - sit scores were significantly lower in the ks group than in either the healthy control or fl groups ( figure 1(a ) ) .
one - way anova revealed a significant main effect of group ( f(2,22 ) = 72.20 , p < .001 ) .
post hoc multiple comparisons using tukey 's hsd revealed significant differences between the ks and healthy controls groups ( mean difference of 6.39 , p .001 ) and also between the ks and fl groups ( mean difference of 5.50 , p .001 ) .
in contrast , the ks and fl patients had similar high scores on the bvrt - v , compared to the healthy age - matched control subjects , whereas only the ks patients performed poorly on the b - sit ; therefore the poor performance of ks patients on the b - sit can not be attributed to a working memory dysfunction , rather it reflects olfactory dysfunction ( figure 1(b ) ) . one - way anova revealed a main effect of group ( f(2,10 ) = 6.00 , p > .005 ) .
post hoc multiple comparisons using tukey 's hsd revealed significant differences interaction between the healthy control and ks groups ( mean difference of 7.10 , p .05 ) and also between the fl and control groups ( mean difference of 6.85 , p .05 ) .
the observed deficits in olfactory function support previous studies [ 5 , 7 ] . in contrast
, fl patients showed normal olfactory function despite evidence of memory impairment similar to ks patients .
this supports our suggestion that assessment of olfactory dysfunction may aid in the differential diagnosis of patients presenting with working memory dysfunction .
chronic alcohol misuse typically results in deficits in memory , planning , organizing , judgment making , and social skills . since these are difficulties usually related to frontal lobe damage , the presentation of these symptoms in ks or fl patients might be easily confused .
the diencephalic pathology seen in ks may be responsible for the selective impairment in olfactory perception observed , which may explain why the loss of perception is characteristic to the disorder . clinical diagnosis of ks is subject to clinical error .
when comparing two surveys conducted on the detection rate of ks , it was revealed that standardised tests can often miss the disorder altogether .
the literature documenting olfactory dysfunction in ks patients is relatively limited and sometimes contradictory [ 4 , 6 ] . in hulshoff 's et
al . ( 2001 ) study , six fl patients and seven ks patients were tested on odour discrimination .
these authors claimed that fl patients were significantly impaired on odour discrimination tasks compared to the ks group , scoring within minimal impairment percentile ranges .
however , patients with traumatic fl damage often present with olfactory problems , due to shearing of the olfactory fibres as they pass through the cribriform plate into the underside of the frontal lobes .
it is possible that their fl patients may have sustained damage to the olfactory pathways in the inferior part of the frontal lobe , during neurosurgical interventions to remove tumours .
in addition , the fl patients ' results only reached statistical significance once subjects who had not reached the odour detection criterion were factored out from analysis .
we acknowledge that the present study involved a relatively small number of patients , but the results suggest that assessment of olfactory function may be an effective tool in the differential diagnosis of ks patients from patients with memory dysfunction due only to frontal lobe damage .
the nature of ks that often keeps patients in long - term nursing care and the potential for treatment if diagnosed early highlight the importance of an early diagnosis .
the b - sit is an uncomplicated , easily administered assessment tool that is simple for ks and fl patients to complete .
the use of the b - sit could aid in diagnosis and referral for appropriate thiamine treatment .
future studies that include a greater number of patients , as well as comparisons to olfactory performance in other neurological disorders , may be useful in further determining the potential value of the b - sit . | whilst olfactory dysfunction has been reported in korsakoff 's syndrome ( ks ) patients , the diagnostic implications of this have not been fully explored .
ks can be difficult to diagnose because cognitive symptoms are similar to other diagnoses .
for instance , patients with frontal lobe ( fl ) syndrome may present with memory impairments that are similar to ks . participants
were given the benton visual retention test - fifth edition ( bvrt - v ) , to identify working memory dysfunction , and a brief smell identification test ( b - sit ) , to evaluate olfactory function .
b - sit scores were found to be significantly lower in the ks group compared to the control and fl groups .
in contrast , the error scores on the bvrt - v were significantly higher in both the ks and fl groups compared to the healthy control subjects .
therefore , we suggest that olfactory function may aid in the differential diagnosis of patients presenting with working memory dysfunction . |
culture - bound syndrome ( cbs ) is a broad rubric that encompasses certain behavioral , affective and cognitive manifestations seen in specific cultures . as per diagnostic and statistical manual for mental disorders - iv(dsm
iv ) ( appendix i , p. 844 ) , they denote recurrent , locality - specific patterns of aberrant behavior and troubling experience that may or may not be linked to a particular dsm - iv diagnostic category . many of these patterns are indigenously considered to be
illnesses , or at least afflictions , and most have local names ... culture - bound syndromes are generally limited to specific societies or culture areas and are localized , folk , diagnostic categories that frame coherent meanings for certain repetitive , patterned , and troubling sets of experiences and observations .
these manifestations are deviant from the usual behavior of the individuals of that culture and are a reason for distress / discomfort .
since these conditions are considered to be specific to certain regions , they are of concern and interest to the practitioners and the researchers from those regions .
because of this inherent assumption of cultural specificity of these conditions , they are considered to be alien to other cultures .
consequently , they hardly ever become area of interest for clinicians and researchers from these settings .
the current classification systems such as international statistical classification of diseases and related health conditions - 10 ( icd-10 ) ( world health organization ( who ) 1992 ) and dsm - iv - tr ( american psychiatric association 1994 ) do not give guidelines to diagnose these culture - bound conditions in the main text .
this is likely to result in arbitrary assumptions by the clinicians and researchers while dealing with them .
in fact , these conditions do not find a place in the main text of dsm - iv and are placed in the appendix .
this article presents a case for inclusion of these conditions in the mainstream diagnostic classification because of theoretical and management implications .
the argument is based on the available literature that demonstrates that the condition is / has been more widespread than assumed ( as shown by the previous workers ) and the available literature does not support the case for a special labeling of these conditions .
dhat syndrome has been chosen for the argument as this condition has been specified to be bound to the culture of author 's training and clinical work .
dhat syndrome has been specified as a cbs specific to the culture of the indian subcontinent , especially india .
dhatu meaning metal and also elixir . in this condition ,
the individual ascribes the symptoms of weakness , fatigability , sexual dysfunction , loss of virility and vitality to the loss of this valuable body fluid through the urine / nocturnal emission or masturbation .
it is assumed that dhat syndrome is a condition that is prevalent in the indian subcontinent . however , sumathipala et al .
have reviewed the history of the condition and have found it to be prevalent in different geographical regions of the world .
it has been described in literature from china , europe and americas and russia at different points of time in history .
mention of semen as a soul substance could be found in the works of galen and aristotle who have explained the physical and psychological features associated with its loss .
have cited the works of beard , hare and maudsley linking semen loss with mental illness .
over the years , the cases of the semen loss anxiety and the theories regarding the implication of the loss of seminal fluid due to indulgence in masturbation / sexual activity have gone into disrepute in many regions of the world .
one could easily correlate this with the increasing advances in the field of medical science .
an increasing scientific rigor with consequent increase in understanding and awareness into various issues and phenomenon has helped modify many locally held beliefs .
people in many developed countries now have more scientifically based and plausible explanation for their bodily / psychic discomfort .
it would be erroneous to say that the discomfort has disappeared from the lives of the people in these countries .
conditions such as somatoform disorders are now recognized as validated conditions present across all regions of the world .
in fact , the case of unexplained somatic symptoms presents an interesting scenario in this context . while depression and anxiety are quiet common among these individuals , they are not an essential component of clinical presentation .
the associations and attributions made by these patients are based on their ( mis)attributions based on their perceptions and knowledge .
reattribution training has helped the general physicians to improve patient satisfaction and reduces the cost of treatment in some studies .
the members of a particular community , culture or region use the local vocabulary to express their feelings , moods and concerns .
the idioms of expression used by them are based on the adjectives provided by the local language .
locally prevalent cultural norms , societal acceptance of certain issues and the role of the individual in society tend to govern the way in which one expresses himself .
this applies to different feeling states including those of happiness , joy , sorrows or distress .
it has been held that the individuals from the developing countries tend to somatize more than their western counterparts .
this is not entirely correct assumption as presence of somatization among western populations has been well documented over the years.[1315 ] multicenter cross - national studies have shown strong association between somatic symptoms and psychological distress , which did not tend to vary across disparate cultures .
comparable rates of somatic complaints in the developing and the developed world were observed in the international study of somatoform disorders conducted by who .
lipowski has reported three components of the presentation of the complaints by the patients : experiential ; cognitive ; and behavioral .
these components are governed by the locally prevalent attitudes and beliefs , the knowledge and awareness of the residents on the issue and the way the currently prevalent assumptions are reinforced or refuted so as to give way to new and more acceptable explanations .
the authors from developing countries have observed that somatic complaints and symptoms take precedence over the symptoms given in the diagnostic manuals . the lack of appropriate adjectives for the expression of the mood , cognitive symptoms could be a reason for the same .
the local cultures have been found to be lacking in the direct equivalents for the terms depression or anxiety .
it is vital to understand the culture - specific terminology used by patients in such settings .
also it is recommended to assess for mood and cognitive features of underlying depression and anxiety in those presenting with multiple somatic complaints .
this is so because most of these patients do report cognitive and emotional symptoms of on being inquired .
missing the culturally specific idioms of distress could contribute to the under - recognition of depression and anxiety .
labels such as shenjing shuairuo ( neurasthenia ) in china , ghabrahat ( anxiety ) in india , pelo y tata ( heart too much ) in botswana , and nerves in some latin american and south african societies are described as local illness categories that overlap with depression.[1922 ] the widely prevalent cultural beliefs and the overwhelming importance given to certain issues tend to provide the individuals with the avenues to express their mood , cognitive and bodily symptoms . in context of dhat ,
the local beliefs tend to emphasize the seminal fluid as source of vitality and virility .
hence , loss of this fluid ( albeit non - pathological ) provides individuals from these cultures a plausible explanation for the experiences of distress .
this attribution is strengthened by its acceptance among others from the same cultural background including certain traditional and faith healers .
lack of appropriate knowledge on the physiological processes helps these beliefs to persist in the community .
role of culture has been studied in disorders such as schizophrenia , major depression , anxiety disorders and attention deficit hyperactive disorder . in the context of psychiatric disorders ,
first , culture and society shape the meanings and expressions people give to various emotions .
so , it would not be erroneous to conclude that cultural influence on psychiatric disorders includes conditions other than cbs .
the current view on the dhat syndrome describes it as a culture - bound condition restricted to certain cultures .
however , there is a need to relook at this condition as a functional somatic presentation of universal distribution- a manifestation that has been modified in certain cultures by the improved knowledge and awareness ; and continues to manifest in others in its original form in absence of such knowledge and awareness .
mumford studied the patients of dhat syndrome presenting in the outpatient setting in a developing asian country .
the dhat complaint was reported equally by the men attending medical clinics and by the patients with
it was strongly associated with depressed mood , fatigue symptoms , and a dsm - iii - r diagnosis of depression .
the findings of the study suggest dhat to be a culturally determined symptom associated with depression rather than a cbs .
malhotra and wig reported that respondents in the study belonging to social class i discussed sex freely when compared with lower social classes .
people in social class iv were more likely than those of any other group to see nocturnal emission as abnormal .
they were also least likely to see psychological persuasion as a mode of treatment for it .
the authors explained the symptom as a consequence of the information and knowledge base of the individuals from the different social groups .
chadha in his study found around half of the cases of dhat to be having depressive disorder , 18% to be having anxiety disorder and 32% to be having somatoform disorders .
dewaraja and sasaki found that half of the patients with sexual dysfunction in a clinic - based sample in sri lanka attributing their symptoms to semen loss had somatic symptoms and a third had sexual deficiencies .
this observation also suggests a role of knowledge and locally prevalent beliefs in explanation of the condition .
the authors attempted to replicate the findings in japan but were not able to do so .
a higher awareness of the individuals regarding the role of semen and body vitality was put forth as the explanation for this observation .
bhatia , while studying 60 cases of cbss seen in psychiatry out - patient department from india , found depression to be the most commonly associated psychiatric disorder in cases of dhat syndrome .
it could have been possible that the cases seen by the author were having semen loss anxiety as one of the manifestations of the underlying psychiatric disorder like depression and anxiety
. however , patients knowledge and awareness shaped the semen loss as the presenting complaint and main area of concern .
there is a need to label the condition of dhat syndrome as a functional somatic syndrome .
it should no longer be considered restricted to / as a manifestation of a particular culture .
the findings from the previous literature on the condition suggests that the condition is not restricted to a particular culture type and has been found in different cultures .
culture bound. various other medical conditions have also seen a change in distribution pattern across world .
the frequently associated co - morbidity of the psychiatric disorders like depression , anxiety and somatization disorders reported in the available studies on dhat syndrome suggests that the condition could be a presenting form of these underlying psychiatric disorders .
attribution of the underlying distresses to the easily observable and locally acceptable beliefs should not lead to a specific and distinct labeling of the distress .
one must keep in mind the role of the culture in manifestation and management of such cases .
it seems uncalled for to label certain manifestations of distress as cbs and treat them as exotic / alien phenomenon .
while studying 29 patients with dhat syndrome and 32 medical controls , they found that patients with dhat syndrome have significantly different illness beliefs and behaviors compared to controls and have similarities with other functional somatic syndromes .
thus , one could argue for a case for inclusion of dhat syndrome as a functional somatic disorder rather than restricting it to a cbs .
there is a need to have a relook at the other cbss and place them under rubrics of functional affective , functional somatic and functional behavioral conditions in the upcoming revisions of the diagnostic manuals .
the current version of dsm - iv has included the cultural underpinnings of the presentations of various mental and behavioral conditions in the text descriptions of the individual disorders .
also , it has incorporated the description of the cbs and the outline for assisting the clinicians in systematic evaluation of these conditions in its glossary section .
this approach highlights the acceptance of the importance of the cultural variables in shaping the psychiatric conditions and their management .
authors like wig ( 1994 ) and littlewood ( 1996 ) have proposed the change in the current diagnostic classifications to shift the conditions currently included under the rubric of cbs to the group considered to be more widely prevalent .
one could easily draw the parallels from other psychiatric conditions considered to be prevalent all over the globe .
although these psychiatric disorders would be classified under the same diagnostic categories , there could still be differences in their manifestation and presentation in different cultures .
these differences are shaped by the locally prevalent cultural beliefs , attitudes and knowledge among other factors .
the patient of paranoid schizophrenia would be harboring a delusional belief of being persecuted against .
however , it would not be surprising to find a variation in the content of this delusion .
such variations are shaped by the cultural and individual differences , part of which is governed by the knowledge and the understanding of the condition .
this would not change the diagnosis , although one would need to be aware of the local cultural nuances for the better understanding and the management of the case . with the increasing globalization ,
the culture bound cases are being seen by the clinicians in different cultures and geographical regions . separately categorizing cbss is unlikely to improve the management of these conditions .
rather , such an approach could impede the understanding of these conditions as researchers from other cultures and countries might consider it irrelevant to study these conditions because of their culture specificity. the concerns of the americans and europeans during the 19 century were not much different from the concerns of the asians in the present times .
the limited awareness of some populations on certain issues related to the health and illness coupled with the locally prevalent beliefs and attitudes might have led to persistence of these issues in some societies .
one of the ways forward could be to consider these as manifestations that have not ceased to manifest in certain cultures .
the change observed in certain regions is a result of improved knowledge and changed belief system .
labeling these conditions a culture - bound condition is probably one of the reasons behind limited published literature on them .
this is evident in case of dhat syndrome as there has been no published randomized trial of interventions for this condition .
more research on dhat syndrome would not only help these regions with management of these conditions but also provide a better understanding in the functional somatic conditions .
researching how a once widely prevalent functional somatic condition became restricted to certain cultures is likely to come out with some interesting findings .
the same argument could be extended to other current cbss as the cases of functional somatic / functional affective / functional behavioral disorders .
this would mean a more systematic study of these conditions that would help in improving the understanding and management of these conditions .
description of the many conditions currently labeled as cbs have diagnostic equivalents in dsm - iv [ table 1 ] .
relabeling and inclusion of these manifestations in the mainstream diagnostic categories in the upcoming revisions of the diagnostic manual would pave way for a better understanding and management of these conditions . | culture - bound syndrome is a broad rubric that encompasses certain behavioral , affective and cognitive manifestations seen in specific cultures .
these manifestations are deviant from the usual behavior of the individuals of that culture and are a reason for distress / discomfort .
this entitles these manifestations for a proper labeling and subsequent management .
however , the available information and literature on these conditions suggest that at least some of them are / have been more widely prevalent than being considered .
this article presents a case for possible relabeling and inclusion of these conditions in the mainstream diagnostic systems based on the example of the dhat syndrome- a culture - bound syndrome from india .
these conditions could be relabeled as functional somatic syndromes . |
research on the one - electron reduced
analogue of no , namely nitroxyl ( hno / no ) , has revealed
distinguishing properties regarding its utility as a therapeutic .
however , the fleeting nature of hno requires the design of donor molecules .
metal nitrosyl ( mno ) complexes could serve as potential hno donors .
the synthesis , spectroscopic / structural characterization , and hno
donor properties of a { cono}8 complex in a pyrrole / imine
ligand frame are reported .
the { cono}8 complex [ co(ln4phcl)(no ) ] ( 1 ) does not react with
established hno targets such as feiii hemes or ph3p .
however , in the presence of stoichiometric h+1 behaves as an hno donor .
complex 1 readily
reacts with [ fe(tpp)cl ] or ph3p to afford the { feno}7 porphyrin or ph3p = o / ph3p = nh ,
respectively . in the absence of an hno target ,
the { co(no)2}10 dinitrosyl ( 3 ) is the end product .
complex 1 also reacts with o2 to yield the corresponding
coiii-1-ono2 ( 2 ) nitrato analogue .
this report is the first to suggest an hno donor
role for { cono}8 with biotargets such as feiii - porphyrins . | |
autoimmune channelopathies are becoming one of the exciting areas of neurological diseases in clinical practice because , though relatively uncommon ( collectively perhaps 20 per million per year ) , diagnosis of these conditions usually indicates a significant clinical improvement following immunotherapies that reduce autoantibody levels .
the field stems from three decades of research into myasthenia gravis and the lambert - eaton myasthenic syndrome ; in these conditions , autoantibodies to muscle nicotinic acetylcholine receptors ( achrs ) or voltage - gated calcium channels ( p / q - type ) , respectively , are the main pathogenic agents and cause destruction and/or downregulation of their targets , leading to neuromuscular junction transmission failure ( table 1 ) which can be demonstrated in animal models
. newer disorders of peripheral neurotransmission include ( a ) peripheral nerve hyperexcitability syndromes with antibodies binding to i - dendrotoxin - labelled shaker - type ( kv1 ) voltage - gated potassium channels ( vgkcs ) extracted from mammalian cortex and ( b ) autonomic neuropathies with antibodies to i - epibatidine - labelled ganglionic nicotinic achrs .
achr , acetylcholine receptor ; vgcc , voltage - gated calcium channel ; vgkc , voltage - gated potassium channel . over the last decade or so
, a new family of antibody - associated diseases has emerged that is beginning to overturn previous concepts that regarded the brain as immune - privileged and protected by an impermeable blood - brain barrier .
first , glutamate receptor ( glur3 ) antibodies were present in children with the very rare but devastating form of epilepsy called rasmussen encephalitis , but these findings were not always confirmed in other cohorts of patients , and the main pathology is now thought to be cellular rather than antibody - driven .
the paradigm shift really began with the finding of very high vgkc antibody levels in patients with limbic encephalitis - which includes seizures , psychological disturbance , memory loss and high signal on magnetic resonance imaging ( mri ) in the medial temporal lobes - who responded convincingly to immunotherapies such as plasma exchange ( which removes circulating plasma components such as antibodies and replaces them with substitute plasma proteins ; see figure 1 ) [ 8 - 10 ] . until then , limbic encephalitis was almost always recognised as
paraneoplastic ( that is , associated with a t cell - mediated immune response to a tumour ) and with a poor response to treatments . the vgkc antibody - associated central nervous system ( cns ) phenotypes are now recognised widely , are usually nonparaneoplastic and include patients with some form of epilepsy or morvan syndrome .
however , despite colocalisation with antibodies to different kv1 subtypes on brain tissue and in transfected hela cells expressing different kv1 subtypes , the specificity of the antibodies is not altogether clear ; a new finding is that many of the antibodies bind not to the kv1s themselves but to other juxtaparanodal proteins , such as contactin - associated protein 2 ( caspr2 ) , that form part of the vgkc complex after extraction from brain tissue .
antibodies to caspr2 are particularly frequent in patients with morvan syndrome ( s irani , s alexander , a vincent , unpublished data ) .
( a ) above , magnetic resonance imaging evidence of hippocampal inflammation and swelling ( arrows ) in a patient with voltage - gated potassium channel ( vgkc ) antibodies . below , graph of dramatic decrease in the antibodies after successful treatment in another patient .
( b ) in the images above , antibodies ( red anti - human igg ) bind to human embryonic kidney cells expressing n - methyl - d - aspartate receptors ( nmdars ) ( coexpressed with enhanced green fluorescent protein [ egfp ] ; some cells express egfp without detectable nmdars ) in a young girl who developed an encephalopathy with mutism and catatonia and made a complete recovery after immunotherapies ( see and the video available online ) . in the image
below , nmdar antibodies ( green ) bind to unpermeabilised hippocampal neurons in culture , which were then fixed , permeabilised and stained for synaptophysin ( blue ) and nr1 ( red ) ( the binding of antibodies to the live cell surface does not colocalise well with the intracellular nr1 which appears throughout the neuron ) .
( c ) in the images above , antibodies to aquaporin-4 ( aqp4 ) in a patient with neuromyelitis optica ( nmo ) bind to cells transfected with aqp4-egfp . in this case
, the directly tagged aqp4-egfp can also be solubilised from the cells and , as seen in the graph , the antibodies can be measured quantitatively by counting the fluorescence in the immunoprecipitates . in these assays ,
images courtesy of s irani , l zuliani , mi leite , p waters and b lang .
some patients presenting with symptoms of cognitive problems , psychiatric disturbance or epilepsy were found to have antibodies that bound to the proximal dendrites of the hippocampal neuropil , distinct from the binding of vgkc antibodies more distally ; many of these antibodies were subsequently shown to be directed against n - methyl - d - aspartate receptors ( nmdars ) ( nr1/nr2b ) , with nr1 as the main target .
most of these patients progressed to a more complex phenotype with movement disorders or catatonia , mutism , sleep disturbance and autonomic dysfunction . at first , the syndrome was associated with ovarian teratomas in young women , but in these cases , unlike the traditional paraneoplastic disorders , the conditions improved when the tumour was removed and immunotherapies given .
now many nonparaneoplastic cases are being identified and the phenotype is widening to include both male and female adults , teenagers , and even young children ( s irani , a vincent , unpublished data ) .
these nmdar antibodies may be different from those measured by binding to linear peptide sequences of nr2a / nr2b seen in neuropsychiatric patients and have the potential to be pathogenic since they target extracellular domains on nr1/nr2b transfected human embryonic kidney cells and substantially reduce the expression of these subunits in primary cultures of hippocampal neurons .
the nmdar antibodies were most easily detected in the cerebrospinal fluid ( csf ) ( at 1:10 ) compared with serum ( at 1:400 ) , and there is substantial intrathecal synthesis of the specific antibody ( table 2 ) ; nevertheless , in absolute terms , serum levels are higher than csf levels .
recently , antibodies to ampar ( -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor ) glur1/glur2 were identified in another form of limbic encephalitis that was mostly cancer - related .
this is calculated as ( cerebrospinal fluid [ csf ] titre of specific antibody / concentration of csf igg ) / ( serum titre of specific antibody / concentration of serum igg ) .
values above 1.5 are usually considered indicative of synthesis within the csf compartment rather than the result of passive leakage .
it needs to be appreciated that the normal serum igg concentration is about 400 times higher than the normal csf igg concentration .
therefore , even in the presence of substantial intrathecal synthesis , the serum concentration of specific antibody will be higher than the csf concentration .
ab , antibody ; ampar , -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor ; aqp4 , aquaporin-4 ; cns , central nervous system ; eaat1 , excitatory amino acid transporter 2 ; glur , glutamate receptor ; glyr , glycine receptor ; nmdar , n - methyl - d - aspartate receptor ; vgkc , voltage - gated potassium channel .
meanwhile , a completely different condition was found to be associated with antibodies to a water channel .
neuromyelitis optica ( nmo , or devic disease ) has usually been considered to be part of the spectrum of inflammatory demyelinating disorders , of which multiple sclerosis is the best known . however , nmo is a distinct inflammatory condition of the optic nerves which involves severe visual failure and inflammation of the spinal cord causing longitudinally extensive transverse myelitis ( at least three spinal cord segments with high signal on mri ) , that leads to para- or tetraparesis , sensory deficits and bladder disturbances .
patients show variable recovery with immunomodulatory treatments but accumulate disability over time , and mortality is high if the disease is not appropriately treated . in 2004 ,
antibodies binding around small vessels , under the pia and in virchow - robin spaces were defined by immunofluoresence , and the target was subsequently identified as aquaporin-4 ( aqp4 ) , the only water channel expressed strongly in the brain ( and also in kidney and stomach ) .
antibodies to aqp4 bind to the astrocyte endfeet that abut cns blood vessels and are thought to be important contributors to the integrity of the blood - brain barrier .
the antibodies lead to substantial loss of surface aqp4 by internalisation and activate complement with formation of the membrane attack complex , leading to cellular damage .
they also reduce astrocyte expression of excitatory amino acid transporter 2 ( eaat2 ) with reduced reuptake of glutamate and hence potential excitotoxic damage .
whether these changes alone lead to the substantial inflammatory infiltrates , areas of demyelination , loss of aqp4 and sometimes necrosis that are found in lesions is not yet clear , but increases in antibody levels are associated with clinical relapses , and aqp4 antibodies decrease in parallel with clinical improvement after immunosuppression ; overall , there seems little doubt that the antibodies contribute to the pathology
. finally , another receptor target is emerging in patients with rare spinal and brain stem syndromes . in one adult male who presented with excessive startle and progressive encephalomyelitis with rigidity and myoclonus ( perm ) , a form of stiff person syndrome , antibodies to glycine receptor alpha 1 pentamers ( glyr1s ) were identified .
these antibodies are now being found in other patients with related disorders ( a vincent , i leite , h - m meinck , unpublished data ) .
once defined , the antibodies are best identified by binding to native proteins extracted from mammalian tissue in mild detergents ( vgkcs ) , or better still to the native protein expressed in an appropriate human cell line ( nmdars , ampars , aqp4 , glyr ) , rather than to short peptides that do not represent the native conformation of the target antigen .
the protein must be expressed on the cell surface and the cells should be unpermeabilised so that only cell surface - binding antibodies are detected ( this ensures that they are potentially pathogenic , in contrast to those antibodies to intracellular components found in paraneoplastic disorders ) .
clustering of the antigen by use of intracellular scaffolding proteins can increase sensitivity and specificity as recently demonstrated for achr antibodies .
in addition , the antibodies should be shown to bind to the extracellular surface of neurons or astrocytes cultured from mammalian tissues and to induce relevant biological changes in such cultures . in the future ,
one hopes that these studies will extend to examining the effects of these recently discovered antibodies on neuronal activity in brain slices in vitro and in animal models in vivo . considering the diversity of ion channels and receptors in the nervous system , it would be strange if there were no other autoimmune channelopathies to be discovered , diagnosed and treated .
until now , most of the target channels have been identified by a candidate approach , but if the target for binding to the cultured cells is sufficiently abundant , as appears to be the case for ampars , it is possible to immunoprecipitate the target using the relatively pure csf igg from the patients ; this technique has potential for identifying new targets in the future .
even the total patient plasma igg can be used to identify antigens by this approach when a suitable cell preparation or cell line is identified . in each of these diseases ,
csf antibodies are found , and there is often evidence of high concentrations of csf - specific antibody relative to csf igg concentration when compared with similar measurements in serum ( intrathecal synthesis , table 2 ) , but the absolute concentration of antibody is still higher in serum than in csf . a major question , therefore , is whether the antibodies that are pathogenic come directly from the blood into the cns parenchyma via a leaky or damaged blood - brain barrier or whether the disorders require the presence of specific antibodies in the csf .
the latter could be the result of passive diffusion across the choroid plexus and/or intrathecal synthesis by b cells that have gained entry to the cns and synthesise the antibodies in the intrathecal compartment .
these considerations are not purely academic . does intrathecal synthesis decrease with current systemic treatments and increase if the patient relapses ? do immune responses ever begin in the cns and remain undetectable in the serum ? and importantly , should drugs and therapies be specifically targeted to the csf compartment rather than to the systemic immune system ?
these are just some of the questions that arise from the identification of these new autoimmune disorders , and the answers will likely come from both focused human studies and animal models .
| contrary to established wisdom , there now appear to be antibody - mediated central nervous system ( cns ) disorders . over the last few years
, a number of patients have been defined with antibodies to voltage - gated ( vgkc ) or ligand - gated ( nmdar , glyr ) ion channels or ungated water ( aqp4 ) channels .
some of the disorders improve spontaneously over time , others may be more chronic and relapsing - remitting , but immunotherapies reduce antibody levels and improve clinical outcomes .
these are exciting developments that herald a new era of immunotherapy - responsive cns diseases , and they raise interesting questions regarding the aetiological and pathogenic mechanisms mediating these conditions . |
while the use of autologous tissue is limited for reconstructing critical - sized defects , the development of biomaterial - based approaches to promote bone formation may abrogate some of those limitations .
regenerative medicine has the potential to revolutionize reconstructive approaches for critical - sized bone defects by providing prefabricated tissue with patient - specific geometry while minimizing donor tissue requirements [ 4 , 13 , 17 ] .
repair of critical - sized bone defects with cadaver - derived bone graft , biomaterials such as titanium , hydroxyapatite , and ceramics , autologous vascularized tissue transfers , or prosthetic rehabilitation have limitations and thus the use of biodegradable bone - conducting biomaterials is being explored .
ectopically grafted periosteum and silk fibroin - chitosan ( sfcs ) biomaterial reportedly regenerate bone in an in vivo sheep model [ 4 , 7 , 8 , 16 ] .
these studies suggest an approach to bone repair by prefabricating biomaterial - based transferable bone tissue flaps is a potential alternative .
stimulation of cellular processes for tissue - engineering applications has focused primarily on delivery of proteins or nucleic acids , antisense oligonucleotides , adenoviral - based delivery , and hammerhead ribozymes , which have been used to modulate the expression of proteins [ 6 , 10 ] .
these methods , however , have been plagued with oligonucleotide stability or nonspecific global suppression .
short - interfering rna ( sirna ) is a gene - silencing process involving the introduction of 21- to 25-nucleotide double - stranded rna into a cell that results in the degradation of the complementary mrna via a multiple enzyme complex called rna - induced silencing complex .
a short nucleotide can be introduced to target multiple pathways / factors , thus providing a means to guide diverse and interlinked cellular processes simultaneously .
sirna that targets the hypoxia pathway turns on the angiogenic protein expression via the hypoxia - inducible factor 1 ( hif-1 ) and sirna that targets the bone differentiation pathway turns on the core binding factor alpha 1 ( cbfa1 ) pathway to activate expression of bone differentiation proteins .
cbfa1 is a transcript factor important in differentiation of bone precursor cells into osteoblasts and subsequent bone formation .
the activity of cbfa1 is regulated by the chain of the heterotrimeric g protein , gs , and is transcribed by the guanine nucleotide - binding protein ( g protein ) alpha - stimulating activity polypeptide 1 ( gnas1 ) gene ( fig .
the downregulation of the gnas1 gene via sirna leads to the increased expression of cbfa1 that would stimulate the production of bone - differentiating proteins such as osteopontin , collagen i , and osteocalcin [ 3 , 15].fig .
the blocking of the gnas1 gene via signas1 results in the activation of the cbfa1 pathway and expression of proteins such as osteopontin , collagen i , and others .
gtp = guanosine triphosphate ; gdr = guanosine diphosphate ; camp = cyclic adenosine monophosphate .
( b ) a schematic shows the hypoxia pathway regulated by hif-1 ( hif-1 and hif-1 ) and targeted to regulate angiogenesis by blocking of phd2 via siphd2 , which allows the binding of hif-1 to hif-1 and expression of vegf , epo , sdf-1 , and inos .
the blocking of the gnas1 gene via signas1 results in the activation of the cbfa1 pathway and expression of proteins such as osteopontin , collagen i , and others .
gtp = guanosine triphosphate ; gdr = guanosine diphosphate ; camp = cyclic adenosine monophosphate .
( b ) a schematic shows the hypoxia pathway regulated by hif-1 ( hif-1 and hif-1 ) and targeted to regulate angiogenesis by blocking of phd2 via siphd2 , which allows the binding of hif-1 to hif-1 and expression of vegf , epo , sdf-1 , and inos .
hif-1 is a transcription factor and a heterodimer composed of two subunits : hif-1 and hif-1 ( aryl hydrocarbon receptor nuclear translocator ) , whose pathway is targeted to control angiogenesis via the silencing of prolyl hydroxylase domain - containing protein 2 ( phd2 ) ( fig .
hif-1 expression is tightly regulated by oxygen tension . phd2 hydroxylates the key proline residues of hif-1 , which targets hif-1 for destruction via the ubiquitin - proteasome pathway , in normoxia . under hypoxic conditions , hif-1 accumulates leading to its stabilization and it partners with hif-1 to bind to the hif response elements on its target genes to turn on transcription of genes important to vasculogenesis , such as vascular endothelial growth factor ( vegf ) , erythropoietin ( epo ) , stromal - derived factor 1 ( sdf1 ) , leptin , and inducible nitric oxide synthase ( inos ) [ 11 , 18 ] .
the sirna against phd2 that blocks the binding of phd2 with hif-1 promotes the expression of angiogenic proteins via the hypoxia pathway .
we hypothesized blocking of the bone differentiation pathway via sirna against gnas1 ( signas1 ) and the hypoxia pathway via sirna against phd2 ( siphd2 ) would affect msc proliferation and expression of collagen , osteopontin , and alkaline phosphatase ( alp ) .
we therefore determined ( 1 ) whether the administration of signas1 and siphd2 in mscs would promote osteogenic phenotype ( cell proliferation and collagen , osteopontin , and alp expression ) ; ( 2 ) the dose - dependent effects of signas1 on msc differentiation to osteogenic phenotype ( alp assay ) and the concentration of signas1 needed for the in vivo bone formation study ; and ( 3 ) whether the two sirnas promote the formation of bone in vivo ( bone volume and density with time ) .
this study was designed to investigate bone differentiation in sfcs scaffolds via the administration of sirna in in vitro and in vivo assays ( fig . 2 ) .
the effect of signas1 dosage on msc differentiation to bone phenotype was determined using alp assay ( n = 3/treatment group / concentration / time point ) .
the sfcs scaffolds were embedded with either a single sirna or both together and bone volume and density with time were determined using micro - ct in an in vivo sheep model ( n = 4/treatment group / time point , except at day 70 [ n = 2]).fig .
2a bflow diagrams show the setup of the ( a ) in vitro and ( b ) in vivo studies .
the in vitro study involved the transfection of mscs with signas1 and siphd2 and evaluation of cell proliferation , collagen expression , osteopontin expression , and dose - dependent alp expression .
the in vivo study involved embedding either signas1 or siphd2 or both in sfcs scaffolds and implanting them over periosteum engrafted on the latissimus dorsi muscle to fabricate ectopic bone .
flow diagrams show the setup of the ( a ) in vitro and ( b ) in vivo studies .
the in vitro study involved the transfection of mscs with signas1 and siphd2 and evaluation of cell proliferation , collagen expression , osteopontin expression , and dose - dependent alp expression .
the in vivo study involved embedding either signas1 or siphd2 or both in sfcs scaffolds and implanting them over periosteum engrafted on the latissimus dorsi muscle to fabricate ectopic bone . for in vitro transfection with sirna , human mscs ( sciencell research laboratories , carlsbad , ca , usa ) were maintained in msc medium , supplemented with 5% fetal bovine serum , 1% msc growth supplement , and 1% penicillin / streptomycin solution .
mscs were seeded in six - well tissue culture plates at ~ 333,333 cells / well ( n = 3 wells / treatment group ) .
we had three time points with n = 3/group for cell proliferation , collagen expression , and osteopontin expression .
thus , there were 12 wells / time point for cell proliferation and 15 wells / time point for collagen and osteopontin expression .
overall , we had 36 wells for cell proliferation and 45 wells for collagen and osteopontin expression .
cells were allowed to adhere overnight . to transfect the cells with the sirna , it was mixed with 5 l ambion siport amine transfection agent ( life technologies , grand island , ny , usa ) and incubated at room temperature for 10 minutes .
cells seeded in the wells were transfected with 5 g / ml signas1 ( 5-ggcaaccaaagugcaggactt-3 ; applied biosystems / ambion inc , austin , tx , usa ) , siphd2 ( 5-aacgggttatgtacgtcatgt-3 ) , signas1-siphd2 ( qiagen inc , valencia , ca , usa ) , silamin ( 5-cuggacuuccagaagaacatt-3 ; applied biosystems / ambion inc ) , or nothing ( control ) at time 0 .
after incubation for 1 , 2 , or 4 days , the cells were ready for 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide ( mtt ) assay ( cell proliferation ) , western blot analysis ( osteopontin ) , and collagen assay . to count the number of cells
, we used an mtt assay from bioassay systems ( hayward , ca , usa ) .
the reagents were incubated for 5 hours and measured by spectrophotometer . for the osteopontin assay
, the cells grown in the six - well tissue culture plates were lysed at the previously mentioned time points in 100 l lysis buffer containing 2.5 l phenylmethylsulfonyl fluoride , 2.5 l sodium orthovandate , 2.5 l protease inhibitor cocktail , and 1455 l radioimmunoprecipitation assay buffer ( all from santa cruz biotechnology , santa cruz , ca , usa ) .
briefly , cell pellets were suspended in lysis buffer and kept on ice for 10 minutes .
the suspensions were centrifuged at 4500 rpm for 10 minutes and supernatants were collected for analysis .
cell lysis aliquots equivalent to 50,000 cells were taken from each sample , mixed with similar volume of sodium dodecyl sulfate ( sds ) buffer ( biorad , hercules , ca , usa ) containing 5% -mercaptoethanol ( sigma - aldrich corp , st louis , mo , usa ) , and heated at 100 c for 5 minutes .
samples were separated on a 10% sds - polyacrylamide gel and then blotted for western analyses .
the nitrocellulose membrane was stained with antibodies specific for osteopontin ( santa cruz biotechnology ) .
intensities of various bands on western blot films were scanned and analyzed using gel - pro analyzer software ( media cybernetics , silver spring , md , usa ) .
we performed the sircol soluble collagen assay ( biocolor ltd , carrickfergus , uk ) on the sirna - transfected mscs .
measurements were compared to standards and statistical analyses were performed appropriately . for the alp assay , we cultured mscs in 96-well plates at 1000 cells / well overnight .
signas1 or silamin at 10 , 50 , and 100 g / ml ( n = 3 wells / concentration ) was added to 6-mm 75:25 sfcs scaffolds and placed over the mscs in the wells .
we used the tracp & alp assay kit ( takara bio , inc , otsu , japan ) to assess alp in the sirna - transfected mscs .
seven adult sheep raised and housed at the university of texas m. d. anderson cancer center s
michael e. keeling center for comparative medicine and research at bastrop , tx , usa , were used for the in vivo study .
laws on animal experimentation were strictly followed and all protocols involving the animals were preapproved by the institutional animal care and use committee .
sheep were anesthetized with diazepam 0.2 mg / kg ( intravenously ) , ketamine 4 mg / kg ( intravenously ) , and 3% isoflurane ( endotracheal via ventilator ) . once the sheep were fully anesthetized , 0.02 mg / kg succinyl choline chloride was administered via an intravenous drip during the first 30 minutes of an approximately 90-minute procedure to reduce the occurrence and intensity of muscular spasms during surgery . before surgery ,
the skin above the animal s lateral chest wall was sheared , sterilized using iodine , and draped .
the sheep was given an intramuscular injection of 1.0 g cefazolin and an incision was made overlying and parallel to the seventh or eighth rib to expose the two adjacent ribs ( fig .
two pieces of periosteum from each of the seven sheep were carefully harvested ( to avoid damage to the cambium layer ) from the outer table of two adjacent ribs , each measuring 12 to 15 cm in length and the entire width of each rib ( approximately 11.5 cm ) .
each piece was sectioned into either four or five segments that were 3 cm long to make a total of nine pieces .
each segment was then transplanted to the latissimus dorsi with the cambium layer facing up and sutured to the underlying muscle using 5.0 prolene polypropylene suture ( ethicon , inc , somerville , nj , usa).fig .
( a ) two pieces of periosteum ( 1215 cm ; width approximately 11.5 cm ) were harvested from sheep ribs using a scalpel and periosteal elevator dissection , ( b ) cut into eight to nine 3-cm pieces , and ( c ) autografted over the latissimus dorsi muscle of the same sheep at 3 cm apart .
( d ) pmma chambers , one chamber each for pure sfcs scaffold , bone graft , and empty as controls and two chambers each for signas1-sfcs , siphd2-sfcs , and signas1-siphd2-sfcs , were created and ( e ) implanted on top of the grafted periosteum on the latissimus dorsi muscle using pdms to suture and 5.0 prolene suture .
( a ) two pieces of periosteum ( 1215 cm ; width approximately 11.5 cm ) were harvested from sheep ribs using a scalpel and periosteal elevator dissection , ( b ) cut into eight to nine 3-cm pieces , and ( c ) autografted over the latissimus dorsi muscle of the same sheep at 3 cm apart .
( d ) pmma chambers , one chamber each for pure sfcs scaffold , bone graft , and empty as controls and two chambers each for signas1-sfcs , siphd2-sfcs , and signas1-siphd2-sfcs , were created and ( e ) implanted on top of the grafted periosteum on the latissimus dorsi muscle using pdms to suture and 5.0 prolene suture .
a total of two sheep in each time point gave the study 85% power to detect an increase in maximal bone volume for signas1-sfcs from day 7 to day 36 and 99% power from day 7 to day 70 .
it gave 99% power to detect an increase in maximal bone volume for signas1-sfcs from day 7 to day 70 and day 21 to day 70 .
the study had 97% and 84% power to detect a signas1-siphd2-sfcs increase from day 7 to day 70 and day 36 to day 70 , respectively .
we fabricated chambers ( inner dimensions : 1 3 1.25 cm ) with polymethylmethacrylate ( pmma ) and glued polydimethyl siloxane ( pdms ) to the edges of the open end of the chamber .
chambers containing sfcs scaffolds with signas1 ( n = 4 ) , siphd2 ( n = 4 ) , and signas1-siphd2 ( n = 4 ) ( 75:25 blend with 50 g / ml of the sirna ) were implanted on top of the grafted periosteum over the latissimus dorsi muscle using the pdms to suture the chamber to the muscle with 5.0 prolene suture . each of the seven sheep had nine chamber implants ( two for each of the four experimental groups and one for each control ) .
chambers were harvested after euthanasia ( beuthanasia 0.22 ml / kg intravenously ) at days 7 , 21 , and 36 with harvests from two sheep at each time point .
empty chambers ( negative control , n = 2 ) , chambers filled with bone graft ( clinically relevant positive control , n = 2 ) , and chambers filled with sfcs ( n = 2 ) were controls for the time points above .
bone chips were used as fresh bone graft by dissecting bone from the ribs and crushing in a bone mill ( medtronic , inc , fort worth , tx , usa ) .
a similar procedure was used for implantation and harvest at day 70 , although the sample sizes for controls and experimental conditions were n = 1 and n = 2 , respectively , since there was only one sheep harvested . to assess the extent of vascularization in sfcs scaffold after bone formation at the time of harvest , the lateral thoracic artery was perfused with india ink using an intravenous injection . at the time of harvest ,
samples were preserved in 10% formalin and sent for micro - ct scans and then histologic staining .
we performed micro - ct scans to assess bone formation in the chambers at the harvest time points at 92-m sections / slices at the m. d. anderson small animal imaging core facility using a model rs-9 tabletop ct scanner ( general electric medical systems , london , on , canada ) .
this scanner has a gantry that is rotated by a servo mechanism around the object bed , imaging the entire object in one pass .
the gantry features a tungsten anode x - ray source , operated at 80 vp and 450 ia , which is fixed opposite a detector composed of a cesium iodide scintillator and a charge - coupled camera .
we used ge healthcare s microview software ( version 2.2 ) to analyze the regenerated bone .
regions of interest were outlined , three - dimensional isosurface images were generated , and bone volume and bone mineral densities were calculated .
the threshold was set to 100 for these measurements . of the total four samples for the experimental groups and two samples for the control groups
subsequent to micro - ct imaging , samples were demineralized and dehydrated using a series of ethanol solutions and embedded in paraffin .
serial sections ( 4 m ) were cut , set on glass slides , and baked at 56c overnight .
different sections from each sample were then deparaffinized , rehydrated , and stained with hematoxylin and eosin ( h&e ) or movat s pentachrome and imaged with bright - field light microscopy ( olympus ix50 ; olympus america inc , center valley , pa , usa ) .
two - way anova with bonferroni post hoc test was conducted to determine the effect of the two sirnas ( independent variables ) by assessing differences in dependent variables with time ( independent variable ) : cell number , collagen expression , and osteopontin expression .
dose ( independent variable)-dependent effects on alp expression ( dependent variable ) with time ( independent variable ) were assessed using two - way anova with bonferroni post hoc test . one - way anova with tukey post hoc test was used to assess differences in bone volume ( dependent variable ) and bone density ( dependent variable ) with time ( independent variable ) among the sirna treatment groups ( independent variables ) .
all data are reported as mean standard error of the mean ( sem ) with the number of samples averaged per group ( n ) given in parentheses for each condition .
we used graphpad prism 5 ( graphpad software , la jolla , ca , usa ) to determine differences in cell proliferation and protein expression in vitro and bone volume and bone density in vivo between different groups ( type of sirna ) with time .
the sample size for bone volume and bone mass density measurements at day 70 for sfcs controls was n = 1 and was not used for comparisons .
we performed post hoc power analysis to determine the power in the study using nquery advisor 7.0 ( statistical solutions , saugus , ma , usa ) .
cells proliferated from day 1 to day 4 with and without administration of sirna , although the relative increase in cell number by day 4 for cells with no sirna was higher than for cells with signas1 or siphd2 or signas1-siphd2 ( fig . 4 ) . for signas1-administered mscs ,
collagen i synthesis increased by day 2 and then decreased by day 4 ( fig .
osteopontin expression was maximal for signas1-administered cells at day 1 and was higher than that in cells administered siphd2 , signas1-siphd2 , or silamin ( fig .
4cellular proliferation was measured using mtt assay for mscs transfected with signas1 , siphd2 , signas1-siphd2 , and no sirna .
msc proliferation decreased with the administration of sirna within 24 hours whereas there was an increase in cell number without any sirna administration .
comparison among groups with time shows differences : * = p < 0.05 , * * = p < 0.01 , and * * * = p < 0.001 compared to day 4 in the same group ; = p < 0.05 and = p < 0.01 compared to gnas1 ; = p < 0.05 , = p < 0.01 , and = p <
5collagen i expression was determined for mscs transfected with signas1 , siphd2 , signas1-siphd2 , silamin , and no sirna using western blot .
comparison among groups with time shows differences : * * * = p < 0.001 compared to day 1 ; # # = p < 0.01 compared to day 4 .
6osteopontin expression was determined for mscs transfected with signas1 , siphd2 , signas1 and siphd2 , silamin , and no sirna using western blot .
osteopontin expression was maximal within 24 hours of signas1 administration and decreased with time by day 4 , reaching the same expression level as siphd2 .
comparison among groups with time shows differences : * = p < 0.05 , * * = p < 0.01 , and * * * = p < 0.001 compared to signas1 within the respective time point ; = p < 0.01 and
= p < 0.001 compared to day 1 signas1 ; = p < 0.05 compared to day 2 signas1 .
cellular proliferation was measured using mtt assay for mscs transfected with signas1 , siphd2 , signas1-siphd2 , and no sirna .
msc proliferation decreased with the administration of sirna within 24 hours whereas there was an increase in cell number without any sirna administration .
comparison among groups with time shows differences : * = p < 0.05 , * * = p < 0.01 , and * * * = p < 0.001 compared to day 4 in the same group ; = p < 0.05 and = p < 0.01 compared to gnas1 ; = p < 0.05 , = p < 0.01 , and = p <
collagen i expression was determined for mscs transfected with signas1 , siphd2 , signas1-siphd2 , silamin , and no sirna using western blot .
comparison among groups with time shows differences : * * * = p < 0.001 compared to day 1 ; # # = p < 0.01 compared to day 4 .
osteopontin expression was determined for mscs transfected with signas1 , siphd2 , signas1 and siphd2 , silamin , and no sirna using western blot .
osteopontin expression was maximal within 24 hours of signas1 administration and decreased with time by day 4 , reaching the same expression level as siphd2 .
comparison among groups with time shows differences : * = p < 0.05 , * * = p < 0.01 , and * * * = p < 0.001 compared to signas1 within the respective time point ; = p < 0.01 and = p <
0.001 compared to day 1 signas1 ; = p < 0.05 compared to day 2 signas1 .
values are expressed as mean sem . at day 1 , cells administered with 100 g / ml signas1 had lower alp expression than the cells administered 10 and 50 g / ml signas1 ( fig . 7 ) .
alp expression was higher for cells treated with signas1 at concentrations of 10 and 50 g / ml signas1 on day 1 as compared to untreated cells cultured on petri dishes with no scaffold and no signas1 .
the presence of scaffold increased the alp expression compared to no - scaffold controls , with no signas1 administration in both cases . by day 2
there was no change in alp expression from day 2 to day 4 for 10 and 50 g / ml although it remained lower than day 1 for both concentrations . at day 4 , administration of signas1 at 100 g / ml and silamin at 10 g / ml had higher alp expression than administration of signas1 at 10 g / ml .
cells administered silamin at 10 g / ml and signas1 at 100 g / ml had higher alp expression than no - scaffold / no - signas1 controls.fig .
7alp assay was used to determine the effect of signas1 concentrations at 10 , 50 , and 100 g / ml and compared to silamin ( 10 , 50 , and 100 g / ml ) , scaffold only ( no sirna ) , and no scaffold ( no sirna ) controls .
the maximal alp expression was observed for 10 and 50 g / ml at day 1 , which decreased with time .
the alp expression for 100 g / ml increased from day 1 to day 4 .
comparison among groups shows differences : * = p < 0.05 , * * = p < 0.01 , * * * = p < 0.001 compared to 10 g / ml signas1 ; = p < 0.001 and = p < 0.01 compared to 50 g / ml gnas1 ; = p < 0.05 compared to scaffold only ; $ $ = p < 0.01 and $ = p < 0.05 compared to 100 g / ml signas1 ; @@ = p < 0.01 compared to 10 g / ml silamin ; # # # = p < 0.01 compared to day 1 for 10 g / ml signas1 ; + + = p < 0.01 and + = p < 0.05 compared to day 1 for 50 g / ml signas1 .
alp assay was used to determine the effect of signas1 concentrations at 10 , 50 , and 100 g / ml and compared to silamin ( 10 , 50 , and 100 g / ml ) , scaffold only ( no sirna ) , and no scaffold ( no sirna ) controls .
the maximal alp expression was observed for 10 and 50 g / ml at day 1 , which decreased with time . the alp expression for 100
comparison among groups shows differences : * = p < 0.05 , * * = p <
0.01 , * * * = p < 0.001 compared to 10 g / ml signas1 ; = p < 0.001 and = p < 0.01 compared to 50 g / ml gnas1 ; = p < 0.05 compared to scaffold only ; $ $ = p < 0.01 and $ = p < 0.05 compared to 100 g / ml signas1 ; @@
= p < 0.01 compared to 10 g / ml silamin ; # # # = p < 0.01 compared to day 1 for 10 g / ml signas1 ; + + = p < 0.01 and + = p < 0.05 compared to day 1 for 50 g / ml signas1 .
representative micro - ct imaging of bone in the chambers showed differences in bone formation for the three sirna groups ( signas1 , siphd2 , and signas1-siphd2 ) from day 7 to day 70 ( fig .
bone formation was observed at the periosteum interface ( bottom of the chamber ) as early as day 7 . by day 21
, there appeared to be an increase in bone formation at the interface and into the chamber in siphd2-sfcs and signas1-siphd2-sfcs .
there was further filling of the chamber with bone at days 36 and 70 for all three conditions .
quantification of the bone volume indicated no difference in the empty chamber negative control with time and a decrease in bone volume of bone graft - positive controls with time ( table 1 ) .
there was also a difference in the bone volume between bone graft and empty chambers at days 7 and 21 .
there was no change in bone mass density with time for bone graft and empty chambers ( table 1).fig .
8micro - ct imaging of bone regeneration in pure sfcs scaffolds , signas1-sfcs , siphd2-sfcs , and signas1-siphd2-sfcs scaffolds at days 7 , 21 , 36 , and 70 after implantation shows bone formation in white .
bone formation is observed at the periosteum - chamber interface as early as day 21 in sfcs , siphd2-sfcs , and signas1-siphd2-sfcs .
bone filled into the chamber from day 36 to day 70 for signas1-sfcs , siphd2-sfcs , and signas1-siphd2-sfcs.table 1bone volume and bone mass density of bone graft and empty chambersvariableday 7day 21day 36day 70bone volume ( mm )
bone graft1436.0 185.31036.8 246.0
531.7 77.9 * ,
50.8 * , ,
empty135.0 129.4 * 195.2 178.0
2.9 2.8
1.6bone density ( mg / cm ) bone graft628.0 6.1646.2 17.1667.7 42.3708.1 empty165.3 65.4212.7 6.5116.4 45.0177.2values are expressed as mean standard error of the mean ; * p < 0.0001 , p
< 0.01 , as compared to bone graft at day 7 ; p < 0.001 , as compared to bone graft at day 21 ; p < 0.0001 , p < 0.01 , as compared to bone graft at day 36 .
micro - ct imaging of bone regeneration in pure sfcs scaffolds , signas1-sfcs , siphd2-sfcs , and signas1-siphd2-sfcs scaffolds at days 7 , 21 , 36 , and 70 after implantation shows bone formation in white .
bone formation is observed at the periosteum - chamber interface as early as day 21 in sfcs , siphd2-sfcs , and signas1-siphd2-sfcs .
bone filled into the chamber from day 36 to day 70 for signas1-sfcs , siphd2-sfcs , and signas1-siphd2-sfcs .
bone volume and bone mass density of bone graft and empty chambers values are expressed as mean standard error of the mean ; * p < 0.0001 , p < 0.01 , as compared to bone graft at day 7 ; p < 0.001 , as compared to bone graft at day 21 ; p < 0.0001 , p < 0.01 , as compared to bone graft at day 36 .
h&e staining of empty chamber microsections showed heterotopic bone formation within periosteum at the muscle - periosteum interface by day 21 ( fig .
histologic movat s pentachrome - stained microsections showed bone formation in sfcs scaffolds with signas1 , siphd2 , and siphd2-signas1 at days 21 and 36 ( fig . 10).fig .
9h&e staining of a 4-m section of tissue harvested at day 21 shows heterotopic bone formation at the muscle - periosteum interface with mineralizing bone in reddish - pink surrounding the newly forming marrow spaces in the middle.fig .
staining of 4-m sections of tissue harvested at days 21 and 36 shows mineralizing bone in deep yellowish - brown and mineralizing silk fibroin fibrils in pinkish - red . at day 36
, black india ink perfusion shows the vasculature in black for signas1 , siphd2 , and signas1-siphd2 .
staining of a 4-m section of tissue harvested at day 21 shows heterotopic bone formation at the muscle - periosteum interface with mineralizing bone in reddish - pink surrounding the newly forming marrow spaces in the middle .
movat s pentachrome staining of 4-m sections of tissue harvested at days 21 and 36 shows mineralizing bone in deep yellowish - brown and mineralizing silk fibroin fibrils in pinkish - red . at day 36
, black india ink perfusion shows the vasculature in black for signas1 , siphd2 , and signas1-siphd2 . at day 7
, there was no difference in bone volume among the sirna - embedded sfcs scaffolds ( fig .
, siphd2-sfcs and signas1-siphd2-sfcs tended to have higher bone volumes than sfcs and signas1-sfcs . at day 36
, the maximal bone volume was observed for siphd2-sfcs and was higher than day 7 . by day 70 ,
bone volume for signas1-sfcs increased compared to days 7 and 21 , that for siphd2-sfcs increased compared to day 7 , and that for signas1-siphd2-sfcs increased compared to days 7 and 36 .
there was no difference in bone mass density among the sirna - sfcs groups ( fig .
11bone volume was plotted against time to assess bone formation for sfcs scaffolds modified with signas1 , siphd2 , and signas1-siphd2 .
there was an overall increase in bone volume from day 7 to day 70 for sirna - embedded scaffolds .
for the signas1 group , * * * = p < 0.0001 compared to day 70 ; for the siphd2 group , = p < 0.01 compared to day 36 and = p < 0.01 compared to day 70 ; for the signas1-siphd2 group , # # = p < 0.01 compared to day 70 .
12bone mass density was plotted against time for sfcs scaffolds modified with signas1 , siphd2 , and signas1-siphd2 .
bone volume was plotted against time to assess bone formation for sfcs scaffolds modified with signas1 , siphd2 , and signas1-siphd2 .
there was an overall increase in bone volume from day 7 to day 70 for sirna - embedded scaffolds .
for the signas1 group , * * * = p < 0.0001 compared to day 70 ; for the siphd2 group , = p < 0.01 compared to day 36 and = p < 0.01 compared to day 70 ; for the signas1-siphd2 group , # # = p < 0.01 compared to day 70 .
bone mass density was plotted against time for sfcs scaffolds modified with signas1 , siphd2 , and signas1-siphd2 .
the cbfa1 and hif-1 pathways can be modulated to direct bone formation via signas1 and siphd2 , respectively .
we assessed the use of these sirnas for modulating the bone formation pathway to provide a tool for promoting and directing the growth of functional bone for repair and reconstructive surgery applications .
we determined ( 1 ) how the administration of signas1 and siphd2 in mscs affects cell proliferation and collagen , osteopontin , and alp expression ; ( 2 ) how the dose of signas1 affects alp expression in mscs as a bone differentiation marker and what concentration of signas1 was needed for the in vivo bone formation study ; and ( 3 ) whether the two sirnas affect in vivo bone formation by measuring bone volume and density with time .
first , while we observed bone formation , the model provided no mechanical loading apart from the mechanical simulation from movement of the latissimus dorsi muscle .
however , mechanical loading affects the bone formation with growth factor signaling in a clinical setting .
such constructs could be moved as a bone tissue flap with the vascular supply from latissimus dorsi to a mechanically loaded defect site where the vascular supply could be connected but would have to be supported with metallic plates and screws initially until this prefabricated bone remodeled to specifications of the mechanical loading at the defect repair site .
second , we had only two experimental samples at day 70 although we noted an increase in bone volume at day 70 compared to day 7 .
third , siphd2 and signas1 both guide bone formation via complex and different intercellular mechanisms that were not explored in this study .
fourth , the overall concentration of the loaded sirna in the sfcs scaffold and the rate of release of sirna relative to the cells migrating into the scaffold , degradation of scaffold , differentiation of cells into osteogenic phenotype , and remodeling of new tissue in the sheep model need further investigation .
fifth , with the in vivo sheep model , no cells were seeded in the scaffold before implantation and the cell source for in vivo bone formation was limited to the periosteum . since the vascular supply to the periosteum and the newly forming bone in the scaffold are from the latissimus dorsi muscle , cells would migrate in with the nutrient supply from the vasculature .
although it is well established that the cambium layer of the periosteum provides bone precursor cells , other cells that vascularize the scaffold may either migrate from the periosteum connective tissue or from the adjacent muscle .
we found osteogenic differentiation of mscs can be mediated via signas1 as indicated by increased expression of collagen i , osteopontin , and alp .
the administration of both signas1 and siphd2 slowed the proliferation of cells as compared to no - sirna controls .
cellular proliferation was decreased by the presence of sirna probably due to guidance toward the bone differentiation pathway .
the cell cycle arrest in mscs has been observed by others as the cells are directed toward a differentiation pathway . in this study ,
collagen i is the primary component of the bone extracellular matrix and its expression is regulated by the cbfa1 transcriptional regulator [ 2 , 5 ] .
signas1 promoted the expression of the proteins in the cbfa1 pathway , while there was no effect of siphd2 on the collagen i expression .
osteopontin expression would be expected to increase as a marker for the osteogenic phenotype as observed in this study within 1 day of exposure to signas1 in mscs .
osteogenic differentiation of mscs marked by increase in cbfa1 expression due to administration of antisense gs , which simulates inactivation of the gnas1 gene similar to sirna administration , has been studied .
additionally , coadministration of sirnas against gnas1 and bmp2 reportedly accelerates the differentiation of human adipose tissue - derived stem cells toward osteogenic lineage .
we performed the signas1 dose assessment to determine the signas1 concentration needed for the in vivo study .
lower concentrations of signas1 exhibited higher alp at day 1 , and as the sirna depleted by days 2 and 4 , the alp was reduced .
while 10 and 50 g / ml signas1 resulted in maximal alp expression after day 1 , the alp expression decreased considerably for 10 g / ml and intermediate effects were observed for 50 g / ml as compared to 10 and 100 g / ml .
for the higher - concentration of signas1 , maximal alp expression was not observed until day 4 , probably due to the effect of sirna administration on cellular proliferation since the concentration values were normalized with overall cell number .
the effect of sirna in the cellular system on the cellular proliferation may have dominated the effect of signas1 on the bone differentiation phenotype at short time points for 100 g / ml . alp detected at day 1 was the lowest compared to 10 and 50 g / ml , and as the cells proliferated by day 4 and the signas1 was utilized by the cells , the overall alp expression increased for 100 g / ml . since the signas1 targeted cellular proliferation and differentiation into an osteogenic phenotype in mscs , the dose - dependent effects evident in this study were a function of the proliferation / differentiation balance that the cells try to achieve with sirna administration to their system . increased alp activity , collagen i expression , and osteocalcin expression with increased in vivo bone formation
have also been reported for cbfa1 adenovirus - transfected mscs . in vivo data suggested an overall increase in bone volume from day 7 to day 70 in signas1 , siphd2 , and signas1-siphd2 . at day 21 , signas1-administered cells had a lower volume of bone , which increased by day 70 .
an increase in bone volume was observed for signas1 , siphd2 , and signas1-siphd2 from day 7 to day 70 .
no change in bone density for any of the conditions indicated consistent bone density was maintained in the newly forming bone in the sfcs scaffolds with time up to 70 days .
the sequence of siphd2 used has been well tested in our laboratory to show it results in downregulation of phd2 and upregulation of angiogenic factors such as inos .
it also promotes vacuole formation in endothelial cells at short time points ( within 24 hours ) and microvascular network formation by 96 hours . in this study , in vivo bone formation
was aided by cells incoming from the periosteum and the adjacent latissimus dorsi muscle providing the vascular supply to the periosteum .
thus , siphd2 had the opportunity to act on other cells besides the bone precursors , although the siphd2-driven vascular phenotype may support the regeneration of bone by supporting the nutrient supply to the growing bone .
the silencing rna mechanism allowed us to control the fate of cell proliferation and differentiation by targeting the expression of multiple proteins via a single sirna administration .
since sirna affects the proliferative capacity of cells , for bone formation of critical sizes , it is important to consider the type of cells with respect to the type of sirna , time of sirna introduction , amount or concentration of sirna with respect to the number of cells in the system , and sustained release of needed amount with time , so as to not to promote premature differentiation of cells without adequate proliferation to fill or repair a defect .
this article is distributed under the terms of the creative commons attribution license which permits any use , distribution , and reproduction in any medium , provided the original author(s ) and the source are credited . | backgroundour ability to guide cells in biomaterials for in vivo bone repair is limited and requires novel strategies .
short - interfering rna ( sirna ) allows the regulation of multiple cellular pathways .
core binding factor alpha 1 ( cbfa1 ) and hypoxia - inducible factor 1 ( hif-1 ) pathways can be modulated to direct bone formation via sirna against guanine nucleotide - binding protein alpha - stimulating activity polypeptide 1 ( signas1 ) and prolyl hydroxylase domain - containing protein 2 ( siphd2 ) , respectively.questions/purposeswe determined whether the administration of signas1 and siphd2 in mesenchymal stem cells ( mscs ) promotes osteogenic phenotype , the dose - dependent effects of signas1 on msc differentiation to osteogenic phenotype , and whether the two sirnas promote bone formation in vivo.methodssirnas were administered to mscs at day 0 , and protein expression of bone - specific markers was assessed at days 1 , 2 , and 4 ( n = 3/group / time point ) . in an in vivo model using seven sheep , chambers containing silk fibroin - chitosan ( sfcs ) scaffolds with sirna were implanted over the periosteum and harvested at days 7 , 21 , 36 , and 70 ( n = 4/group / time point , except at day 70 [ n = 2 ] ) to assess bone formation.resultssignas1 promoted collagen i and osteopontin expression , whereas siphd2 had no effect in vitro .
dose - dependent effects of signas1 on alp expression were maximal at day 1 for 10 g / ml and day 4 for 100 g / ml
. in vivo , by day 70 , mean bone volume increased compared to day 7 for signas1-sfcs ( 47.8 versus 1.8 mg / ml ) and siphd2-sfcs ( 61.3 versus 1.5 mg / ml).conclusionsboth siphd2 and signas1 support bone regeneration in vivo , whereas only signas1 regulates bone phenotype in mscs in vitro.clinical relevancewhile the use of autologous tissue is limited for reconstructing critical - sized defects , the development of biomaterial - based approaches to promote bone formation may abrogate some of those limitations . |
as the most common malignant tumor in the gastrointestinal tract , gastric carcinoma is the second leading cancer in china , with an incidence of 400,000/per year ( approximately 42% of worldwide incidence ) .
although diagnostic and treatment technology has progressed , the prognosis of gastric cancer is still unfavorable , mainly due to the occurrence of lymph node or distal metastasis by the time of first diagnosis , making radical resection impractical .
moreover , the predictive power of prognosis using currently available methods is not high enough .
early diagnosis and accurate prediction of prognosis is , therefore , of critical importance for improving treatment efficacy .
microrna ( mir ) is a type of single - stranded non - coding rna molecule 22 nucleotides in length .
it can inhibit or down - regulate the target gene translation by specific binding to the 3-untranslated region ( 3-utr ) .
it is now commonly believed that mir plays a vital role in both tumor pathogenesis and tumor suppression .
various mir molecules have been found to be related with gastric carcinoma , thus improving the diagnostic and treatment efficacy .
studies have revealed the existence of a gene polymorphism locus at + 60 of mir-421 .
this locus is within the seeding sequence and can regulate the expression of mir-421 , suggesting a close correlation between up - regulation of mir-421 and gastric cancer occurrence . the relationship between mir-421 polymorphism and gastric cancer ,
the present study investigated the distribution of mir-421 genotypes and its role in occurrence of gastric cancer , in an attempt to strengthen target gene prediction .
a total of 96 gastric carcinoma samples were collected from surgical resection of patients from january 2010 to january 2013 in the affiliated hospital , academy of military medical sciences .
no radio-/chemo- or molecular target therapy had been performed before the surgery . among the 96 patients ,
there were 74 males and 22 females , ages 42 to 78 years old ( average age=61.34 years ) . according to lauren type
, there were 68 cases of intestine type carcinoma and 28 cases of diffused cancer .
the number of cases with high , moderate , and low differentiation was 12 , 46 , and 38 , respectively . using the uicc staging system ,
the locations of tumors were the gastric antrum in 38 cases , the body in 16 cases , the cardia in 21 cases , and the whole stomach in 21 cases .
this study was pre - approved by the ethics committee of the affiliated hospital , academy of military medical sciences , and we obtained written consent from all participants in accordance with the declaration of helsinki .
pathological typing of tumor tissues was performed to evaluate the infiltration degree based on the uicc standard .
post - operative follow - up was performed on all patients once per month for a maximum duration of 5.5 years ( average=3.6 years ) .
total rna was extracted by use of an rna extraction kit ( sigma , usa ) from all gastric cancer tissues .
the relative expression level of mir-421 was determined by taqman mirna assay kit ( santa cruz , usa ) using rnu6 as the internal reference in 2-step reactions . in brief , cdna was synthesized using stem - loop primers in an in vitro reverse transcription system using 10 ng rna as the template ( 2 ng/l ) .
the gradient denaturing reaction was performed as follows : 94c for 30 s , 60c for 30 s , 72c for 60 s , ending with 72c for 7 min . in the second step , mir-421-specific primers ( forward , 5-cacag uaggc cucaa auguu uguug auga-3 ; reverse : 5-guguc guccg gguuu acaga caacu acu-3 ) were added into a 2pcr taqman universal mixture .
pcr parameters were : 94c for 30 s , 60c for 30 s , and 72c for 60 s , repeated for 40 cycles , ending with 72c for 7 min .
the reaction was quantified using fluorescent real - time quantitative pcr equipment ( rotor - gene , australia ) .
the relative level of mir-421 was determined by 2 method ( ct = ctmir421ctrnu6 ) .
pearson chi square analysis was used to determine if genotype distribution fit hardy - weinberg equilibrium .
data that fit the normal distribution are presented as mean standard deviation ( sd ) .
analysis of variance ( anova ) was used to compare the differences in genotype frequencies of mir-421 , with snk correction coefficient for between - group comparison .
the paired t test was used to compare the differential expression of mir-421 between tumor and normal tissues . logistic regression analysis detected if gg / gc genotypes were risk factors for gastric cancer .
a kaplan - meier survival curve was plotted to analyze the postoperative survival of patients .
a total of 96 gastric carcinoma samples were collected from surgical resection of patients from january 2010 to january 2013 in the affiliated hospital , academy of military medical sciences .
no radio-/chemo- or molecular target therapy had been performed before the surgery . among the 96 patients ,
there were 74 males and 22 females , ages 42 to 78 years old ( average age=61.34 years ) . according to lauren type
, there were 68 cases of intestine type carcinoma and 28 cases of diffused cancer .
the number of cases with high , moderate , and low differentiation was 12 , 46 , and 38 , respectively . using the uicc staging system ,
the locations of tumors were the gastric antrum in 38 cases , the body in 16 cases , the cardia in 21 cases , and the whole stomach in 21 cases .
this study was pre - approved by the ethics committee of the affiliated hospital , academy of military medical sciences , and we obtained written consent from all participants in accordance with the declaration of helsinki .
pathological typing of tumor tissues was performed to evaluate the infiltration degree based on the uicc standard .
post - operative follow - up was performed on all patients once per month for a maximum duration of 5.5 years ( average=3.6 years ) .
total rna was extracted by use of an rna extraction kit ( sigma , usa ) from all gastric cancer tissues .
the relative expression level of mir-421 was determined by taqman mirna assay kit ( santa cruz , usa ) using rnu6 as the internal reference in 2-step reactions . in brief , cdna was synthesized using stem - loop primers in an in vitro reverse transcription system using 10 ng rna as the template ( 2 ng/l ) .
the gradient denaturing reaction was performed as follows : 94c for 30 s , 60c for 30 s , 72c for 60 s , ending with 72c for 7 min . in the second step ,
mir-421-specific primers ( forward , 5-cacag uaggc cucaa auguu uguug auga-3 ; reverse : 5-guguc guccg gguuu acaga caacu acu-3 ) were added into a 2pcr taqman universal mixture .
pcr parameters were : 94c for 30 s , 60c for 30 s , and 72c for 60 s , repeated for 40 cycles , ending with 72c for 7 min .
the reaction was quantified using fluorescent real - time quantitative pcr equipment ( rotor - gene , australia ) .
the relative level of mir-421 was determined by 2 method ( ct = ctmir421ctrnu6 ) .
pearson chi square analysis was used to determine if genotype distribution fit hardy - weinberg equilibrium .
data that fit the normal distribution are presented as mean standard deviation ( sd ) .
analysis of variance ( anova ) was used to compare the differences in genotype frequencies of mir-421 , with snk correction coefficient for between - group comparison .
the paired t test was used to compare the differential expression of mir-421 between tumor and normal tissues . logistic regression analysis detected if gg / gc genotypes were risk factors for gastric cancer .
a kaplan - meier survival curve was plotted to analyze the postoperative survival of patients .
demographic information , such as age and sex distribution , was not significantly different between gastric cancer and control individuals ( p>0.05 , table 1 ) .
the liver and kidney functions , however , were significantly compromised in gastric cancer patients ( p<0.05 , table 1 ) .
a preliminary survey of genotype ( gg and gc ) distribution showed the existence of hardy - weinberg equilibrium . in gastric cancer patients with lymph node metastasis , the frequency of gg genotype of mir-421 was higher than that in patients without lymph node metastasis , suggesting that the gg genotype might be a risk factor for lymph node metastasis .
moreover , age and liver / kidney dysfunctions were shown to be major risk factors for the metastasis of gastric cancer ( table 2 ) .
the frequency of gg and gc genotypes in gastric cancer patients were significantly higher than those in the control group ( 46.88% vs. 7.69% ; 29.17% vs. 12.82% ) , suggesting that both genotypes are risk factors for gastric cancer .
logistic regression analysis showed that or values of gc and gg genotypes were 2.998 ( 95% ci : 1.176~7.639 ) and 2.936 ( 95% ci : 1.198~7.246 ) , respectively .
the occurrence of gastric cancer was increased by 16% in gc carriers and by 28% in gg carriers . when we checked tumor metastasis , or values of gc and gg genotypes were 2.936 ( 95% ci : 0.576~1.896 ) and 1.846 ( 95% ci : 1.148~2.965 ) , respectively ( table 3 ) .
the average relative expression level of mir-421 in all 96 gastric carcinoma tissues was 1.436 ( 95% ci : 0.323~0.534 ) .
there were 42 cases with higher than average expression values and 54 cases had mir-421 below the average value .
when comparing the prognosis of those 2 groups of patients , we found that those with high expression levels had a median survival time of 37.34 months , while mir-421 low - expression patients had a median survival time of 54.23 months .
therefore , a relationship existed between disease prognosis and gg genotype frequency ( r=5.16 , p<0.05 ) but not gc genotype ( r=2.86 , p>0.05 , figure 1 ) .
demographic information , such as age and sex distribution , was not significantly different between gastric cancer and control individuals ( p>0.05 , table 1 ) .
the liver and kidney functions , however , were significantly compromised in gastric cancer patients ( p<0.05 , table 1 ) . a preliminary survey of genotype ( gg and gc ) distribution showed the existence of hardy - weinberg equilibrium .
in gastric cancer patients with lymph node metastasis , the frequency of gg genotype of mir-421 was higher than that in patients without lymph node metastasis , suggesting that the gg genotype might be a risk factor for lymph node metastasis .
moreover , age and liver / kidney dysfunctions were shown to be major risk factors for the metastasis of gastric cancer ( table 2 ) .
the frequency of gg and gc genotypes in gastric cancer patients were significantly higher than those in the control group ( 46.88% vs. 7.69% ; 29.17% vs. 12.82% ) , suggesting that both genotypes are risk factors for gastric cancer .
logistic regression analysis showed that or values of gc and gg genotypes were 2.998 ( 95% ci : 1.176~7.639 ) and 2.936 ( 95% ci : 1.198~7.246 ) , respectively .
the occurrence of gastric cancer was increased by 16% in gc carriers and by 28% in gg carriers . when we checked tumor metastasis , or values of gc and gg genotypes were 2.936 ( 95% ci : 0.576~1.896 ) and 1.846 ( 95% ci : 1.148~2.965 ) , respectively ( table 3 ) .
the average relative expression level of mir-421 in all 96 gastric carcinoma tissues was 1.436 ( 95% ci : 0.323~0.534 ) .
there were 42 cases with higher than average expression values and 54 cases had mir-421 below the average value .
when comparing the prognosis of those 2 groups of patients , we found that those with high expression levels had a median survival time of 37.34 months , while mir-421 low - expression patients had a median survival time of 54.23 months .
therefore , a relationship existed between disease prognosis and gg genotype frequency ( r=5.16 , p<0.05 ) but not gc genotype ( r=2.86 , p>0.05 , figure 1 ) .
due to the insidious onset of gastric cancer at early stage , most patients are already at the late or terminal stage by the time of first diagnosis , causing unfavorable prognosis and lower survival rate for patients with gastric cancer .
mirna can exert pluripotent biological functions via modulating multiple target gene expressions , and is thus a popular research focus in oncology . with the advancement of bioinformatics
, mirna has been found to be related to tumor growth , development , proliferation , invasion , and apoptosis .
the expressional spectrum of mirna also determines the genotype of tumor cells and may play a vital role in subtyping of tumors and prediction of metastasis , especially for the post - operative survival time and prognosis , as suggested by previous studies showing the relationship between mirna expressional level and tumor prognosis . in various gastrointestinal tumors ( e.g. , gastric and colon cancer ) , metastatic lymph node ratio is reported to be an important prognostic factor . in recent years , high - throughput analysis has identified mir-421 as a gastric cancer - related molecule . as an important family of human irna-200
, the mir-421 coding gene is located on human chromosome 1p34.23 , where multiple tumor - related genes have been identified [ 1518 ] .
a close correlation exists between up - regulation of mir-421 and gastric cancer occurrence , because single - nucleotide polymorphism ( snp ) at certain locations causes higher risk of disease .
certain snp loci have been suggested to exist at the + 60 locus of the mir-421 coding sequence .
this locus localizes within the seeding sequence of mir-421 , and its expression can be regulated [ 2023 ] . the relationship between mir-421 polymorphism and gastric cancer , however , has not been reported .
this study thus investigated the correlation between gastric occurrence / prognosis and g / c polymorphism within the mir-421 gene , in an attempt to further illustrate the role of mir-421 in gastric cancer .
we performed an analysis using sequencing technique to determine the alleles involved in snp of mir-421 from tumor samples obtained from surgical resection .
multi - variant logistic regression analysis showed that both gc and gg genotypes may be risk factors for gastric cancer pathogenesis .
gg genotype carriers had higher incidence of lymph node metastasis , suggesting it may be a risk factor for metastasis .
age and liver / kidney dysfunctions are also identified as major risk factors for tumor metastasis .
gg carriers had significantly higher incidence of gastric carcinoma occurrence and lymph node metastasis . in a further survival analysis
, we found that gg carriers had shorter survival time compared to gc genotype carriers .
this study demonstrated the possible involvement of mir-421 gg - genotype in the invasion and metastasis of gastric carcinoma , indicating it might be a potential drug target .
we also revealed a correlation between mir-421 polymorphism and gastric cancer , suggesting the potential facilitation of gastric cancer pathogenesis by gg genotype via up - regulating mir-421 expression .
however , this study has certain limitations due to the relatively small samples size and lack of long - term follow - up .
we believe that more mir - related modulatory mechanisms could be identified , thus providing novel methods for diagnosis , treatment , and prognostic prediction of gastric cancer . | backgroundas a common malignant tumor , gastric carcinoma requires early diagnosis to improve treatment efficacy .
microrna ( mir ) molecules have highly conserved nucleotide sequences and can negatively regulate target gene expression at the translational level .
mir-421 has been suggested to be related with gastric cancer occurrence .
the gene polymorphism of mir-421 , however , has not been reported .
this study thus investigated the g / c polymorphism of mir-421 and its role in progression and prognosis of gastric cancer.material/methodsa total of 96 gastric cancer patients were recruited in this study and tumor samples were collected from surgical resection .
single - nucleotide polymorphism ( snp ) of mir-421 was determined by dna sequencing for analyzing the correlation between lymph node metastasis and mir-421 genotypes .
logistic regression analysis was used to determine the relationship between genotype and risk factors of gastric cancer .
kaplan - meier survival analysis was also performed to compare gg and gc carriers.resultsdifferential expression patterns existed between gastric cancer tissues and normal gastric mucosa .
logistic regression analysis showed gc and gg genotypes were risk factors for gastric cancer .
patients with lymph node metastasis had higher gg genotype frequency compared to those without metastasis . in survival analysis , gg carriers had shorter survival time than gc carriers . furthermore , gg genotype was correlated with tumor prognosis ( p<0.05).conclusionsg allele of mir-421 is a risk factor for gastric cancer .
gg genotype is correlated with lymph node metastasis and prognosis , indicating it is a risk factor for gastric cancer . |
nurses who provide end of life and bereavement care to infants and their families are potentially at great risk for developing stress - related health problems . the emotional strain associated with end of life and bereavement care not only affects a nurse 's health but can also affect relationships at home and with coworkers . the stress experienced by a nurse can even affect the quality of care provided to patients and parents [ 1 , 2 ] . moral distress is recognized as one of the major sources of stress for nurses who provide end of life care to infants .
factors that induce moral distress in nurses can result from providing care to infants who have withdrawal of treatment followed by death or extending futile treatment that induces unnecessary suffering .
there has been an increase in the proportion of deaths associated with decisions to forgo intensive care treatment from 23% in 1987 to 1988 to 64% in 1998 to 1999 .
more than half of neonatal deaths are associated with withdrawal of treatment , and treatment - related stressors to health care professionals have no doubt increased over the years as a result of advanced treatment options [ 6 , 7 ] .
nurses need to be knowledgeable about bereavement and end of life issues and need to be comfortable in their interactions in order to provide quality bereavement and end of life care [ 8 , 9 ] .
it is a nurse 's obligation to act on behalf of the patients and their families to ensure that the care provided is congruent with their preferences .
a key to fulfilling this obligation is to cultivate relations with patients and their families .
the american academy of pediatrics committee on bioethics and its committee on hospital care stated in their integrated model of palliative care that palliative care should be offered at diagnosis and continued throughout the course of illness , whether care results in cure or death .
it is recommended that an uncertain prognosis should be a signal to initiate , rather than to delay , palliative care discussion .
palliative care is not limited to end of life care ; however , it ensures quality of end of life care .
however , studies have indicated that neonatal nurses often are not comfortable with providing end of life care to infants .
some of the reasons associated with this discomfort are inadequate nursing education related to bereavement care in nursing school or in their place of employment [ 13 , 14 ] .
other factors that may elevate the stress levels of nurses who provide end of life care to infants are provider 's age , life experience , and clinical experiences that involve management of ethical dilemmas .
it has been suggested that nurses ' prior knowledge of end of life care is correlated with nurses ' comfort in providing related care .
nurses ' moral distress can affect communication with parents , thus leading to changes in parents ' perceptions of the quality of nursing care .
however , communication problems and a lack of awareness of cultural issues can serve as barrier that can cause parents to feel abandoned and that nurses lack sensitivity .
in addition , noneffective communication between health care professionals and infant 's parents has been identified as one of the barriers that could cause unnecessary conflict in providing infant 's care .
issues related to the parents ' cultural background can profoundly influence medical ethical decision making for infant 's care [ 19 , 20 ] and can result in moral distress in nurses .
the literature indicates that nurses experience stress and moral discomfort in the provision of end of life and bereavement care .
ethical issues related to withdrawal of treatment and futile treatments are major contributors to nursing stress .
communication and awareness of cultural needs are also recognized as important in the care of patients and families .
this study contributes to the literature because it is an intervention study which evaluates nurses ' perceptions about their role , comfort , and involvement in end of life and bereavement care following an educational seminar .
in recognition of the stress experienced by nurses in providing end of life care to infants , the women 's center of a level ii hospital in the southeast offered a bereavement seminar to nurses who work in areas that experience infant deaths .
the purpose of this study was to evaluate the effectiveness of the bereavement seminar on the attitudes of nurses regarding end of life care of neonates .
the research questions were as follows : ( 1 ) what are the characteristics and attitudes of nurses who provide end of life / bereavement care to infants ? ( 2 ) is there a difference in nursing role , comfort , and involvement in providing end of life care between pre- and posttest scores of nurses who attended a bereavement seminar ? ( 3 ) will nurses who attend the educational seminar have higher scores in the domains of comfort , role , and involvement than the control group ?
a pre- and posttest design with a nonequivalent control group and intervention group was used to compare the effect of the bereavement seminar on role , comfort , and involvement .
a convenience sample of nurses was drawn from a community hospital in the southeastern united states .
nurses were eligible to participate in the study if they worked in labor and delivery , nicu , mother baby , pediatrics , or the emergency department and worked directly with patients and were not in an administrative position .
nurses who attended the one - day education program were enrolled in this study as the intervention group .
the control group comprised those who did not attend the education program , but worked in areas that provide end of life or bereavement care to infants .
nurses voluntarily signed up to attend the bereavement seminar and participate in the study .
bereavement / end of life attitudes about care of neonatal nurses scale ( beaconns ) was the instrument used for this study .
permission to use the beaconns scale was obtained from engler and associates , the researchers who had developed the instrument .
reliability for the instrument when used in a descriptive study ranged between 0.81 and 0.95 ; it has not yet been used in quasiexperimental design .
there are three domains in the beaconns scale : ( 1 ) the comfort level of handling end of life and bereavement care , ( 2 ) role of propensity in providing end of life and bereavement care , ( 3 ) tendency of allowing family involvement in providing end of life care .
the comfort scale measures nurses ' perceptions about the degree of comfort they felt with various aspects of bereavement / end of life care .
there are 19 likert - scale items in this subscale , with scores ranging from 1 ( very uncomfortable ) to 5 ( very comfortable ) .
the 18-item likert - type role scale measures nurses ' perceptions of their roles with families of critically ill and/or dying infants ; scores range from 1 ( strongly disagree ) to 5 ( strongly agree ) . some items required reverse scoring .
higher total scores indicate more supportive roles in facilitating families ' involvement in end of life / bereavement care .
the 14-item involvement scale measures nurses ' ratings of the importance of various factors relative to their involvement with patients and families .
the likert - type scale ranges from 1 ( very unimportant ) to 5 ( very important ) , and the items are summed for a total score .
the higher the total score , the more the involvement nurses thought they should have with their patients and families .
the reliability for the involvement scale cronbach is 0.85 . in the beaconns instrument , there are additional items that acquire information about a nurse 's educational background , ethnicity , prior experience with infant death , and significant personal loss .
institutional approval for conducting the study was obtained prior to the study from the professional development council from the institution where all nurses were recruited .
an eight - hour end of life / bereavement seminar was developed by a planning committee of staff representatives from the areas that experience fetal and infant deaths . the seminar was entitled
it refers to the physiological and psychological impact of loss on both family and staff .
the objective of the seminar was to alleviate this impact and reduce staff 's moral distress .
the seminar focused on developing strategies to support the caregiver and family in dealing with their grief and to utilize support services in the community and hospital .
flyers and brochures were sent out to the labor and delivery , mother - baby , nicu , pediatrics , and the emergency departments advertising the workshop .
nurses who were direct patient care providers were invited to participate in the study through distribution of a flyer and communication at staff meetings . the morning of the bereavement seminar prior to the start of the program the nurses who wished to participate in the study signed a consent form and completed the beaconns survey .
this group represented the intervention group and the survey completed before the seminar was the pretest survey .
the beaconns survey and a consent form were also distributed to these selected departments and all staff nurses were invited to complete the survey as the control group .
two months after the bereavement program , the intervention group completed a second beaconns scale as the posttest survey .
independent and paired t - tests were conducted to test for statistical mean differences between the pre- and the posttest group .
an independent t - test was used to compare the differences between the intervention and the control groups . a total of 63 nurses participated in the study .
the majority was married ( 83% ) , protestant ( 34.4% ) , and caucasian ( 82.3% ) and had children ( 84% ) .
most held a bsn ( 46.8% ) or an adn ( 41.9% ) degree and worked in the nicu ( 38.7% ) .
the average age of the nurses who participated in the study was 39.42 years ( sd 10.5 ) .
the average years of working experience as a nurse were 5.29 years ( sd 8.2 ) .
seventy - one percent had end of life / bereavement care in their basic nursing education .
less than half ( 43.5% ) were satisfied or very satisfied with their prior end of life nursing education .
slightly more than a fourth ( 28.6% ) had attended continuing education courses on end of life / bereavement care . over three - fourths
had cared for dying infants . when asked who was most important in providing support to families of critically ill infants on a daily basis participants indicated that nurses ( mean = 4.70 ) were most important ( 5 indicates the most important role , and 1 indicates the least important role ) , followed by physicians ( mean = 4.43 ) and chaplains ( mean = 4.05 ) . to answer the research question was there a difference between pre- and posttest scores of nurses who attended the bereavement seminar ? , a paired t - test was used .
data from nurses prior to the seminar and two months after the seminar were compared .
nineteen participants completed the pretest questionnaire ; only 14 participants returned the posttest questionnaire of nurses ' comfort level with end of life / bereavement care being significantly increased ( t = 3.37 , p = 0.01 ) .
however , postrole scores were not significantly different from the prerole scores ( t = 1.09 , p = 0.30 ) .
likewise , postinvolvement scores were not significantly different from the preinvolvement scores ( t = 0.19 , = 0.84 ) .
boxplots and paired ladder plots were used to provide illustrative support for these findings .
an independent t - test was performed in order to answer the third research question , is there a difference in the three domains between the nurse groups who attended the workshop and the group that had not attended the workshop ?
no differences were found between these two groups in comfort level ( t = 0.77 , p = 0.44 ) , role ( t = 0.09 , p = 0.92 ) , and involvement ( t = 0.24 , p = 0.82 ) .
further , no difference was detected in those who signed up for the class and those who did not sign up for the class in whether they had previous training in end of life care / bereavement care .
however , the difference in satisfaction with previous end of life / bereavement care training was significant in those who came to the workshop and those who did not come to the workshop .
those who came to the workshop were less satisfied with their previous end of life / bereavement care training than those who did not come ( t = 2.21 , p = 0.03 ) .
the majority of the sample was caucasian , christian , married , and had children .
these demographic characteristics were similar to the participants in a study by engler et al . that looked at neonatal staff and advanced practice nurses ' perceptions of bereavement / end of life care .
however , the nurses in engler 's study had worked on average over twice as long ( 16.4 versus 5.29 years ) and had a greater percentage of nurses who had experienced a significant loss in the past year ( 31 versus 19 percent ) .
the difference in the number of years worked may reflect the transient nature of the southeast population and the location of several nursing programs near the hospital .
the greater percentage of nurses who had experienced loss in the engler study may be due to that sample being slightly older than the nurses in the current study .
most nurses had received end of life care in their basic nursing programs ; interestingly , less than half were pleased with these offerings .
those who came to the workshop were less satisfied with their previous end of life nursing education training , which may have been a motivator for their attendance at the bereavement workshop .
these nurses in the study have been working in areas that experience neonatal deaths ; surprisingly only a third had attended continuing education on end of life / bereavement care .
another important finding was that the intervention group 's comfort level had increased at the posttest which was 2 months after the seminar .
this supports the findings of fredrickson et al . who found that a nurse 's prior education about end of life care increases comfort and decreases moral distress .
the comfort level increments could be the result of the seminar content as well as the interactions among nurses during the daylong seminar .
a noticeable observation from this seminar is that nurses are gaining new knowledge while they were sharing their own experience and acknowledging their peers .
the participants felt that their role in providing support to families of critically ill infants on a daily basis was more important than that provided by physicians .
a lack of statistical difference between the control and intervention group may have been due to the small sample size .
also , nurses who did not attend the seminar may have already felt at ease in the areas of comfort , role , and involvement .
in addition , the reliability of the involvement scale may have been affected by the phrasing of the directional stem on the likert scale ; many nurses found the involvement scale confusing .
the results of this study supported the use of a bereavement seminar to increase nurse comfort levels in the provision of end of life / bereavement care to infants and families in the intervention group .
nurses overall were not satisfied with the end of life / bereavement education they had received in their undergraduate nursing programs .
this suggests a need for nursing schools to ensure their curricula should include content on caring for critically ill and dying neonates and their families .
nurses who work on units with neonates need supportive continuing education , since three - fourths of the nurses had experienced neonatal deaths , yet only one - fourth had attended continuing education on end of life / bereavement care .
future intervention studies are needed with larger nurse populations in order to evaluate the effectiveness of end of life / bereavement education on comfort and involvement in end of life care .
studies should also assess whether greater nurse comfort scores impact family satisfaction with end of life / bereavement of their neonate . | background . nurses who provide end of life and bereavement care to neonates and their families are potentially at risk for developing stress - related health problems .
these health problems can negatively affect nurses ' ability to care for their patients .
purpose .
nurses need to be knowledgeable about end of life and bereavement issues to provide quality care .
this study sought to evaluate the effect of a bereavement seminar on the attitudes of nurses regarding end of life and palliative care of neonates .
design .
a convenience sample of fourteen neonatal nurses completed a bereavement / end of life attitudes about care of neonatal nurses scale after a bereavement seminar designed to provide information on end of life care .
a pre- and posttest design with an intervention and control group was used to assess changes in nurse bereavement attitudes in relationship to comfort , role , and involvement . results .
after bereavement seminar , the seminar attendees had higher levels of comfort in providing end of life care than nurses in the control group ( t = 0.214 ; p = 0.04 ) .
discussion .
nurses ' comfort levels can be improved by attending continuing education on end of life care and having their thoughts on ethical issues in end of life care acknowledged by their peers . |
gastric cancer is the fifth most common cancer worldwide , with an estimated occurrence of almost one million new cases every year , with half of the world total occurring in east asia . in terms of mortality
, it remains one of the most common causes of cancer related mortality , only second to lung cancer , as less than 50% of patients are eligible for complete resection , and , in spite of recent progress , expected survival duration with palliative treatment barely exceeds 1 year .
her2 amplification or overexpression is known to occur in approximately 15%-20% of patients with gastric cancer .
the pivotal trastuzumab for gastric cancer ( toga ) trial demonstrated that patients with her2-positive gastric or gastroesophageal junction ( gej ) cancer may benefit from trastuzumab , an anti - her2 monoclonal antibody . in the toga trial , patients assigned to receive trastuzumab in combination with chemotherapy showed significant improvement in overall survival ( os , 13.8 vs. 11.1 months ) , progression - free survival ( pfs , 6.7 months vs. 5.5 months ) , and overall response rate ( orr , 47% vs. 35% ) .
in addition , an exploratory analysis demonstrated that patients with strong protein expression ( immunohistochemistry [ ihc ] 3[+ ] or ihc 2[+]/fluorescence in situ hybridization [ fish ] [ + ] ) were more likely to have favorable os with the addition of trastuzumab ( 16.0 months vs. 11.8 months ) . with these results ,
it is well - known that ethnicity matters in gastric cancer . not only in terms of epidemiology , histology , or pharmacodynamics , certain treatments which were effective for a western population did not show similar efficacy in an asian population , as in the avagast trial .
in fact , in the subset analysis of the toga trial , os benefit was not observed in the asian population , while patients from america and europe did benefit from the treatment .
in addition , according to the subset analysis of the toga trial , patients with diffuse - type cancer who seldom harbor her2 overexpression did not show os benefit from trastuzumab - based treatment . in the current study
, we conducted a multicenter , retrospective analysis on her2-positive gastric cancer patients from 12 tertiary institutes in korea , who were treated with trastuzumab - based treatment in order to confirm whether the clinical outcomes are affected by asian ethnicity or histology , and to provide further understanding of the disease nature of her2-positive gastric cancer .
patients diagnosed with her2-positive unresectable or metastatic gastric cancer from 2009 to 2014 were included in the analysis .
the inclusion criteria were as follows : ( 1 ) a pathologically confirmed diagnosis of gastric cancer ; ( 2 ) her2 positivity confirmed according to the criteria used in the toga trial , that is , either ihc 3(+ ) or ihc 2(+)/fish ( + ) ; ( 3 ) treated with trasutuzumab - based chemotherapy as the first - line treatment .
( 4 ) complete medical records , including demographics , site of primary tumor , and pathologic reports .
the study was approved by the institutional review board of the primary investigator s institute , samsung medical center .
clinicopathologic parameters including age , sex , performance status , primary tumor site , lauren s classification , world health organization ( who ) histologic classification , measurability of lesions , methods for her2 confirmation , backbone chemotherapeutic regimen , and baseline laboratory findings were collected retrospectively from patients medical records .
os was measured from the date of diagnosis of an unresectable or metastatic disease to the date of death or last follow - up , and pfs was measured from the date of diagnosis of a metastatic or unresectable disease to the date of progressive disease , death , or last follow - up .
all survival parameters were calculated using the kaplan - meier method and were compared using a log - rank test .
p - values less than 0.05 were considered statistically significant , and all p - values corresponded to two - sided significance tests . to assess whether higher her2/cep17 ratio affected pfs , patients were dichotomized according to good survival group ( median pfs 6 months ) versus poor survival group ( median pfs < 6 months ) .
the optimal cutoff for her2/cep17 ratio for predicting a favorable pfs was determined using receiver operating characteristic ( roc ) curve analyses , and further evaluation was performed using the area under the curve ( auc ) .
patients diagnosed with her2-positive unresectable or metastatic gastric cancer from 2009 to 2014 were included in the analysis .
the inclusion criteria were as follows : ( 1 ) a pathologically confirmed diagnosis of gastric cancer ; ( 2 ) her2 positivity confirmed according to the criteria used in the toga trial , that is , either ihc 3(+ ) or ihc 2(+)/fish ( + ) ; ( 3 ) treated with trasutuzumab - based chemotherapy as the first - line treatment .
( 4 ) complete medical records , including demographics , site of primary tumor , and pathologic reports .
the study was approved by the institutional review board of the primary investigator s institute , samsung medical center .
clinicopathologic parameters including age , sex , performance status , primary tumor site , lauren s classification , world health organization ( who ) histologic classification , measurability of lesions , methods for her2 confirmation , backbone chemotherapeutic regimen , and baseline laboratory findings were collected retrospectively from patients medical records .
os was measured from the date of diagnosis of an unresectable or metastatic disease to the date of death or last follow - up , and pfs was measured from the date of diagnosis of a metastatic or unresectable disease to the date of progressive disease , death , or last follow - up .
all survival parameters were calculated using the kaplan - meier method and were compared using a log - rank test .
p - values less than 0.05 were considered statistically significant , and all p - values corresponded to two - sided significance tests . to assess whether higher her2/cep17 ratio affected pfs , patients were dichotomized according to good survival group ( median pfs 6 months ) versus poor survival group ( median pfs < 6 months ) .
the optimal cutoff for her2/cep17 ratio for predicting a favorable pfs was determined using receiver operating characteristic ( roc ) curve analyses , and further evaluation was performed using the area under the curve ( auc ) .
the median age of patients was 60 years ( range , 27 to 85 years ) and 118 patients ( 70.2% ) were male .
regarding the location of the primary tumor , 161 cases ( 95.8% ) were stomach cancer and seven cases ( 4.2% ) were gej cancer .
as many pathologists in korea do not interpret lauren s classification with biopsied specimens , only 78 cases had been confirmed using lauren s classification , and among them , 44 ( 56.4% ) , 22 ( 28.2% ) , and 12 cases ( 15.4% ) were found to be intestinal - type , diffuse - type , and mixed - type cancer , respectively .
however , who histologic classification was confirmed in all cases , with 14 ( 8.3% ) , 63 ( 37.5% ) , 75 ( 44.6% ) , and 11 cases ( 6.5% ) found to be well - differentiated , moderately - differentiated , poorly - differentiated tubular adenocarcinoma , and signet ring cell carcinoma , respectively . in terms of methods for her2 confirmation , in 129 patients disease was confirmed by ihc 3(+ ) , and by ihc 2 - 3(+)/fish ( + ) in 59 patients whose median value of her2/cep17 ratio was 3.34 ( range , 2.09 to 20.00 ) .
other details are described in table 1 . regarding the backbone regimen of the trastuzumab - based therapy , 145 patients ( 86.3% ) had received capecitabine plus cisplatin ,
14 patients ( 8.3% ) had received 5- fluorouracil ( 5-fu ) plus cispaltin , and nine patients had received other agents .
median number of cycles was six ( range , 1 to 62 ) for both trastuzumab and chemotherapy .
tumor responses were as follows : 12 ( 7.1% ) complete response , 73 ( 43.5% ) partial response , 48 ( 28.6% ) stable disease , 17 ( 10.1% ) progressive disease , and 18 ( 10.7% ) cases were not evaluable , resulting in 50.6% of orr ( table 2 ) . with a median follow - up duration of 32.9 months ( 95% confidential interval [ ci ] , 30.0 to 35.8 ) ,
median pfs was 10.2 months ( 95% ci , 8.7 to 11.7 ) , and median os was 18.5 months ( 95% ci , 16.4 to 50.6 ) .
considering that asian patients usually achieve approximately 12 months of os and 6 months of pfs , the numerical data of the current study may suggest that even in an asian population , trastuzumab - based therapy could be beneficial for patients with her2-positive gastric cancer .
patients with well - differentiated and moderately - differentiated tubular adenocarcinoma were categorized as the well - differentiated histologic ( wdh ) group , and poorly - differentiated tubular adenocarcinoma and signet ring cell carcinoma as poorly - differentiated histologic ( pdh ) group .
consequently , 77 ( 45.8% ) and 86 ( 51.2% ) patients were classified as the wdh and pdh group , respectively .
the median pfs of the wdh group was slightly longer than that of the pdh group , which did not reach statistical significance ( 11.5 months [ 95% ci , 8.1 to 14.9 ] vs. 8.9 months [ 95% ci , 7.2 to 10.6 ] , p=0.16 ) ( fig .
however , the median os of two groups was significantly different ; 19.0 months ( 95% ci , 14.2 to 23.8 ) for the wdh group ; 14.6 months ( 95% ci , 11.9 to 17.3 ) ( p=0.025 ) for the pdh group ( fig .
2 ) . again , in numerical perspectives , these data of pdh patients , that is , pfs of 8.9 months and os of 14.6 months , are better than the data from the typical asian trial , which might suggest that even patients of pdh could benefit from trastuzumab - based therapy .
other than histologic differentiation , eastern cooperative oncology group ( ecog ) status and serum albumin level were associated with survival ( table 3 ) .
roc analysis was performed for 59 patients who were confirmed as fish ( + ) if a certain her2/cep17 ratio could predict favorable pfs for trastuzumab - based treatment .
higher ratios ( 3 or higher and 4 or higher ) were not associated with prolonged pfs ( table 4 ) .
the median age of patients was 60 years ( range , 27 to 85 years ) and 118 patients ( 70.2% ) were male .
regarding the location of the primary tumor , 161 cases ( 95.8% ) were stomach cancer and seven cases ( 4.2% ) were gej cancer .
as many pathologists in korea do not interpret lauren s classification with biopsied specimens , only 78 cases had been confirmed using lauren s classification , and among them , 44 ( 56.4% ) , 22 ( 28.2% ) , and 12 cases ( 15.4% ) were found to be intestinal - type , diffuse - type , and mixed - type cancer , respectively .
however , who histologic classification was confirmed in all cases , with 14 ( 8.3% ) , 63 ( 37.5% ) , 75 ( 44.6% ) , and 11 cases ( 6.5% ) found to be well - differentiated , moderately - differentiated , poorly - differentiated tubular adenocarcinoma , and signet ring cell carcinoma , respectively . in terms of methods for her2 confirmation , in 129 patients disease was confirmed by ihc 3(+ ) , and by ihc 2 - 3(+)/fish ( + ) in 59 patients whose median value of her2/cep17 ratio was 3.34 ( range , 2.09 to 20.00 ) .
regarding the backbone regimen of the trastuzumab - based therapy , 145 patients ( 86.3% ) had received capecitabine plus cisplatin , 14 patients ( 8.3% ) had received 5- fluorouracil ( 5-fu ) plus cispaltin , and nine patients had received other agents .
median number of cycles was six ( range , 1 to 62 ) for both trastuzumab and chemotherapy .
tumor responses were as follows : 12 ( 7.1% ) complete response , 73 ( 43.5% ) partial response , 48 ( 28.6% ) stable disease , 17 ( 10.1% ) progressive disease , and 18 ( 10.7% ) cases were not evaluable , resulting in 50.6% of orr ( table 2 ) . with a median follow - up duration of 32.9 months ( 95% confidential interval [ ci ] , 30.0 to 35.8 ) , median pfs was 10.2 months ( 95% ci , 8.7 to 11.7 ) , and median os was 18.5 months ( 95% ci , 16.4 to 50.6 ) . considering that asian patients usually achieve approximately 12 months of os and 6 months of pfs , the numerical data of the current study may suggest that even in an asian population , trastuzumab - based therapy could be beneficial for patients with her2-positive gastric cancer .
patients with well - differentiated and moderately - differentiated tubular adenocarcinoma were categorized as the well - differentiated histologic ( wdh ) group , and poorly - differentiated tubular adenocarcinoma and signet ring cell carcinoma as poorly - differentiated histologic ( pdh ) group .
consequently , 77 ( 45.8% ) and 86 ( 51.2% ) patients were classified as the wdh and pdh group , respectively .
the median pfs of the wdh group was slightly longer than that of the pdh group , which did not reach statistical significance ( 11.5 months [ 95% ci , 8.1 to 14.9 ] vs. 8.9 months [ 95% ci , 7.2 to 10.6 ] , p=0.16 ) ( fig .
however , the median os of two groups was significantly different ; 19.0 months ( 95% ci , 14.2 to 23.8 ) for the wdh group ; 14.6 months ( 95% ci , 11.9 to 17.3 ) ( p=0.025 ) for the pdh group ( fig .
in numerical perspectives , these data of pdh patients , that is , pfs of 8.9 months and os of 14.6 months , are better than the data from the typical asian trial , which might suggest that even patients of pdh could benefit from trastuzumab - based therapy . other than histologic differentiation , eastern cooperative oncology group ( ecog ) status and serum albumin level were associated with survival ( table 3 ) .
roc analysis was performed for 59 patients who were confirmed as fish ( + ) if a certain her2/cep17 ratio could predict favorable pfs for trastuzumab - based treatment .
higher ratios ( 3 or higher and 4 or higher ) were not associated with prolonged pfs ( table 4 ) .
although several combinations have been introduced in treatment of unresectable or metastatic gastric cancer patients , their prognosis remains poor . as a significant interest in targeted therapies
has emerged , a number of targeted agents have been evaluated in gastric cancer . however , most of them fell short of success [ 7 - 9 ] , and trastuzumab is the only agent with a known predictive biomarker and proven efficacies .
clinical efficacy of ramucirumab and apatinib was recently demonstrated in refractory gastric cancer patients ; however , their predictive biomarkers have not been found .
her2 is a member of the epidermal growth factor receptor family which promotes cell proliferation and inhibits apopapoptosis , and its overexpression has been described in various human malignancies .
trastuzumab , a recombinant humanized anti - her2 monoclonal antibody , was first developed as a targeted agent for her2-positive breast cancer , and its unprecedented successes heralded the era of target therapy and relevant biomarkers in the field of oncology . as the carcinogenic roles of her2 in gastric cancer had been suggested , a randomized phase iii trial commenced to assess the clinical efficacy and safety of trastuzumab , which was followed by a great success .
not only the nationwide screening policies , epidemiology , pathogenesis , anatomic location or dominant histology , treatment modalities including surgical approaches and adjuvant treatment also differ between asian and western countries .
in addition , several pharmacokinetic studies demonstrated that efficacy and tolerability of fluoropyrimidine , a major antineoplastic agent in management of gastric cancer , differ between asian and western people . owing to these factors ,
the clinical outcomes of asian gastric cancer patients are superior to those of western patients .
therefore , mindfulness of ethnicity is important in managing patients and in planning clinical trials as well . given that her2 overexpression is frequently found in gej and intestinal - type cancer , her2 overexpression rate might be lower in asians , who tend to have distal and diffuse - type cancers
. however , several series of surveys demonstrated that her overexpression rate was not low in asians .
as prevalence of gastric cancer is high in asia , many gastric cancer trials have been conducted with asians . in the toga trial , among 584 patients , 319 ( 54.6% )
were asians , and , as described above , asians per se , were not associated with os benefit with trastuzumab - based treatment .
however , this could result from the fact that asian gastric cancer patients already have sufficient survival outcomes with chemotherapy alone , probably due to subsequent treatment which may attenuate the impact of effective combinations as shown in the avagast trial
. the benefit of trastuzumab - based therapy in asians might be suggested in two recent asian trials . in a japanese study ,
53 patients were treated with trastuzumab combined with s-1 plus cisplatin , and their median pfs and os were estimated as 7.8 months and 16.0 months . and , in a korean study , 55 patients treated with trastuzumab combined with capecitabine and oxaliplatin ( xelox ) showed a median pfs of 9.8 months and a median os of 21.0 months .
along with these data , our retrospective analysis showed a median pfs and os of 10.2 months and 18.5 months , respectively ( table 5 ) . considering that asians usually achieve pfs of 5 to 6 months and os of 12 to 13 months with the first - line fluoropyrimidine and platinum combinations ,
these numerical data imply that asians may also benefit from trastuzumab - based therapy in the same manner with western people
. however , prospective head - to - head comparative analyses are warranted in order to confirm the efficacy of trastuzumab - based therapy in this unique population .
gastric cancer can be categorized according to two distinct histologic subtypes , intestinal and diffuse , which are distinct in their microscopic and gross appearance , epidemiology , pathogenesis , and prognosis . among them , diffuse - type gastric cancer is associated with poor response to treatment and early relapse , thereby a worse survival . in the current study , we used who classification as a substitute for lauren s classification , as many pathologists in korea do not interpret lauren s classification with endoscopic specimens due to fear of misreading , yet a high concordance between two systems has been observed .
compared to the patients with wdh , the patients with pdh showed worse clinical outcomes in both pfs ( 8.9 months vs. 11.5 months , p=0.16 ) and os ( 14.6 months vs. 19.0 months , p=0.025 ) . as these data are not head - to - head comparisons , it is difficult to make a firm conclusion whether patients with pdh can benefit from trastuzumab - based therapy or not .
nonetheless , considering the numerical clinical data usually reported in asians , our data may imply that even patients harboring pdh cancer could benefit from trastuzumab - based therapy , although the degree is somewhat limited compared to patients with wdh . in the perspective of frequency of her2 overexpression or amplification ,
it is more commonly found in intestinal - type cancer than diffuse - type cancer . in the toga trial ,
51 out of 584 patients ( 8.7% ) had diffuse - type cancer , and in the logic trial , the differential became even more remarkable ; 3.9% of patients ( 19/487 ) had diffuse - type cancer .
this dominance of intestinal - type cancer in the two trials looks suitable considering its disease nature .
however , in our cohort , the number of patients with pdh ( n=86 , 51.2% ) was slightly higher than that of patients with wdh ( n=77 , 45.8% ) .
several large studies from asia demonstrated that diffuse - type or poorly - differentiated cancer was more predominant .
although these types of gastric cancer do not harbor her2 overexpression very often , out - numbering of patients could draw the results .
similar to our finding , in the tytan trial , which recruited her2-positive gastric cancer patients confined to asia , only 43% of patients had intestinal - type gastric cancer .
a her2/cep17 ratio was recently suggested as a novel predictive marker for trastuzumab - based treatment . in a spanish trial ,
66 fish positive patients were evaluated and her2/cep17 higher than 4.7 was identified as the cutoff value for prolonged os . in another report by ock et al .
, a her2/cep17 ratio of 4.48 clearly discriminated clinical outcomes . in our study , however , we failed to find optimal discriminating values for favorable pfs .
this could have resulted from the small number of samples , and our fish data did not come from a single laboratory .
this could draw somewhat inconsistent interpretation regarding the favorable tumor response , such as complete or partial responses .
however , as we assessed pfs and os , we believe the validity of the conclusive data and rather , it may reflect real world clinical outcomes .
although the backbone regimens are varied , most of them ( 98.2% ) are the combination of fluoropyrimidine ( 5-fu and capecitabine ) and platinum ( cisplatin , oxaliplatin ) which have similar efficacies and can be used interchangeably . and ,
as we focused on the efficacy of the treatment , the current analysis does not provide ethnic or histologic difference of safety issue .
in conclusion , despite the result of subset analysis of the toga trial , we might conclude that irrespective of ethnicity or histologic subtypes , patients with her2-positive gastric cancer can benefit from trastuzumab - based chemotherapy . further analysis to find predictive biomarkers for these patients should be continued and ethnic difference and histologic subtypes should be considered for future clinical trials on gastric cancer patients . | purposewhile the trastuzumab for gastric cancer ( toga ) trial demonstrated the efficacy and safety of trastuzumab - based chemotherapy in her2-positive metastatic gastric cancer , the overall survival ( os ) benefit was not found in asian and diffuse - type cancer patients .
the aim of the study is to investigate predictive markers for trastuzumab - based chemotherapy.materials and methodsdata of patients with her2-positive gastric cancer treated with trastuzumab - based chemotherapy were analyzed retrospectively.resultsa total of 168 asian patients were included .
the median age was 60 years ( range , 27 to 85 years ) and the male : female ratio was 118 ( 70.2%):50 ( 29.8% ) .
fourteen ( 8.3% ) , 63 ( 37.5% ) , 75 ( 44.6% ) , and 11 ( 6.5% ) patients had well , moderately , poorly - differentiated tubular adenocarcinoma and signet ring cell carcinoma , respectively .
with 14 complete responses and 73 partial responses , the response rate was 50.6% .
the median progression - free survival ( pfs ) was 10.2 months ( 95% confidence interval [ ci ] , 8.7 to 11.7 ) , and the median os was 18.5 months ( 95% ci , 16.4 to 50.6 ) .
next , we investigated the effect of poorly - differentiated histology ( pdh , poorly - differentiated tubular adenocarcinoma+signet ring cell carcinoma ) on clinical outcomes .
the median pfs ( 8.9 months vs. 11.5 months , p=0.16 ) was slightly inferior in pdh patients , and the median os was significantly shorter in pdh patients ( 14.6 months vs. 19.0 months , p=0.025).conclusionwhile subset analysis of the toga trial demonstrated that trastuzumab - based chemotherapy may not be beneficial for asians and patients with pdh , our data may suggest that even in asian patients and patients with pdh , trastuzumab - based chemotherapy could be associated with improved clinical outcomes in patients with her2-positive gastric cancer . |
severe acute pancreatitis ( sap ) , characterized by atrocious
progression , multicomplications , and difficult treatment
[ 13 ]
is an acute abdomen that ranges from a mild illness to a
life - threatening condition .
clinically ,
the overall mortality rate for sap is approximately
38.4% . in spite of
decades of intensive study , we are still in lack of etiological
therapy .
sap causes such a hypermeolic state that adequate
supply of nutrients plays a decisive role to reverse systemic
malnutrition , potentiate resistance against infection , block the
pathological deterioration , and facilitate recovery
[ 5
7 ] . in the past ,
it was
believed that early
enteral nutrition ( een ) in patients with sap might exacerbate
the clinical as well as pathological conditions and provoke
serious complications .
the high mortality rate of sap is often a result of multiple
organ dysfunction syndrome ( mods ) and appears to be especially
related to systemic inflammatory response syndrome ( sirs )
or even to infections of the necrotic pancreas .
since mods and
sirs are apt to be facilitated
with gut mucosal dysfunction and abundant gram - negative
bacteria as well as other gastrointestinal pathogens are
commonly detected in pancreatic
infections , the gut is considered to be the main source of sap - related septic
complications , so that maintaining gut barrier is equally as important as
resting the pancreas when the inflammation within the gland resolves .
though total parenteral nutrition ( tpn ) has been the standard practice
for providing exogenous nutrients during the early stage of sap , long - term administration
of tpn can produce catheter - related complications , weaken systemic immunity ,
and induce secondary enterogenous infection . in comparison to tpn
, een helps
enhance the immunological and mechanical functions of intestinal mucosa ,
maintain the balance of gut microflora , reduce oxidative stress , prevent
secondary infections , and improve therapeutic results .
emodin , the main
effective component of rhubarb , which is a traditional chinese herbal medicine
and has the function of purgation , heat - clearing , and detoxicating as well as
promoting blood circulation to remove blood stasis , has proved to be a beneficial
pharmacy for sap [ 812 ] . because of the liver 's
special position in the gut
circulation and the unsubstitutable role played in body metabolism , it becomes
a major target for extrapancreatic damage of sap . and liver injury , in turn ,
deteriorates sap and amplifies the systemic damage caused by mods .
therefore ,
effective prevention of liver injury is a key to shortening the course of sap
and decreasing the related mortality .
een and emodin have been fully affirmed by most scholars as an important part of therapy for
sap .
however , it is still controversial what substance and dosage in een and
what timing and route of administration of een associated with emodin are more
appropriate and whether the combined treatment is more effective than either
one alone . in the present experiment
, we tried to explore possible mechanisms
of een and emodin in the treatment of sap as well as its secondary hepatic
injury , and to demonstrate the feasibility and effectiveness of
emodin - assisted early enteral nutrition ( eaeen ) .
sixty healthy male wistar rats , weighing 190250 g ,
were purchased from animal center , the first clinical hospital
of harbin medical university ( harbin , china ) .
emodin was
obtained from pharmacy , the first clinical hospital of harbin
medical university .
sodium taurocholate was obtained
from sigma ( st . louis , mo , usa ) .
sixty male wistar rats , housed in
cages with a controlled temperature of 26c
and 12-hour light - dark cycles , were fed
standard laboratory chow as well as water ad libitum , and
allowed to acclimatize
for at least a week .
surgical anesthesia was performed
by intraperitoneal
injection of 1% pentobarbital sodium ( 40 mg / kg body weight ) .
sap was induced by
retrograde infusion of 5.0% sodium taurocholate
( 0.15 ml/100 g body weight ) into the
common pancreatic duct .
the out
end of the enteral nutrient tube was placed at 7 cm
from the far point of treitz ligament , while the in
end was dragged
backward through the body wall and fixed on the back .
the experimental protocol
was undertaken in accordance with the guideline for the care and use of
laboratory animals in research and was approved by the ethical
and research
committee of the first clinical hospital ,
harbin medical
university .
sixty rats were randomly divided into
four groups ( n=15 ) after sap induction :
control group ( group a ) , emodin group ( group
b ) , een group ( group c ) , and eaeen group ( group d ) .
control group was allowed
free access to water without any intervention ; emodin group
received emodin ( 3.0 mg/100 g
body weight ) through enteral nutrient tube at 12 h , 22 h ,
32 h , 42 h , 52 h , and 62 h ,
respectively ; een group received pepti-2000
variant through enteral nutrient tube at 15 h , 25 h ,
35 h , 45 h , 55 h , and 65 h ,
respectively .
the enteral nutrient solution was continuously
infused at each
time point and the dosage at 15 h , 25 h , and 35 h
was 120 ml / kg ( body weight )
followed by that increasing to 240 ml / kg ( body weight )
at 45 h , 55 h , and 65 h ;
eaeen group received emodin ( 3.0 mg/100 g body weight )
at 12 h , 22 h , 32 h , 42 h ,
52 h , and 62 h and pepti-2000
variant at 15 h , 25 h , 35 h ,
45 h , 55 h , and 65 h. the dosage and
infusion method of
pepti-2000 variant at each time point in eaeen group were
the same as those in een group .
the remaining living animals in each
group were reanesthetized 72 hours after sap induction
using intraperitoneal
injection of 1% pentobarbital sodium ( 40 mg / kg body weight ) .
a portion of the pancreatic tail and
hepatic and ileal tissues from each rat was incised
and the pancreatic as well as hepatic tissues were fixed in 10%
neutral - buffered formalin , embedded in paraffin , and stained with hematoxylin
and eosin ( he ) for light microscopy .
two experienced
pathologists who were
blinded to the experimental protocol scored the pancreatic tissue
on a scale from 0 to 4 for the degrees of edema , inflammation ,
hemorrhage , and necrosis in 20 fields .
the jejunum and ileum were excised and the intestinal mucus
was collected to measure the level of siga by radio
immunoassay with the kit ( institute of
atomic energy physics , chinese academy
of sciences , china ) .
the values of mpo activity in pancreas and liver were measured
to assess polymorphonuclear leukocyte ( pmn ) infiltration .
a portion of the pancreatic and hepatic tissues was obtained
to determine pancreatic and hepatic mpo activity by chromatometry
with the kit ( invitrogen , calif , usa )
and ascitic fluid was
quantified .
after removal , fresh samples of
pancreas and ileum were immediately weighed and dried at
100c for 24 hours and reweighed .
the wet - dry weight ratio of pancreas ( pww / dw ) and
ileum ( iww / dw ) were calculated to determine the
degrees of pancreatic edema and ileal edema .
blood samples were collected from abdominal aorta and
centrifuged at 3000 rpm for 5 minutes .
tumor necrosis factor - alpha ( tnf- )
and angiotensin ii ( angii ) were measured by radio
immunoassay with the kits ( institute
of atomic energy physics , chinese academy of sciences , china ) .
maleic dialdehyde ( mda ) ,
c - reactive protein ( crp ) ( jiancheng biotech ltd .
,
nanjing , china ) ,
and plasma d - lactate ( sigma , usa ) were assessed by
enzymatic - spectrophotometric method .
plasma endotoxin ( shanghai
med & chem institute , shanghai , china )
was tested by chromogenic limulus amebocyte lysate technique .
glutamic - pyruvic transaminase ( alt ) , glutamic - oxaloacetic
transaminase ( ast ) , and amylase were determined by automated hitachi-7150
analyzer .
the significance of differences in histopathologic scores was
assessed by kruskal - wallis test .
statistical analysis
was finished by spss10.0 statistical program and difference
was considered statistically significant
when
p<.05 .
before sampling
72 hours after the induction of sap , the ratio of survival to death was 7 to 8 in control group , 9 to 6 in emodin group , 8 to 7 in een group , and 12 to 3 in eaeen group .
compared with control
group , there were significant differences in the following data as described in
emodin , een , and eaeen groups ( p<.05 , tables
14 ,
figures 15 ) . in group
a , severe edema , diffuse
hemorrhage , and necrosis in pancreas were observed .
there was
much bloody ascites in the peritoneal cavity .
compared with group a
, the severity of macroscopic
changes was significantly abated in
groups b , c , and d. in contrast to emodin and een , eaeen significantly reduced
the levels of tnf- , angii , mda , alt , ast , crp ,
and amylase in serum as well as the
levels of plasma endotoxin and d - lactate
( p<.05 , tables 1 , 2 , and 4 , figures 1 and 5 ) .
differences in these data were not considered statistically
significant between group b and group c. the levels of mpo in pancreas and liver , pww / dw , iww / dw , and
the amount of ascitic fluid were significantly lower , while the
level of siga was obviously higher in group d than those in
group b and group c ( p<.05 ,
tables 2 ,
3 , and 4 ,
figures 24 ) .
however , no significant differences were found in
these data between group b and group c. the degrees of edema , inflammatory infiltration , hemorrhage , and
necrosis and the scores of histopathologic alterations
in pancreas were significantly reduced in group d as
compared with those in
group b and group c ( p<.05 ,
table 3 ,
figures 69 ) .
eaeen could
also attenuate the severity of secondary liver injury obviously
( figures 1014 ) .
the villus height of ileum was significantly increased
by eaeen compared with emodin and
een ( p<.05 ,
table 3 ) .
there were
no significant differences in the scores of pancreatic histopathologic
alterations as well as the ileal villus height between group
b and group c.
emodin characterized by the main active monomer of
rhubarb is a derivative of anthraquinone
( 3-methyl-1 , 6 , 8-trihydroxyanthraquinone ) .
studies have proved that emodin is a potent agent in
the management of clinical
and experimental acute pancreatitis
( ap ) .
zhang et al . , and wu et al .
found
emodin had significant therapeutic effects on sap rats by
correcting intestinal flora disturbances , promoting intestinal
peristalsis , inhibiting inflammatory cytokine release and
pancreatin activity , and scavenging oxygen free radicals ( ofr ) .
wu et al . proved the mechanisms
of emodin in the treatment of sap included
modulation of abnormal eicosanoid metabolism , promotion of pancreatic
cytoprotection , and improvement of pancreatic microcirculation .
found emodin was a potent inhibitor of nuclear
factor - kappab ( nf-b ) activation as well as expression of adhesion molecules and could be
useful in treating various inflammatory diseases .
in a word , emodin has multiple beneficial effects on sap . in our experiment ,
administration of eaeen could significantly abate the
severity of sap as
compared with other groups , which was reflected by the
reductions in levels of
serum amylase , the amount of ascitic fluid ,
pancreatic mpo activity , pww / dw ,
and the pathologic scores of pancreas .
our
results indicated that the advantages of eaeen for the
treatment of sap existed
in ( 1 ) een alone could enhance
gut immunity , promote hyperplasia of intestinal mucosa , improve
microcirculation of intestinal mucosa , decrease the
permeability of intestinal mucosa , and reduce the incidence
of translocation of intestinal bacteria ; ( 2 ) pepti-2000 variant ,
which is an elemental diet with low - fat content and can
be absorbed easily , contains adequate nutritional ingredients ,
scanty residue and has low viscosity .
administration of
pepti-2000 variant by enteral nutrient tube at different time
points conformed to the daily dietary habit , placed the pancreas at a
full rest , and inhibited rebound
of the inflammatory reaction ; ( 3 ) emodin could clear the intestinal
bacteria and toxin , promote intestinal peristalsis and
recovery of the intestinal function , so as to reduce the
incidence of secondary enterogenous infection .
taken together , this
combined strategy was methodologically more rational and
purposive , with undoubtedly higher efficacy . a vicious cycle of pancreatic microcirculatory changes such
as capillary stasis and vasoconstriction has been shown to occur
in the course of sap .
renin - angiotensin system ( ras ) is an important stress system
in pancreatic microcirculatory disturbances .
ras in systemic circulation is activated to a
great extent and produces massive angiotensinii ( angii ) ,
which subsequently combines with its receptors
predominantly localized to the epithelia of pancreatic ducts , vascular
endothelia , and pancreatic acinar cells
, results in the strong
vasoconstriction of pancreas , and aggravates the pancreatic
ischemia and necrosis in sap . in this study ,
our results proved
that administration of eaeen enhanced colloid osmotic pressure ,
prevented excessive peripancreatic and retroperitoneal fluid
exudation followed by hypovolemic shock , and inhibited the
activation of pancreatic enzymes [ 9 ,
12 ] so as to eliminate the main
factors that induced the abnormal activation of ras ,
further significantly
reduced the level of serum angii and ameliorated pancreatic
microcirculatory disturbances .
gut barrier dysfunction and intestinal immunosuppression are
directly related to the severity of sap , so that how to
protect the intestinal
mucosa from being damaged might play a key role
in preventing bacterial and
toxin translocation and deterioration of sap .
normally , the level of blood d - lactate
is quite low and the measurement of the increased
plasma d - lactate level may be a useful marker to assess
the intestinal injury .
siga , the predominant immunoglobulin present in
mucosal secretions , is the major ingredient of the
intestinal immunological function
on mucosal surfaces . in this study , the levels
of intestinal siga , plasma d - lactate , the ileal villus
height , and iww / dw
showed obvious differences in eaeen group as compared with
those in other
groups .
our data demonstrated that administration of een
associated with emodin
could significantly attenuate the severity of gut damage
and enhance the intestinal immunity by restoring
the microecology of the intestinal flora , maintaining the intestinal
epithelia integrity , abating edema of the intestinal
wall , and promoting the
secretion of intestinal immune substances so as to reduce
the incidences of
enterogenous infection and endotoxemia . in sap ,
bacterial and endotoxin
translocation through the damaged gut barrier is the culprit for the
development of secondary septic complications and a second
peak of mortality .
endotoxin is the lipopolysaccharide
( lps ) present in the gram - negative bacterial wall .
abundant plasma endotoxin intensively
remotivates mononuclear - macrophage system and accelerates
the production of many
cytokines as well as inflammatory mediators , which induce septic shock and
amplify sirs followed by the development of mods after releasing into the
blood .
atrociously amplified sirs is a hallmark for
sap with elevated circulating proinflammatory cytokines including tumor
necrosis factor- ( tnf- ) and interleukin-1 .
cytokine in sirs and plays a central role in
the pathogenesis of ap and related systemic
complications .
tnf- can induce infiltration and activation of neutrophils ,
upregulate cellular adhesion
molecules , promote the secretion of ofr , initiate and
aggravate the cascade of other proinflammatory
cytokines and inflammatory mediators .
mpo , a member of
peroxidases secreted by
neutrophils , is an indicator for the count and activity
of neutrophils . in our study ,
administration of eaeen significantly reduced
the levels of plasma endotoxin , serum tnf- , and mpo
in pancreatic tissue , so as to inhibit both local and systemic
inflammatory reaction as well as
oxidizing reaction and abate the severity of pancreatic damage .
we enumerated the following explanations as the rationales : ( 1 )
emodin could inhibit the overgrowth of pathogenic
organisms including gram - negative bacteria in the
intestinal tract
and accelerate their excretion by promoting intestinal
peristalsis , so that the direct source of endotoxin
was eradicated and the level of plasma endotoxin depressed ; ( 2 )
een could potentiate plasma protein synthesis , increase the blood
perfusion in portal system , promote hepatoenteral circulation ,
and enhance hepatic detoxification , which in turn
greatly reduced the level of plasma endotoxin ; ( 3 )
een improved the gut barrier function and maintained
the balance of gut microflora so as to decrease the production of
endotoxin and to prevent it from entering the blood ,
which consequently abated the remotivation of mononuclear - macrophage
system , so that the releases of tnf- and
other cytokines were reduced ; ( 4 )
emodin inhibited the activation of pancreatic enzyme
so as to prevent mononuclear - macrophage system ,
activated by pancreatic enzyme after its entering the plasma ,
from producing tnf- ; ( 5 )
angii is capable of promoting the releases of
proinflammatory cytokines and stimulating the migration
and infiltration of monocytes and neutrophils [ 15 ,
19 , 20 ] .
administration of eaeen significantly
reduced the level of serum angii , which further
decreased the concentration of
serum tnf- and pancreatic mpo activity
so as to attenuate the
severity of inflammation in pancreas ; ( 6 ) tnf-
induces aggregation and activation of neutrophils in the pancreas .
administration of eaeen could significantly reduce
blood tnf- level , which consequently reversed
this process and abated
the pancreatic inflammatory reaction .
secondary liver injury ( sli ) has a remarkable incidence
in sap , and the severity of hepatocyte
injury is proportional to the severity of sap .
several mechanisms are involved
in the initiation of sli : ( 1 ) in the acute stage ,
massive proinflammatory
factors excessively activate the liver kupffer cells ,
which then intensely
release even more profactors , so that a cascade amplifying
cycle inside and
outside the liver is formed , bringing heavy inflammatory
injury to the liver ; ( 2 ) tnf- and endotoxin induce massive
neutrophils infiltration and activation , producing great
amount of ofrs , which
initiate intensive lipid peroxidations aggravating liver
injury ; ( 3 ) intestinal mucosal damage leads to a remarkable
increase of endotoxin level in the portal circulation
bringing enormous damage to the liver .
found that lps could
significantly elevate serum tnf-
activity and induce more severe liver damage in ap rats ,
which suggested that both lps and tnf- played
critical roles in the development of secondary hepatic
injury during ap .
found that activated
neutrophils and macrophages produced large
amount of ofrs in sap and they were closely involved in
the secondary liver
disease .
mda is the product of lipid peroxidation of ofr ,
which indicates the amount
of ofr and indirectly reflects the severity of tissue damage .
crp is an acute
phase reactive protein produced by the liver
especially in the context of sap .
the
increase of serum crp is proportional to the severity of
hepatocyte injury .
our
experiment demonstrated that eaeen could remarkably decrease
tnf- , mda , and
endotoxin levels in the blood and mpo activity in the liver ,
so that protect
the liver from the tremendous inflammatory and oxidizing
injuries brought upon by sap , as manifested
by the decreases of alt , ast , and crp levels in the blood . in a word , administration of emodin
prior to een formed a firm base for subsequent een therapy of
sap by clearing bacteria and toxin , promoting exsufflation and
defecation , abating abdominal distension and systemic
inflammatory reaction , so
that the enteral nutrient solution could be fully absorbed .
compensate for
the massive loss of nutrients
caused by administration of emodin , so they two could benefit
each other in
combination and obviously improve the therapeutic efficacy .
in this experiment , administration of emodin and een in the early stage
of sap was safe and rational , and the combined strategy was
more effective than
either one alone .
our results showed that administration of
eaeen could control multiple deteriorating factors
of sap and provided an effective treatment for sap and
its secondary hepatic
injury by protecting intestinal mucosal barrier ,
enhancing body immune
competence , inhibiting systemic inflammatory reaction and reversing
microcirculatory dysfunction , and so forth .
this study suggested a broad
potential value for eaeen in the clinical treatment of sap .
however , further
studies are needed to determine whether administration of eaeen can also
protect other organs from being damaged in sap . | severe acute pancreatitis ( sap ) characterized by atrocious progression and numerous complications often leads to a high mortality rate due to hypermetabolism , systemic inflammatory response syndrome ( sirs ) , and multiple organs dysfunction syndrome ( mods ) .
studies have revealed that both early enteral nutrition ( een ) and emodin are potent agents in the management of sap . however , whether the combined strategy is rational and more effective than either one alone remains unknown . in this
regard , wistar rats were treated with emodin - assisted een ( eaeen ) through enteral nutrient tubes after induction of sap by retrograde infusion of 5.0% sodium taurocholate into the common pancreatic duct .
serum levels of amylase , tumor necrosis factor - alpha ( tnf- ) , angiotensin ii ( angii ) , maleic dialdehyde ( mda ) , glutamic pyruvic transaminase ( alt ) , glutamic oxaloacetic transaminase ( ast ) and c - reactive protein ( crp ) , intestinal secretory iga ( siga ) , pancreatic and hepatic myeloperoxidase ( mpo ) activity as well as plasma levels of d - lactate and endotoxin were measured .
in addition , pathologic alterations of pancreas and liver were observed microscopically .
we found that eaeen could significantly ameliorate these parameters and prevent pancreas and liver from serious damage . in conclusion
, our results indicated that eaeen could exert beneficial effects on experimental sap and obviously abate the severity of secondary hepatic injury .
the combined strategy was safe and more effective than either one alone in the acute stage of sap .
this study also provided an experimental base for the clinical treatment of sap patients with eaeen . |
to a 2 dram , clear glass
vial equipped
with a teflon - coated magnetic stir bar were added 4,4-di - tert - butyl-2,2-bipyridine ( 13.4 mg , 0.050 mmol )
and [ nicl2(dme ) ] ( 11 mg , 0.050 mmol ) .
the vial was sealed
and evacuated three times via an inlet needle and purged with argon .
the resulting suspension
was heated briefly with a heat gun until the nickel and ligand were
fully solubilized , yielding a pale green solution .
solvents were then
evaporated in vacuo to give a fine coating of the ligated nickel complex .
once dry , a phenol derivative ( 134 mg , 0.5 mmol , 1.0 equiv ) ( liquid
phenol derivatives were added with solvent ) , alkylsilicate ( 335 mg ,
0.75 mmol , 1.5 equiv ) , and [ ru(bpy)3]2pf6 ( 8.6 mg , 0.01 mmol ) were added in succession . under an inert
atmosphere , dmf ( 5 ml )
the cap was sealed with parafilm ,
and the solution was irradiated in front of a 26 w cfl bulb or blue
leds .
the temperature of the reaction was maintained at approximately
27 c via a fan .
once judged to be complete , the solution was transferred to a separatory
funnel and diluted with deionized h2o ( 20 ml ) and
et2o ( 20 ml ) .
the layers were separated , and the
aqueous layer was extracted with et2o ( 3 20
ml ) .
the combined organic layers were washed with 1 m naoh ( 30
ml ) , 1 m hcl ( 30 ml ) , and brine ( 50 ml ) . the organic
layer was dried ( mgso4 ) , and the solvent was removed in vacuo by rotary evaporation .
further purification was
accomplished via column chromatography , eluting with hexane / etoac
to give the desired compound as a light yellow oil ( 102 mg , 95% ) .
h nmr ( cdcl3 , 500 mhz ) : 7.89 ( d , j = 8.3 hz , 2h ) , 7.28 ( d , j = 8.3 hz , 2h ) ,
4.09 ( t , j = 6.5 hz , 2h ) , 2.75 ( t , j = 7.6 hz , 2h ) , 2.59 ( s , 3h ) , 2.05 ( s , 3h ) , 2.021.93 ( m ,
2h ) .
c { h } nmr ( cdcl3 , 125 mhz ) :
197.8 , 171.2 , 147.1 , 135.4 , 128.7 , 128.7 , 63.7 , 32.4 , 29.9 ,
26.7 , 21.0 . obtained as a colorless
oil ( 104 mg ,
94% ) .
h nmr ( cdcl3 , 500 mhz ) : 8.02
( d , j = 8.1 hz , 1h ) , 7.85 ( d , j =
7.9 hz , 1h ) , 7.72 ( d , j = 7.9 hz , 1h ) , 7.557.44
( m , 2h ) , 7.39 ( t , j = 7.3 hz , 1h ) , 7.32 ( d , j = 7.0 hz , 1h ) , 4.16 ( t , j = 6.5 hz , 2h ) ,
3.15 ( t , j = 7.7 hz , 2h ) , 2.152.03 ( m , 5h ) .
c { h } nmr ( cdcl3 , 125 mhz ) :
171.2 , 137.3 , 134.0 , 131.9 , 128.9 , 127.0 , 126.1 , 126.0 , 125.6 , 125.6 ,
123.7 , 64.2 , 29.6 , 29.4 , 21.1 . obtained
as a colorless oil ( 57 mg ,
50% ) .
7.837.74
( m , 3h ) , 7.62 ( s , 1h ) , 7.487.38 ( m , 2h ) , 7.32 ( dd , j = 8.4 , 1.8 hz , 1h ) , 4.13 ( t , j = 6.6
hz , 2h ) , 2.85 ( t , j = 7.6 hz , 2h ) , 2.092.01
( m , 5h ) . c { h } nmr ( cdcl3 , 125
mhz ) : 171.2 , 138.8 , 133.7 , 132.2 , 128.1 , 127.7 , 127.5 , 127.2 ,
126.6 , 126.1 , 125.3 , 64.0 , 32.5 , 30.2 , 21.1 . obtained
as a colorless oil ( 31 mg , 30% ) .
h nmr ( cdcl3 , 500 mhz ) : 7.58 ( d , j = 7.3 hz , 2h ) , 7.27
( d , j = 7.7 hz , 2h ) , 4.09 ( t , j =
6.2 hz , 2h ) , 2.75 ( t , j = 7.7 hz , 2h ) , 2.04 ( s , 3h ) ,
2.041.93 ( m , 2h ) . c { h } nmr ( cdcl3 , 125 mhz ) : 171.3 , 146.9 , 132.4 , 129.3 , 119.0 , 110.2 ,
63.5 , 32.5 , 29.8 , 21.0 .
ir ( neat ) : = 2957 , 2227 , 1734 , 1608 ,
1505 , 1453 , 1415 , 1387 , 1366 , 1234 , 1178 , 1038 , 915 , 878 , 846 , 815 ,
733 , 633 , 606 , 559 cm .
hrms ( esi ) : m / z calcd for c12h13no2na [ m + na ] 226.0844 , found 226.0834 . obtained
as a colorless
oil ( 61 mg , 42% ) .
h nmr ( cdcl3 , 500 mhz ) :
7.56 ( d , j = 8.0 hz , 2h ) , 7.31 ( d , j = 7.9 hz , 2h ) , 4.10 ( t , j = 6.5 hz , 2h ) , 2.76 ( t , j = 7.8 hz , 2h ) , 2.06 ( s , 3h ) , 2.031.95 ( m , 2h ) .
c { h } nmr ( cdcl3 , 125 mhz ) :
171.2 , 145.4 , 128.8 , 128.6 ( q , j = 32.3 hz ) , 125.5
( q , j = 3.8 hz ) , 122.3 ( q , j = 271.9
hz ) , 63.7 , 32.2 , 30.0 , 21.1 .
h nmr ( cdcl3 , 500 mhz ) : 7.497.34
( m , 4h ) ,
4.10 ( t , j = 6.5 hz , 2h ) , 2.75 ( t , j = 7.5 hz , 2h ) , 2.05 ( s , 3h ) , 2.031.93 ( m , 2h ) .
c { 1h } nmr ( cdcl3 , 125 mhz ) : 171.2 , 142.2 , 131.9 ,
129.0 , 130.9 ( q , j = 32.0 hz ) , 125.2 ( q , j = 3.7 hz ) , 124.3 ( q , j = 272.2 hz ) , 123.1
( q , j = 3.9 hz ) , 63.7 , 32.2 , 30.1 , 21.0 .
ir ( neat ) :
= 1737 , 1450 , 1388 , 1367 , 1328 , 1236 , 1199 , 1161 , 1120 , 1072 , 1039 ,
1002 , 951 , 904 , 799 , 733 , 702 , 661 , 631 , 606 cm . hrms ( esi ) :
m / z calcd for c12h13f3o2na [ m + na ] 269.0765 , found 269.0754 . obtained
as a white solid ( 63 mg , 63% ) ;
mp , 5758 c .
h nmr ( cdcl3 , 500
mhz ) : 7.89 ( d , j = 8.3 hz , 2h ) , 7.29 ( d , j = 8.2 hz , 2h ) , 2.58 ( m , 4h ) , 1.921.81 ( m , 4h ) ,
1.801.72 ( m , 1h ) , 1.501.34 ( m , 4h ) , 1.331.18
( m , 1h ) .
c { h } nmr ( cdcl3 , 125
mhz ) : 197.9 , 153.8 , 135.2 , 128.6 , 127.1 , 44.8 , 34.2 , 26.8 ,
26.6 , 26.1 . obtained as a yellow oil ( 92 mg , 92% ) .
h nmr ( cdcl3 , 500 mhz ) : 7.87 ( d , j = 8.5 hz , 2h ) , 7.30 ( d , j = 8.3 hz , 2h ) ,
2.832.75 ( m , 1h ) , 2.57 ( s , 3h ) , 2.422.34 ( m , 2h ) ,
1.841.70 ( m , 1h ) , 1.691.48 ( m , 3h ) , 1.411.34
( m , 1h ) , 1.331.18 ( m , 3h ) .
c { h }
nmr ( cdcl3 , 125 mhz ) : 197.9 , 153.5 , 134.8 , 128.5 ,
127.3 , 47.6 , 42.8 , 39.2 , 37.0 , 36.3 , 30.7 , 28.9 , 26.6 .
ir ( neat ) :
= 2949 , 2869 , 1679 , 1604 , 1568 , 1454 , 1411 , 1357 , 1308 , 1267 ,
1212 , 1185 , 1139 , 1014 , 955 , 848 , 822 , 605 , 591 , 570 cm .
hrms ( esi ) : m / z calcd for c15h19o [ m + h ] 215.1436 , found 215.1436 . obtained as a colorless oil ( 76 mg , 74% ) .
h
nmr ( cdcl3 , 500 mhz ) : 7.88 ( d , j = 8.3 hz , 2h ) , 7.27 ( d , j = 8.2 hz , 2h ) ,
2.692.62 ( t , j = 7.7 hz , 2h ) , 2.58 ( s , 3h ) ,
1.671.58 ( m , 2h ) , 1.381.24 ( m , 6h ) , 0.920.82
( m , 3h ) .
c { h } nmr ( cdcl3 , 125
mhz ) : 197.9 , 148.9 , 135.0 , 128.7 , 128.5 , 36.1 , 31.8 , 31.2 ,
29.0 , 26.6 , 22.7 , 14.2 . obtained as a colorless
oil ( 69 mg , 78% ) .
h nmr ( cdcl3 , 500 mhz ) :
7.87 ( d , j = 8.2 hz , 2h ) , 7.23 ( d , j = 8.3 hz , 2h ) ,
2.58 ( s , 3h ) , 2.53 ( d , j = 7.2 hz , 2h ) , 1.90 ( m ,
1h ) , 0.91 ( d , j = 6.6 hz , 6h ) .
c { h } nmr ( cdcl3 , 125 mhz ) : 198.0 , 147.7 ,
135.1 , 129.4 , 128.4 , 45.5 , 30.2 , 26.6 , 22.4 . obtained as a light yellow oil ( 62 mg ,
55% ) .
h nmr ( cdcl3 , 500 mhz ) : 7.88
( d , j = 8.1 hz , 2h ) , 7.27 ( d , j =
8.1 hz , 2h ) , 5.715.62 ( m , 2h ) , 2.71 ( t , j = 7.8 hz , 2h ) , 2.58 ( s , 3h ) , 2.201.99 ( m , 3h ) ,
c { h }
nmr ( cdcl3 , 125 mhz ) : 197.9 , 148.9 , 135.1 , 128.7 ,
128.6 , 127.2 , 126.4 , 38.2 , 33.4 , 33.2 , 31.9 , 28.9 , 26.6 , 25.2 . ir
( neat ) : = 3021 , 2912 , 2836 , 1680 , 1605 , 1433 , 1412 , 1356 ,
1302 , 1265 , 1181 , 1017 , 954 , 871 , 847 , 818 , 689 , 653 , 596 , 581 cm .
hrms ( esi ) : m / z calcd for c16h21o [ m + h ] 229.1592 ,
found 229.1590 . obtained as a colorless oil ( 72 mg , 75% ) .
h nmr ( cdcl3 , 500 mhz ) : 7.89 ( d , j = 8.2 hz , 2h ) , 7.28 ( d , j = 8.2 hz , 2h ) ,
3.38 ( t , j = 6.3 hz , 2h ) , 3.34 ( s , 3h ) , 2.75 ( t , j = 7.8 hz , 2h ) , 2.58 ( s , 3h ) , 1.951.85 ( m , 2h ) .
c { h } nmr ( cdcl3 , 125 mhz ) :
197.9 ,
147.9 , 135.2 , 128.8 , 128.6 , 71.7 , 58.7 , 32.4 , 31.0 , 26.6 .
ir ( neat ) :
= 2926 , 2867 , 1679 , 1606 , 1430 , 1412 , 1386 , 1357 , 1266 , 1207 ,
1181 , 1115 , 1073 , 1017 , 955 , 886 , 845 , 814 , 597 , 584 cm . hrms ( esi ) : m / z calcd for c12h17o2 [ m + h ] 193.1229 ,
found 193.1231 . obtained as a colorless oil ( 86 mg , 57% ) .
h nmr ( cdcl3 , 500 mhz ) : 9.35 ( br s , 1h ) ,
7.88 ( d , j = 8.3 hz , 2h ) , 7.29 ( d , j = 8.2 hz , 2h ) , 4.033.93 ( m , 2h ) , 3.32 ( q , j = 5.7 hz , 2h ) , 2.772.66 ( m , 4h ) , 2.58 ( s , 3h ) , 1.961.86
( m , 2h ) , 1.811.67 ( m , 6h ) .
c { h }
nmr ( cdcl3 , 125 mhz ) : 197.9 , 179.6 , 155.0 , 147.4 ,
135.2 , 128.7 , 128.7 , 43.9 , 40.0 , 39.9 , 33.3 , 30.8 , 29.2 , 28.4 , 26.7 ,
23.6 .
ir ( neat ) : = 1695 , 1680 , 1651 , 1606 , 1526 , 1453 , 1435 ,
1397 , 1358 , 1333 , 1267 , 1213 , 1179 , 1163 , 1082 , 969 , 844 , 729 , 645 ,
595 cm .
hrms ( esi ) : m / z calcd for c18h25n2o3 [ m + h ] 317.1865 , found 317.1877 . obtained
as a yellow semisolid ( 69 mg , 63% ) .
h nmr ( cdcl3 , 500 mhz ) : 8.11 ( d , j = 8.5 , 1h ) , 7.84 ( d , j = 7.8 , 1h ) , 7.68 ( d , j = 8.0 , 1h ) , 7.527.35
( m , 4h ) , 3.343.30
( m , 1h ) , 2.101.80 ( m , 5h ) , 1.751.49 ( m , 4h ) , 1.401.28
( m , 1h ) .
c { h } nmr ( cdcl3 , 125
mhz ) : 143.9 , 134.0 , 131.5 , 129.0 , 126.3 , 125.8 , 125.7 , 125.3 ,
123.3 , 122.4 , 39.4 , 34.3 , 27.4 , 26.7 . obtained as a colorless oil ( 80 mg , 72% ) .
h nmr ( cdcl3 , 500 mhz ) : 8.09 ( d , j = 8.3 hz , 1h ) , 7.83 ( d , j = 8.0 hz , 1h ) ,
7.67 ( d , j = 7.5 hz , 1h ) , 7.537.43 ( m , 2h ) ,
7.437.34
( m , 2h ) , 3.433.32 ( m , 1h ) , 2.59 ( d , j = 3.5
hz , 1h ) , 2.38 ( s , 1h ) , 2.021.94 ( m , 1 h ) , 1.791.58
( m , 4h ) , 1.571.22 ( m , 3h ) .
c { h }
nmr ( cdcl3 , 125 mhz ) : 143.2 , 134.2 , 132.1 , 128.9 ,
126.1 , 125.6 , 125.5 , 125.4 , 124.4 , 121.7 , 43.4 , 41.7 , 39.7 , 37.1 ,
36.7 , 30.6 , 29.4 .
ir ( neat ) : = 2948 , 2868 , 1739 , 1596 , 1509 ,
1452 , 1396 , 1372 , 1311 , 1297 , 1239 , 1139 , 1045 , 950 , 907 , 793 , 775 ,
731 , 648 , 560 cm .
hrms ( esi ) : m / z calcd for c17h18 [ m ] 222.1409 , found 222.1414 . obtained as a yellow oil ( 167 mg ,
97% ) .
h nmr ( cdcl3 , 500 mhz ) : 7.26
( d , j = 8.6 hz , 2h ) , 7.19 ( d , j =
8.6 hz , 2h ) , 4.09 ( t , j = 6.4 hz , 2h ) , 2.72 ( t , j = 7.6 hz , 2h ) , 2.05 ( s , 3h ) , 2.001.92 ( m , 2h ) .
c { h } nmr ( cdcl3 , 125 mhz ) :
171.2 , 148.0 , 141.9 , 130.2 , 121.4 , 118.8 ( q , j =
319.5 hz ) , 63.6 , 31.7 , 30.1 , 21.0 .
ir ( neat ) : = 1737 , 1501 , 1419 ,
1367 , 1246 , 1205 , 1179 , 1135 , 1039 , 1017 , 884 , 846 , 813 , 734 , 638 ,
606 , 568 , 535 , 517 , 498 cm .
hrms ( esi ) : m / z calcd for c12h13f3o5sna [ m + na ] 349.0333 , found
349.0334 . | photoredox / nickel
dual catalysis via single electron transmetalation
allows coupling of csp3csp2 hybridized centers under mild conditions .
a procedure
for the coupling of electron - deficient aryl triflates , -tosylates ,
and -mesylates with alkylbis(catecholato)silicates is
presented .
this method represents the first example of the use of
phenol derivatives as electrophilic coupling partners in photoredox / nickel
dual catalysis . |
the herniation can be classified into spontaneous , post - traumatic , and iatrogenic herniations .
only four cases of post - traumatic cord herniation into pseudomeningocele after nerve root avulsion injury have been reported in literature . in all cases ,
spinal cord herniations are best visualized on computed tomography ( ct ) myelography or magnetic resonance imaging ( mri ) .
we used a three dimensional ( 3-d ) turbo spin echo sequence sampling perfection with application optimized contrasts using different flip angle evolution ( space ) in our patient that provided optimal visualization of the pseudomeningocele and the herniated cord .
3-d sequences such as space or constructive interference in steady - state ( ciss ) are non - invasive alternatives to ct myelography for visualization of spinal cord herniation .
a 40-year - old male patient presented with history of progressive weakness of the right lower limb .
four years back , he had sustained injuries in a road traffic accident and was admitted with head trauma .
clinically , brachial plexus injury was suspected and nerve conduction studies suggested involvement of right c5d 1 nerve roots .
four years later , the patient developed progressive weakness of the right lower limb and started walking with a limp
. on examination , there was wasting and weakness of the right upper limb muscles .
the power in the right hip and knee flexors was grade 4/5 medical research council ( mrc ) .
mri of the patient was done on magnetom avanto 1.5 t mri ( siemens , erlangen , germany ) .
axial , sagittal , and coronal t1- and t2-weighted images of the cervical spine were acquired , which revealed the presence of lateral pseudomeningoceles at c7d1and d1d2 levels [ figure 1 ] .
dedicated 3-d space sequence and oblique coronal inversion recovery sequence of the right brachial plexus were also acquired .
there was lateral herniation of a knuckle of cord into the pseudomeningocele at d1d2 neural foramen [ figures 2 and 3 ] .
reconstruction of data set in any specified plane and superior spatial resolution of the 3-d sequence resulted in better appreciation of dura , dural defect , and the cord herniation .
coronal reconstruction of space sequence showing post - traumatic pseudomeningocele at right c7-d1 and d1-d2 neural foramina ( white arrows ) coronal reconstruction of space sequence showing herniation of knuckle of cord into the pseudomeningocele ( white arrow ) .
the dura is also well appreciated ( black arrow ) axial image of space sequence showing pseudomeningocele ( white arrow ) , cord herniation through dural defect ( black arrow ) and the dura ( black arrowhead ) due to progressive neurological deficit , a decision to treat the patient surgically was made . through a posterior approach
the essential prerequisite for extradural spinal cord herniation is the presence of a dural defect .
depending on the cause of this defect , cord herniations are classified as idiopathic , iatrogenic , and post - traumatic .
idiopathic spinal cord herniation is a fairly well - defined entity occurring predominantly in middle - aged women , typically involving the thoracic cord .
iatrogenic herniations have been reported secondary to failure of c1c2 wiring and pseudomeningocele formation secondary to surgery for cervical spine trauma and cervical spine degenerative disease .
post - traumatic herniations have been noted secondary to nerve root avulsion , penetrating injury to the dura , and after vertebral fractures .
in contrast to idiopathic herniation , diffuse myelopathy or unilateral pyramidal symptoms are the commoner presenting symptoms of post - traumatic spinal cord herniations .
iatrogenic herniation presents with either myelopathy of myeloradiculopathy . our patient presented with progressive unilateral pyramidal symptoms .
idiopathic herniations commonly occur in the dorsal cord through a ventral or venterolateral dural defect .
most of the iatrogenic herniations have been reported in the cervical spine with a dorsal or dorsolateral direction of herniation .
most of the post - traumatic cord herniations have been reported in the dorsal and lumbar spine , and the direction of herniation is either dorsal or dorsolateral .
cord herniation into nerve root avulsion pseudomeningocele is a very rare subset of post - traumatic spinal cord herniation .
only four such cases have been reported , three involving the lower cervical or upper dorsal cord and one involving the conus at upper lumbar level .
dasilva et al . , reported the first such case , in a 40-year - old male .
nineteen years after traumatic brachial plexus injury , the patient suffered brown - sequard syndrome , and herniation of cord into a pseudomeningocele arising from the right d1d2 intervertebral foramen was noted on imaging .
yokota et al . , reported a 33-year - old patient presenting with brown - sequard syndrome 14 years after traumatic left brachial plexus injury .
mri demonstrated herniation of the spinal cord into a large pseudomeningocele inside the c7d1 intervertebral foramen . in this patient ,
tanaka et al . , reported the case of a 22-year - old man presenting with a 2-year history of quadriplegia .
imaging revealed lateral pseudomeningocele arising from the right c6c7 and c7d1 intervertebral foramen and cervical spinal cord herniation into this pseudomeningocele .
ijiri et al . , reported a 72-year - old patient with spinal cord herniation associated with pseudomeningocele in the lower conus medullaris region , after nerve avulsion .
ciss imaging showed the attachment of the spinal cord to the wall of a herniated pseudomeningocele and associated syringomyelia at the level of d12 .
all the above cases presented with progressive neurological deficits years after the primary injury , which suggests that the herniation of cord was a progressive process .
etiologically , it has been suggested that contact of cord with dural defect can lead to adhesion formation , and subsequently , cerebrospinal fluid ( csf ) pulsations could progressively squeeze a segment of the cord through the dural defect .
radiologically , spinal cord kinking and neural tissue prolapse are observed in extradural cord herniations .
spinal cord atrophy , cord signal change , and syrinx formation have been also observed .
3-d sequences such as space and ciss are especially useful due to superior spatial resolution and ability to reconstruct the dataset in any specified plane .
we used 3-d space sequence with an excellent depiction of the dura , dural defect , and the herniated cord .
the diagnostic information was adequate for surgical planning , and further evaluation by ct myelography was deemed unnecessary .
we present a rare case of progressive neurological deficit due to cord herniation into post - traumatic pseudomeningocele , who was managed surgically and improved .
3-d t2-weighted sequence is a non - invasive alternative to ct myelography in the investigation of spinal cord herniation . | we present a patient with old traumatic right brachial plexus injury , who developed progressive neurological deterioration 4 years after the initial injury . on magnetic resonance imaging ( mri ) ,
herniation of the upper dorsal cord was noted into a post - traumatic pseudomeningocele .
though the herniation of cord into a post - traumatic pseudomeningocele is very rare , it should be suspected in cases of delayed progressive myelopathy .
a three dimensional ( 3-d ) t2-weighted sequence such as sampling perfection with application optimized contrasts using different flip angle evolution ( space ) or constructive interference in steady state ( ciss ) provides optimal visualization of the herniated cord and helps in surgical planning . |
premedication plays an important role in allowing a smooth separation of the child from the parent .
inadequate premedication can result in the child experiencing turbulent anaesthetic induction and adverse behavioural sequelae .
midazolam is the most common oral premedication in children and is reportedly safe and effective both at separation and induction of anaesthesia .
nasal premedication with midazolam and dexmedetomidine has been studied as alternatives to oral premedication with comparable results .
however , it has poor oral bioavailability and absorption is better through the mucosal routes .
the primary objective of our study was the comparison of sedation scores at separation and induction between orally administered midazolam and intranasal dexmedetomidine .
this was a prospective randomised , double - blinded , comparative study conducted in sixty children of the american society of anesthesiologist ( asa ) physical status 1 and 2 , aged 212 years undergoing elective surgical procedures , during february 2012april 2014 . in a previous study by yuen et al .
, 21.9% patients in midazolam group and 75% in dexmedetomidine group had satisfactory sedation scores at separation .
targeting the same difference , with 95% confidence level and 80% power , minimum sample size was calculated as 17 in each group .
after approval from the institutional ethics committee , sixty children presenting for surgery were randomised to receive either dexmedetomidine or midazolam . parental refusal for consent , children with a significant history of allergic disorders , asa iii or higher , those on long - term therapy with theophylline or hepatic enzyme inducing drugs were excluded .
the premedication was administered 60 min before induction of anaesthesia , in the holding area in the presence of one of the parents ; after a random allocation to one of the two groups using a computer - generated sequence of random numbers in blocks of five . group
a received midazolam 0.5 mg / kg ( in 15 mg / kg acetaminophen syrup ) and intranasal placebo ( 0.4 ml normal saline ) .
group b received intranasal dexmedetomidine 1 g / kg 0.4 ml normal saline and 15 mg / kg oral acetaminophen syrup . owing to the non - availability of oral midazolam at the time of this study ,
paracetamol syrup ( 250 mg/5ml ) was used to mask the bitterness of the study medication and was used at a dose of 15 mg / kg .
group b received paracetamol syrup at 15 mg / kg with distilled water to a volume of 5 ml ( 1 tsp ) .
intranasal dexmedetomidine was prepared from the 100 g / ml of parenteral preparation of the drug .
normal saline was added to the calculated dose to make a final volume of 0.4 ml .
intranasal drug was dripped into both nostrils at 0.2 ml per nostril using 1ml syringe with child in recumbent position .
individuals who were not involved in observation or administration of anaesthesia for children prepared the study drugs .
heart rate ( hr ) , oxygen saturation and blood pressure were measured before and every 15 min after intranasal drug administration until transfer to the operation room .
the sedation status and behaviour scores were assessed every 15 min by a blinded observer using a 6-point sedation scale and a 4-point behaviour score [ table 1 ] . at the time of induction of anaesthesia , sedation and
behaviour score at the time of awakening was evaluated using a 4-point wake up score [ table 1 ] . for statistical analysis
sedation , behaviour and wake up behaviour scores data analysis was performed using statistical package for the social sciences spss statistics for windows , version 20.0 software ( ibm , bengaluru , india ) .
student 's t - test was applied to compare age and weight , and the chi - square test was applied to compare sex distribution .
both groups were comparable with respect to age , sex , weight , duration and type of surgical procedures [ table 2 ] .
the average time to induction from premedication was also comparable in groups a and b ( 55.8 6.2 min vs. 56.3 5.7 min ) .
comparison of baseline mean arterial blood pressure ( map ) , hr and oxygen saturation in groups a and b showed no difference between groups .
however , intraoperatively , significantly higher values of map and hr were seen in group a at 30 , 45 and 60 min [ figures 1 and 2 ] .
comparison of demography and types of surgery distribution of mean arterial pressure distribution of mean heart rate the median sedation scores were higher in group a at separation ( 5 vs. 2 ) and at the time of induction ( 6 vs. 3 ) which was statistically significant ( p < 0.001 ) . compared to group a , more patients in group b were satisfactorily sedated at the time of separation ( 93.3% vs. 26.7% ) and induction [ 83.3 vs. 23.3% , table 3 and figure 3 ] .
comparison of sedation , behaviour and wake up scores distribution of sedation and behaviour scores the median behaviour score in group a was higher than group b at separation and induction ( 2 vs. 1 ) .
they were both in the range considered satisfactory ( 2 ) and were not significantly different between the groups at separation and induction [ table 3 and figure 3 ] .
the median wake up score at the time of extubation was similar in both groups .
73.3% of patients of group a and 80.0% of group b had satisfactory scores and were comparable among groups [ table 3 and figure 3 ] .
fear of painful or unpleasant procedures and separation from parents may result in untoward psychological effects .
although oral premedication is effective , the bitterness may cause retching and vomiting resulting in inadequate drug dosing .
midazolam has been established as an effective oral premedication in children . in doses of 0.5 mg / kg , it has been found to offer adequate sedation at separation and induction . it does not prolong recovery from anaesthesia or discharge from day care procedures .
yuen et al . had concluded that an oral dose of 0.5 mg / kg midazolam was satisfactory as a premedicant and that the effect correlated more closely with 1 g / kg dexmedetomidine rather than with 0.5 g / kg dexmedetomidine intranasally .
akin et al . compared the effects of intranasally administered midazolam versus dexmedetomidine administered intranasally on premedication in children undergoing elective adenotonsillectomy .
they concluded that both drugs were equally effective in decreasing separation anxiety in children although mask induction appeared to be superior in the midazolam group .
dexmedetomidine is a newer selective alpha-2 agonist with a site of action at the locus coeruleus .
it inhibits presynaptic release of norepinephrine that is responsible for its sedative and hypnotic effects .
the analgesic effects occur on account of activation of alpha-2 adrenoceptor in the descending medulla - spinal noradrenergic pathway .
bradycardia and hypotension occur on account of post - synaptic activation of alpha-2 receptors in the central nervous system .
the finding of electroencephalogram activity similar to natural sleep supports the easy arousability from its effects .
while there is sufficient literature supporting the use of oral midazolam , less information is available on the use of dexmedetomidine .
we found that a dose of 1 g / kg dexmedetomidine resulted in better sedation scores and behavioural scores at separation and induction in comparison to oral midazolam .
dexmedetomidine has limited bioavailability administered orally as compared to the nasal route ; hence , we chose to compare the nasal administration of dexmedetomidine with the oral midazolam which has been accepted as a standard premedicant .
although dexmedetomidine has good bioavailability through the mucosal route allowing the use of oral preparation , the effects would be manifested only if the patient made an effort to retain the drug in the mouth without swallowing .
the nasal route of dexmedetomidine is not associated with irritant side effects as with intranasal midazolam .
the advantages of dexmedetomidine are the absence of respiratory depression and analgesic effects ; however , the drawback could be a longer onset time for sedation in comparison with midazolam .
a number of studies have compared the analgesic effects of oral midazolam with hydroxyzine and chloral hydrate .
the utility of midazolam has been well established although some workers have observed better sedation with chloral hydrate in comparison with midazolam .
we evaluated a lower dose of dexmedetomidine versus an accepted dose of oral midazolam to assess its applicability in paediatric premedication .
the average time from drug administration to induction in our study group was 45 min in two - third of our patients .
this is similar to the study by yuen et al . who concluded that at 1 g / kg dose intranasal dexmedetomidine produced satisfactory sedation scores at 30 , 45 and 60 min after administration .
we found a similar profile in our patients ; although we did not measure the onset of sedation , the haemodynamic responses showed lowering of hr and blood pressure at 30 min after drug administration that correlated with the onset of sedation in our patients .
yuen et al . evaluated the efficacy of intranasal dexmedetomidine in two doses 1.0 and 1.5 g / kg in 18 healthy adult volunteers and compared to a placebo .
they concluded that the nasal route is convenient and safe for use and sedation occurred in 45 min with peak effect between 90 and 150 min after administration .
our study was designed to primarily evaluate the efficacy of dexmedetomidine in comparison to midazolam as a premedicant in the profile of paediatric patients presenting to us for surgery .
the objectives were the safety , comfort and ease of separation of the child in the holding area and behaviour at induction .
we did not focus on the recovery characteristics and did not control the type and duration of surgery and anaesthesia .
we also did not assess the analgesic benefits resulting in lesser fentanyl or propofol usage during anaesthesia .
the scoring scales for sedation , behaviour and wake up were incorporated from the study by yuen et al . in this study
, we found that children receiving dexmedetomidine had significantly lower hr and blood pressure at 30 , 45 and 60 min after drug administration in comparison with children receiving midazolam . a previous study comparing the additive effects of dexmedetomidine versus fentanyl with propofol showed that significant lowering in hr and map from baseline occurred in both groups .
a study using intranasal dexmedetomidine premedication in obese adults found that dexmedetomidine reduced the hr but not map in comparison to alprazolam .
the fall in hr due to dexmedetomidine has been explained by its effect on sympathetic outflow and reducing levels of epinephrine and norepinephrine .
the fall in blood pressure and hr was within acceptable limits for the age of the child and did not require the use of chronotropic agents , fluids or inotropes .
this could not be controlled most of the time ; however , the dexmedetomidine group appeared to be sedated from 30 min after drug administration .
schmidt et al . have documented decreased sympathetic stimulation and decreased post - operative pain in the groups receiving dexmedetomidine .
have shown an anaesthetic sparing effect of dexmedetomidine in patients who received infusions of dexmedetomidine undergoing spine surgery .
we did not assess opioid - sparing effects of dexmedetomidine and did not analyse the surgical requirements of analgesic .
the management of intraoperative conditions was at the discretion of the anaesthesiologist who managed the case .
it is possible that intraoperative requirements of fentanyl and propofol were lesser in the dexmedetomidine group .
all children in both groups had an uneventful recovery , and wake up scores were not different between the groups .
cardiovascular stability of dexmedetomidine has been documented in several studies ; doses of 2 g / kg intranasally in children undergoing non - complex cardiac surgeries have been shown to be safe .
oral dexmedetomidine was also found to have better mask acceptability in children undergoing cardiac surgery . although the map and hr were lower in the dexmedetomidine group , this was still within acceptable limits for the age of the child .
the limitations in the study included failure to correlate the effects of dexmedetomidine premedication on the analgesic and anaesthetic requirements during surgery .
the drugs were administered at 4560 min before surgery ; however , scheduling could not take place at the time proposed in some instances .
we also did not monitor the time to onset of sedation in our study group , and the types of surgeries included were variable .
the inclusion of a similar profile of surgeries along with monitoring analgesic requirements can document the additional intraoperative effect of the premedicant drugs .
intranasal dexmedetomidine at a dose of 1 g / kg produces superior sedation and behavioural scores at separation and induction but normal wake up scores in comparison to oral midazolam in paediatric patients .
| background and aims : premedication is an integral component of paediatric anaesthesia which , when optimal , allows comfortable separation of the child from the parent for induction and conduct of anaesthesia .
midazolam has been accepted as a safe and effective oral premedicant .
dexmedetomidine is a selective alpha-2 agonist with sedative and analgesic effects , which is effective through the transmucosal route .
we compared the efficacy and safety of standard premedication with oral midazolam versus intranasal dexmedetomidine as premedication in children undergoing elective lower abdominal surgery.methods:this was a prospective randomised double - blinded trial comparing the effects of premedication with 0.5 mg / kg oral midazolam versus 1 g / kg intranasal dexmedetomidine in children between 2 and 12 years undergoing abdominal surgery .
sedation scores at separation and induction were the primary outcome measures .
behaviour scores and haemodynamic changes were secondary outcomes .
student 's t - test and chi - square were used for analysis of the variables.results:sedation scores were superior in group b ( dexmedetomidine ) than group a ( midazolam ) at separation and induction ( p < 0.001 ) .
the behaviour scores at separation , induction and wake up scores at extubation were similar between the two groups .
the heart rate and blood pressure showed significant differences at 15 , 30 and 45 min in group b but did not require pharmacological intervention for correction.conclusion:intranasal dexmedetomidine at a dose of 1 g / kg produced superior sedation scores at separation and induction but normal behavioural scores in comparison to oral midazolam in paediatric patients . |
study subjects were participants in a comprehensive health examination conducted at the hitachi health care center , ibaraki , japan , during which colorectal cancer screening and , on request , abdominal ct scanning were performed .
abdominal ct scanning was introduced to encourage changes in lifestyle , such as diet and physical activity , by showing examinees a graphic image , together with estimated data , of their own abdominal fat accumulation . in practice
, it was offered mainly to individuals who underwent chest ct scanning for the screening of lung cancer .
nearly one - third of all individuals who underwent screening chose to receive abdominal ct assessment .
compared with men who did not , those who underwent abdominal ct scanning were older ( 53 vs. 46 years ) , were more likely to be past smokers ( 35 vs. 22% ) , and tended to have a higher bmi ( 23.9 vs. 23.6 kg / m ) .
in contrast , the two groups were similar in terms of alcohol drinking ( > 1 go [ 23 g ethanol]/day : 32 vs. 29% ) .
a go is a conventional unit of alcohol intake in japan . during the 3-year period from april 2004 to march 2007 ,
47,224 examinees underwent fecal occult blood testing , which is specified as the standard procedure for colorectal cancer screening in the japanese guidelines . owing to limitations in colonoscopy resources , individuals with a positive blood test
were first invited to receive a barium enema in the health center , and only those with suspected polyp lesions were referred to a medical specialist for detailed examination by colonoscopy . of 3,521 ( 8% ) who had a positive test result ,
of these , 491 ( 28% ) with a finding suggesting colorectal neoplasia were referred to local clinics or hospitals for confirmation .
of the 280 patients who were notified by the physicians consulted that they had colorectal neoplasia , the present case series consisted of the 86 with histologically confirmed adenoma and 22 with early - stage colorectal cancer ( carcinoma in situ or cancer invading within the submucosa ) who received abdominal ct scanning at the time of the health checkup . among patients with adenomas of known size ( n = 82 ) ,
regarding the location of cancer , 5 cases were in the ascending colon , 2 were in the transverse colon , 13 were in the sigmoid colon , 1 was in the rectum , and 1 was not specified .
for each case subject , three control subjects matched by year of examination , sex , and age ( same age ) were randomly selected from among examinees who had undergone abdominal ct measurement and had a negative fecal occult blood test . no case or control
informed consent was obtained from each examinee regarding the use of his or her data for research purposes .
the protocol of the present study was approved by the ethics committee of the hitachi health care center .
measurement of abdominal fat area with a ct scanner has been detailed elsewhere ( 17 ) . in brief ,
single slice imaging was done at the level of the umbilicus in the supine position using a redix turbo ct scanner ( hitachi medico , tokyo , japan ) .
imaging conditions , which have changed since 2004 , were 120 kv , 50 ma , and a 5-mm slice thickness .
visceral fat area , subcutaneous fat area , and waist circumference were calculated using the pc software application fatpointer ( hitachi medico ) .
height and weight were measured using an automated scale ( tanita bf-220 ) with the patient wearing a light gown .
bmi was calculated as the weight in kilograms divided by the square of height in meters .
fasting plasma glucose was measured by the glucose electrode technique using an adams glucose ga-1170 ( arkray ) .
fasting serum immunoreactive insulin ( microunits per milliliter ) was determined by an immunoenzymatic method using the axsym insulin assay ( abbott ) .
homeostasis model assessment of insulin resistance ( homa - ir ) , an index of insulin resistance , was calculated as fasting glucose multiplied by fasting insulin divided by 405 .
participants entered their responses to the questionnaire directly into a computer using a custom - designed data entry system . regarding smoking ,
the questionnaire inquired about smoking status and for ever - smokers it inquired about the duration and intensity of smoking . for alcohol consumption ,
the frequency of drinking and the amount of alcohol consumed per session was assessed in terms of go .
subject characteristics were compared between case subjects with adenomas and their matched control subjects and between case subjects with cancer and their matched control subjects . in control subjects ,
pearson correlation coefficients were calculated to examine the linear associations between visceral fat area and other exposure variables .
conditional logistic regression was used to assess the association of various obesity indexes ( abdominal total fat mass , visceral fat area , subcutaneous fat area , waist circumference , and bmi ) and measures of insulin resistance ( insulin , glucose , and homa - ir ) with colorectal neoplasia . odds ratios ( ors ) and 95% cis for the prevalence of colorectal adenoma or cancer were calculated for the second and third ( highest ) tertiles of exposure , with the lowest tertile used as reference .
cutoff values for the exposure tertile were determined based on the distribution among control subjects for colorectal adenomas and cancer , respectively .
analyses were performed with and without adjustment for smoking ( lifetime nonsmoker , ever - smoker with 1600 cigarette - years , or ever - smoker with > 600 cigarette - years ) , and alcohol consumption ( nondrinker , drinker consuming 1 go / day , or drinker consuming > 1 go / day ) . in analyses for the relation of insulin resistance to visceral fat area and blood markers ,
all analyses were performed using sas ( version 10 ; sas institute , cary , nc ) .
measurement of abdominal fat area with a ct scanner has been detailed elsewhere ( 17 ) . in brief ,
single slice imaging was done at the level of the umbilicus in the supine position using a redix turbo ct scanner ( hitachi medico , tokyo , japan ) .
imaging conditions , which have changed since 2004 , were 120 kv , 50 ma , and a 5-mm slice thickness .
visceral fat area , subcutaneous fat area , and waist circumference were calculated using the pc software application fatpointer ( hitachi medico ) .
height and weight were measured using an automated scale ( tanita bf-220 ) with the patient wearing a light gown .
bmi was calculated as the weight in kilograms divided by the square of height in meters .
fasting plasma glucose was measured by the glucose electrode technique using an adams glucose ga-1170 ( arkray ) .
fasting serum immunoreactive insulin ( microunits per milliliter ) was determined by an immunoenzymatic method using the axsym insulin assay ( abbott ) .
homeostasis model assessment of insulin resistance ( homa - ir ) , an index of insulin resistance , was calculated as fasting glucose multiplied by fasting insulin divided by 405 .
participants entered their responses to the questionnaire directly into a computer using a custom - designed data entry system . regarding smoking ,
the questionnaire inquired about smoking status and for ever - smokers it inquired about the duration and intensity of smoking . for alcohol consumption ,
the frequency of drinking and the amount of alcohol consumed per session was assessed in terms of go .
subject characteristics were compared between case subjects with adenomas and their matched control subjects and between case subjects with cancer and their matched control subjects . in control subjects ,
pearson correlation coefficients were calculated to examine the linear associations between visceral fat area and other exposure variables .
conditional logistic regression was used to assess the association of various obesity indexes ( abdominal total fat mass , visceral fat area , subcutaneous fat area , waist circumference , and bmi ) and measures of insulin resistance ( insulin , glucose , and homa - ir ) with colorectal neoplasia .
odds ratios ( ors ) and 95% cis for the prevalence of colorectal adenoma or cancer were calculated for the second and third ( highest ) tertiles of exposure , with the lowest tertile used as reference .
cutoff values for the exposure tertile were determined based on the distribution among control subjects for colorectal adenomas and cancer , respectively .
analyses were performed with and without adjustment for smoking ( lifetime nonsmoker , ever - smoker with 1600 cigarette - years , or ever - smoker with > 600 cigarette - years ) , and alcohol consumption ( nondrinker , drinker consuming 1 go / day , or drinker consuming > 1 go / day ) . in analyses for the relation of insulin resistance to visceral fat area and blood markers ,
all analyses were performed using sas ( version 10 ; sas institute , cary , nc ) .
table 1 shows patient characteristics for colorectal adenoma and cancer and their respective control subjects .
patients with colorectal adenomas were more likely to smoke and consume alcohol heavily than their matched control subjects .
in contrast , they had levels of obesity and markers of insulin or insulin resistance similar to those of control subjects .
patients with colorectal cancer were more likely to be smokers and alcohol drinkers than their matched control subjects and on average had a greater bmi , waist circumference , and visceral and subcutaneous fat area than control subjects .
markers of insulin resistance were higher among patients with colorectal cancer than among their matched control subjects . in control subjects ,
visceral fat mass was highly correlated with other measures of obesity ( pearson correlation coefficients : waist circumference 0.82 , bmi 0.68 , and subcutaneous fat mass 0.58 ) , moderately with insulin ( 0.44 ) , and weakly with fasting glucose ( 0.18 ) .
characteristics of study subjects data are means sd unless stated otherwise . as shown in table 2 , the odds of having colorectal cancer were increased in subjects with a higher visceral fat mass , with multivariable - adjusted ors ( 95% ci ) for the lowest through highest tertiles of 1 ( reference ) , 2.17 ( 0.4510.46 ) , and 5.92 ( 1.2228.65 ) , respectively ( ptrend = 0.02 ) .
in contrast , subcutaneous fat mass was materially unrelated to colorectal cancer prevalence , with a multivariable - adjusted or for the highest versus lowest tertile of 1.08 ( 0.294.00 ) .
higher levels of bmi or waist circumference were also associated with increased prevalence of colorectal cancer , with multivariable - adjusted ors ( 95% ci ; ptrend ) for the highest versus lowest tertile of visceral fat area of 4.38 ( 0.8223.25 ; 0.09 ) and 2.03 ( 0.577.25 ; >
, no association was seen with any measure of obesity , including visceral fat mass .
associations of measures of obesity with the prevalence of adenoma and cancer in the colorectum * measured by abdominal ct at the umbilical level in supine position .
additionally adjusted for bmi . as shown in table 3 , the odds of colorectal cancer tended to increase with increasing fasting plasma glucose concentration and , to a lesser extent , with increasing fasting plasma insulin concentration and homa - ir .
multivariable ors ( 95% ci ; ptrend ) for the highest versus lowest tertiles of glucose , insulin , and homa - ir were 4.40 ( 0.9919.59 ; 0.04 ) , 1.84 ( 0.477.15 ; >
additional adjustment for bmi attenuated the association with insulin and homa - ir but did not greatly change that with glucose .
in contrast , no measurable association was seen between colorectal adenoma and any of the three blood measurements .
associations of glucose , insulin , and homa - ir with the prevalence of adenoma and cancer in the colorectum adjusted for smoking and alcohol drinking .
among participants in a health screening program who underwent abdominal ct measurement , we found increased odds of early colorectal cancer in subjects with greater visceral fat mass , but not in those with greater subcutaneous fat mass .
in contrast , these associations were not observed for colorectal adenoma . to our knowledge , this study is the first to provide direct evidence of an association between visceral adiposity and colorectal cancer risk .
the present association between greater visceral fat area and increased prevalence of colorectal cancer is consistent with earlier epidemiological data showing a link between colorectal cancer and waist circumference or waist - to - hip ratio ( 4,5 ) .
this finding indicates that visceral but not subcutaneous adipose tissue disposition is involved in the promotion of colorectal carcinogenesis . among studies that have measured visceral fat area using ct scanning in association with colorectal neoplasia ( 610 )
contrary to expectations , this study showed a higher prevalence of colorectal cancer in subjects with low rather than high visceral fat area .
the authors speculated that this finding might have been due to weight loss induced by cancer progression . in our study ,
cancer in subjects included in the analysis was all screening - detected and early stage , and thus the results were unlikely to have been influenced by cancer - induced weight loss .
in contrast to the positive finding for colorectal cancer , we observed no association between any measure of obesity , including visceral fat area , and the prevalence of colorectal adenoma .
findings among studies that have measured abdominal fat area using ct are mixed : a significant positive association with visceral adiposity in a japanese study ( 7 ) was subsequently both supported ( 9 ) and challenged ( 8,10 ) .
further , in an ancillary study to the polyp prevention trial ( 6 ) , visceral fat area measured on ct was not associated with adenoma recurrence .
the reason for this discrepancy among adenoma studies is not clear . given that smoking is a strong determinant of both the prevalence of colorectal adenoma ( 18 ) and body weight ( 19 ) , the null finding in our study might be attributable , at least in part , to the high proportion of subjects with a history of smoking ( 73% ) .
the relation of obesity measures to colorectal adenoma might only be detected in populations with no or low - level exposure to smoking , as suggested by a positive finding among nonsmokers ( 7 ) .
alternatively , if the major role of obesity in colorectal carcinogenesis is to enlarge existing adenomas , the present null finding may be ascribable to the small number of case subjects with large adenomas ( 10 mm : n = 15 ) .
the insulin hypothesis has been proposed to explain the association between obesity or visceral adiposity and colorectal cancer ( 12,13 ) .
prospective studies have shown an increased risk of colorectal cancer among individuals with higher levels of postprandial insulin ( 4 ) , c - peptide ( 20,21 ) , a measure of average insulin secretion , and fasting glucose ( 4 ) at baseline , although the association with fasting insulin was less clear ( 4 ) . in accordance with these data , we observed an increase , albeit without statistical significance , in the odds of colorectal cancer in subjects with higher levels of markers of insulin resistance , particularly fasting glucose . with regard to colorectal adenoma , although some studies have demonstrated an elevated risk among individuals with higher levels of fasting insulin ( 22 ) or fasting glucose ( 23 ) , our data do not support a role of insulin resistance in the development of colorectal adenoma .
recently , tabuchi et al . ( 24 ) reported similar findings in health checkup participants who underwent total colonoscopy : hyperglycemia was associated with an increased risk of colorectal cancer , but not with colorectal adenoma . similarly , chung et al .
( 25 ) demonstrated that glucose concentrations were more strongly associated with colorectal cancer than with adenoma .
further studies are required to determine whether insulin resistance and resulting conditions , including hyperinsulinemia and hyperglycemia , are more strongly involved in the development of cancer than in that of adenoma .
the present study has several methodological advantages over previous studies that directly measured visceral adiposity accumulation using ct .
control subjects were randomly selected from a population of screening participants , among whom the cases arose , and abdominal ct measurement was done before the diagnosis of colorectal neoplasia , precluding the possibility of bias in the selection of control subjects and assessment of exposure , both of which are major concerns in case - control studies .
first , the number of case subjects with colorectal cancer was small ( n = 22 ) .
nevertheless , we were able to detect a statistically significant association with visceral fat area .
second , although the control subjects were selected from among examinees with a negative screening result , they were not confirmed to be polyp free and thus may have included patients with colorectal adenomas , leading to attenuation of the association . given the low probability that the control series included subjects with undetected cancer , however , we believe that the present estimates for cancer were not subject to serious bias .
third , physical activity , a convincing protective factor for colorectal cancer ( 1 ) , was not controlled for in the analysis ; in any case , such control would not be methodologically valid if physical activity exerted an anticarcinogenic effect by decreasing visceral fat .
finally , because the majority of study subjects were male employees working for a large - scale company in japan , the results may not be generalizable to populations with different backgrounds . in summary ,
the present study of screening participants who underwent abdominal ct scanning provides direct evidence for the hypothesis that visceral fat accumulation and insulin resistance promote carcinogenesis of the colorectum . because adipose tissue secretes various hormones that may play a role in the development and progression of cancer not only through their effect on insulin resistance but also by directly controlling cell proliferation
, the biological mechanisms linking visceral fat disposition to cancer risk should be further explored . | objectivealthough abdominal obesity and related metabolic abnormalities are hypothesized to promote colorectal carcinogenesis , direct confirmation of this effect is required . here
, we examined the relation of early - stage colorectal neoplasia to visceral fat area and markers of insulin resistance.research design and methodssubjects were participants in a comprehensive health screening conducted at the hitachi health care center , ibaraki , japan . during a 3-year period ( 20042007 ) , a total of 108 patients with early - stage colorectal neoplasia , including 22 with early cancer , were identified among individuals who received both colorectal cancer screening and abdominal computed tomography scanning .
three control subjects matched to each case subject were randomly selected from those whose screening results were negative .
conditional logistic regression analysis was used to examine the association of measures of obesity and markers of insulin resistance with colorectal neoplasia , with adjustment for smoking and alcohol drinking.resultsvisceral fat area , but not subcutaneous fat area , was significantly positively associated with colorectal cancer , with odds ratios ( 95% ci ) for the lowest to highest tertile of visceral fat area of 1 ( reference ) , 2.17 ( 0.4510.46 ) , and 5.92 ( 1.2228.65 ) , respectively ( ptrend = 0.02 ) . markers of insulin resistance , particularly fasting glucose , were also positively associated with colorectal cancer risk . in contrast , no associations were observed for colorectal adenomas.conclusionsthese results suggest that visceral adipose tissue accumulation and insulin resistance may promote the development of early - stage cancer but not adenoma in the colorectum . |
arguably one of the
most important conceptual breakthroughs in modern structural chemistry
was robson s geometric interpretation of the structures of
coordination polymers as connected nets of nodes and linkers .
the language of reticular chemistry that developed
from that point has enabled the informed design of an extraordinarily
large family of framework materials and coordination polymers , notably
including metal organic frameworks ( mofs ) and covalent organic frameworks ( cofs ) .
the
basic design approach as it is currently used reflects the realization
that specific combinations of node and linker geometries can usually
lead to only a finite number of different possible structures ; hence , control over the former ( e.g. , the choice
of transition - metal elements or clusters with specific coordination
geometry preferences and linkers with characteristic binding modes )
enables control over the latter .
the abstraction of linker molecules to rodlike connections carries
with it the implication that linker shape and chemistry bear little
influence on the resulting framework structure .
certainly this is
sometimes true , but for many coordination polymers
the packing motifs and stereochemical preferences of the organic linkers
often play a defining role in directing structure .
this may be as
simple as linker functionalization preventing network interpenetration
but the effects can also be altogether more drastic .
for example , if the terephthalate linker connecting chains
of corner - sharing alo4(oh)2 octahedra in the
porous breathing
mil-53 ( ref ( 10 ) ) is swapped for the longer
and slightly bent 4,4-azobenzenedicarboxylate then the nonporous
layered mil-129 forms instead , where the ligands now adopt a
stacking motif .
it is in this context
that we have developed a particular interest in coordination polymers
assembled using the dicyanometallate linker [ nc m cn ] ( m = cu , ag , au ) .
the preferred coordination mode of this linker is linear , and
so it combines the nanometre scale of the organic linkers used to
build mofs with the geometric flexibility of an atomically thin linker .
in many ways
, dicyanometallates can be considered the natural extension
of oxide frameworks ( e.g. , perovskites ) and cyanide frameworks ( e.g. ,
prussian blues ) [ figure 1 ] .
increasing scale of oxide ( blue ) , cyanide ( green ) , and dicyanometallate
( red ) architectures illustrated with the perovskite ( a ) and cristobalite
( b ) structures . increasing the length of
the linker dramatically increases
the available volume for interpenetration , extra - framework cations ,
and/or neutral guests .
node metal ( gray ) , oxygen ( red ) , nitrogen ( blue ) ,
carbon ( black ) , and linker metal ( yellow ) . like their oxide and cyanide cousins , dicyanometallate frameworks
examples include
colossal positive and negative thermal expansion in silver hexacyanocobaltate
( co[ag(cn)2]3 ) ; giant negative compressibility and
guest - dependent luminescence in zn[au(cn)2]2 ; piezoelectric behavior in kco[au(cn)2]3 ; tunable room - temperature
luminescence in la[auxag1x(cn)2]3h2o ; vapochromism in cu[au(cn)2]2 ; heat- , light- , and pressure - induced
spin - crossover in fe(pyz)[ag(cn)2]2pyz
( pyz = pyrazine ) ; and strain - free charge
storage in kxfe[ag(cn)2]3 . in each of these
cases , the geometric flexibility of the dicyanometallate linker plays
a key role and
so there is good reason to expect similarly intriguing
functional responses among as - yet - unrealised dicyanometallate frameworks .
yet , in this context of developing new functional dicyanometallates ,
what is conspicuously absent is an overview of the structural chemistry
of the family of the sort that has been so enabling in , e.g. , oxide and cyanide framework chemistry .
table 1 summarizes , to the best of our knowledge ,
the various dicyanometallate frameworks reported to date . for simplicity
,
we have focused on systems expressible in the form ambxn{guest}. in this representation ,
a denotes an extra - framework cation ( if present ) , b is the framework
node cation , x is a dicyanometallate anion , and we allow for the presence
of solvent or other neutral molecules within the structure . what is
immediately clear is that the family is extremely diverse , and encompasses
much of the compositional and topological complexity responsible for
the rich structural chemistry of conventional ceramic frameworks .
in addition , and as in the transition - metal formate
mof perovskites , there is the intriguing capacity to incorporate molecular cations
as a means of balancing framework charge .
there is strong current
interest in these systems as a direct result of the recent discovery
of unconventional semiconductor behavior in the [ ch3nh3]pbi3 family of solar cell materials .
ordering processes involving molecular cations can be fundamentally
different from those involving spherically symmetric or dipolar cations ,
as in conventional oxide ceramics .
the
relationship between molecular geometry and host framework topology
remains relatively unexplored particularly in dicyanometallates .
likewise , the nanometre node separation in dicyanometallate
frameworks potentially allows for the incorporation of molecular cations
capable of complex supramolecular interactions .
formulae
including au / ag indicate that both the pure gold- and silver - containing
compounds are known .
newly synthesized compounds reported as part
of our study are indicated by an asterisk .
abbreviations used : l = n-(3-aminopropyl)-5-bromosalicylaldimine ;
2,2-bpy = 2,2-bipyridine ; et = c2h5 ; 3-cnpy = 3-cyanopyridine ; tpt = 2,4,6-tris(pridyl)triazine ;
me = ch3 ; pmd = pyrimidine ; py = pyridine ; 3-phpy = 3-phenylpyridine ;
deas = 4-diethylaminostilbazole ; dmf = n , n-dimethylformamide ; dmso
= dimethyl sulfoxide ; nit4py = 2-(4-pyridyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide ;
phen = 1,10-phenanthroline ; 2,2-bpe = 2,2-bis(pyridyl)ethene ;
3-fpy = 3-fluoropyridine ; 3-mepy = 3-methylpyridine ; 4-mepy
= 4-methylpyridine ; 3-clpy = 3-chloropyridine ; 3-brpy
= 3-bromopyridine ; 3-ipy = 3-iodopyridine ; bim = benzimidazolate ;
pyz = pyrazine ; 4,4-bpy = 4,4-bipyridine ; bpmp = 1,4-bis(pyridin-4-ylmethyl)piperazine ;
hydeten = n-(2-hydroxyethyl)ethylenediamine ) ;
nit3py = 2-(3-pyridyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide ;
ppn = bis(triphenylphosphine)iminium cation ; bu = n - c4h9 .
the first
aim is to report the structures of a number of new dicyanometallate
frameworks which contain molecular cations . on the one hand ,
these
are of fundamental interest in terms of their structural chemistry ,
but on the other hand they may prove important by virtue of their
conceptual relationship to other molecular analogues of conventional
ceramics , including the semiconducting lead iodide family . the second and
ultimately more important aim of our study is to suggest an
organization of the structures of known dicyanometallates that helps
rationalize the structural diversity of this family . in doing
so we
begin to develop a set of design strategies that will ultimately allow
rational control over framework structure in precisely the way envisaged
by robson s original study .
our starting point is to describe the various new dicyanometallates
we have prepared . motivated by the desire to introduce molecular extra - framework
cations , our typical synthetic strategy was to combine a simple transition - metal
salt with a specially prepared molecular salt of a given dicyanometallate
anion .
the latter acts as the source of molecular cation in the framework
that ultimately forms .
we report eight new compounds , which adopt
a total of five structure types and correspond to the three fundamental
compositions abx2 , a0.5bx2.5 , and
abx3 .
the first
system we describe was actually the fortuitous result of our attempts
to prepare [ net4][ag(cn)2 ] as a dicyanometallate
precursor for subsequent framework formation . the combination of [ net4]cl or [ net4]br with kag(cn)2 yields
the same dicyanometallate framework [ net4]ag[ag(cn)2]2 ( 1 ) in which silver(i )
cations participate as both node and linker .
the dual roles of silver(i )
in this system are distinguishable by both coordination geometry and
cyanide binding orientation : the node cations are tetrahedrally coordinated
by the n cyanide terminus and the linker cations are linearly coordinated
by the c cyanide terminus .
the crystal structure is illustrated in figure 2 , and relevant crystallographic
details are summarized in table 2 .
three distorted diamondoid networks interpenetrate ,
with each [ net4 ] cation contained within a
cavity formed by the combination of all three networks .
representations
of the crystal structures of the various new dicyanometallate frameworks
reported in this study .
the top panels show structures in thermal
ellipsoid representation ( probability = 50% , or 90% where indicated
with an asterisk ) .
each image is represented at the same absolute
scale ; extra - framework cations and solvent molecules have been omitted
for clarity .
the bottom panels show schematic representations of the
corresponding network topologies . in each case
a single extra - framework
cation is shown in space - filling representation to illustrate the
relationship between cation shape and network topology .
an intriguing property of this system is the existence
of a phase transition on cooling below 235 k. this transition which
will be described in detail elsewhere involves the condensation
of a soft phonon mode at an incommensurate wave - vector .
the study
of classical soft - mode transitions in extended frameworks is a relatively
unexplored field ; the purpose of drawing attention
to this transition here is simply to highlight that the dynamical
properties of these materials may , in due course , be of interest in
their own right .
reaction of [ nbu4][ag(cn)]0.5h2o with cadmium nitrate leads to the structurally complex phase [ nbu4]0.5cd[ag(cn)2]2.5 ( 2 ) , as illustrated in figure 2 .
key crystallographic details of 2 are again summarized in table 2 . in the absence of a strong coordination preference
these pentacoordinate nodes connect to give a unique , self - catenating
topology ( topos reference jah1 ; the topology was
detected with topos and deposited in the ttd archive ) that can be related to a linked pair
of interpentrating cds - type nets ( see supporting information ( si ) ) .
mindful that correlation does
not necessarily imply causation , we note nonetheless that there is
again a close match between framework cavity and molecular cation
shape .
this suggests that the form of the [ nbu4 ] cation may be responsible for the unusual topology assumed by this
material .
as in 1 and numerous other dicyanoargentate
frameworks , 2 contains a series of short agag contacts generally considered indicative
of dd argentophilic interactions .
these interactions are often strongly susceptible to changes in external
conditions , such as temperature and pressure .
we determined the thermal expansion behavior of 2 in
order to identify any unusual thermomechanical response , and found
moderately strong positive and negative thermal expansion in directions
that are respectively parallel and perpendicular to these argentophilic
interactions ( = + 138(5 ) mk , = 83.1(1.6 ) mk ; see si for further discussion ) .
an alternative five - connected dicyanometallate
structure that is more obviously related to the molecular perovskite
analogues discussed above can be accessed by reaction of [ ppn ] dicyanometallates with transition - metal salts ( [ ppn ] = bis(trisphenylphosphine)iminium cation ) .
the crystal structures
of [ ppn]0.5cu[au(cn)2]2.5etoh
( 3a ) , [ ppn]0.5mn[ag(cn)2]2.5(etoh ) ( 3b ) , and [ ppn]0.5cd[ag(cn)2]2.5(etoh ) ( 3c ) are shown in figure 2 , and key crystallographic
details are again summarized in table 2 . in these compounds ,
the dicyanometallate network
forms grid - like layers of connected cubes that are then stacked on
top of one another .
the large molecular cations occupy the voids at
the center of each (nm ) cube .
because the layers
are not connected , each transition metal node is coordinated by dicyanometallate
anions in a square pyramidal geometry .
whereas in the case of cu this coordination geometry is straightforwardly understood
in terms of the d electronic configuration ,
for 3b and 3c the mn and cd coordination is actually octahedral and is completed by
one molecule of the ethanol solvent from which the crystals were grown .
the ability to incorporate octahedral node geometries via solvent
coordination means that , in principle at least , this phase may be
accessible for a range of other transition - metal dications . in practice ,
the balance with competing phases ( discussed further below ) is remarkably
subtle . in each of the compounds 3a c , successive layers are offset by one - half of the cube face
diagonal .
this offset has the effect of bringing ag or
au cations at the cube edges into close contact , which
will presumably maximize metallophilic interactions . in this sense
there is a clear distinction from the structural chemistry of oxide
frameworks , where there is a strong repulsion between the linker anions .
in the related n = 2 ruddlesden
popper phases
( e.g. , sr3ti2o7 ) this repulsion is
relieved by presence of additional a - cations as rocksalt - type layers . in our case
, the weak metallophilic forces
holding neighboring layers together may be expected to result in particularly
anisotropic elastic response for this family , while also allowing for the possibility of intercalation
chemistry .
the analogy
between dicyanometallate frameworks and perovskite oxides is clearest
in the last family of compounds we report .
similar
conditions to those described above for 3a c allowed preparation of the abx3 solvate [ ppn]cd[ag(cn)2]33etoh ( 4 ) and the solvent - free
frameworks [ ppn]mn[au(cn)2]3 ( 5a ) and [ ppn]cd[au(cn)2]3 ( 5b ) .
in the case of 4 , crystals grew directly from reaction
mixtures that had originally formed crystals of 3c ; the
relationship between these phases is discussed in more detail below .
the structures of all three compounds 4 , 5a , 5b are clear analogues of perovskites : octahedrally
coordinated mn / cd centers are connected
to form the characteristic cubic network , with [ ppn ] cations
adopting the 12-coordinate a - cation site [ figure 2 ] .
we use the term
super - perovskites to reflect this structural correspondence
and to emphasize that the bb separation across the
perovskite cube edge is now on the nanometre scale . just as
for other perovskite analogues , long - established geometric descriptors
can be applied to these superperovskite frameworks .
for example , we
calculate tolerance factors of 1.02 ( at 150 k ) and
1.00 ( at 300 k ) for 5a and 5b , respectively
( see si ) ; likewise the octahedral
tilt systems active in both structures correspond to the same glazer
notation aac .
while the tilts of compounds 5a and 5b can be described completely using glazer notation , compound 4 shows in - phase tilting of adjacent octahedra correlated
perpendicular to the rotation axis : an impossibility for perovskites ,
such as oxides and halides , with monatomic anions .
the tilt system
of 4 is described by the following tilt tensor ( see si for discussion of this notation ) : because the phenyl groups of the ppn cation extend
up to the perovskite cube faces , there are direct cation cation
interactions that may influence the phase transition behavior of these
systems .
in contrast to the simple monopolar and/or dipolar interactions
between a - site cations in oxide perovskites , these interactions are
inherently multipolar , which in turn may give rise to complex ordering
processes unrealisable in conventional ceramics .
indeed the ppn conformations we observe in compound 3c and
its transformation product 4 are fundamentally different
from those found in 5a and 5b : in the former
the phenyl rings are oriented as if to maximize cation
interactions with the linker ag cation , whereas in the
latter intra- and inter - molecular interactions
dominate ( figure 3a , b ) .
this is entirely consistent with the diversity of ppnppn supramolecular interaction motifs known to occur in the solid
state .
so one alternative view of the
structures of 5a and 5b is that the anionic
dicyanometallate framework acts to impose a particular geometric arrangement
of the ppn cations that may not necessarily be compatible
with the inter - cation interactions .
we suggest that exploration of
this tension between complex multipolar interactions and the geometry
of the lattice supporting those interactions may be a fertile playground
for future research given , for example , the implication of multipolar
dynamics in extending charge - carrier lifetimes in [ ch3nh3]pbi3 ( figure 3c , d ) .
short cation
and interactions in 3c ( a ) and 5b ( b ) .
the phenyl rings are oriented such
that short cation distances are maximized , whereas
in 5b intra- and inter - molecular
interactions dominate .
[ ppn ] cations are inherently
multipolar and their packing in 4 ( c ) corresponds to
ordered hexapoles .
such multipolar interactions are common for framework
materials with molecular cations such as [ ch3nh3]pbi3 ( d ) where the dynamics of these interactions are
implicated in slow exciton recombination ( cation - ordered pnma structure shown ) . framework shown in gray , cation
in teal ( a , b ) , and multipoles in teal and red ( c , d ) ; h atoms not shown
for clarity . given the diversity of
known structures of dicyanometallate frameworks i.e .
, those
reported in the preceding section together with the exhaustive list
given in table 1it
is natural to ask how one might control the adoption of one architecture
or another through choice of chemical composition .
we begin
by organizing all known dicyanometallates into compositional families
of given stoichiometry ambxn .
even if their structures vary , members of any one
family share the same coordination number and charge state of the
b cation two
aspects over which the synthetic chemist has some control .
coordination
number usually fixes the value of n and so constrains
the number and type of relevant network topologies .
likewise , cation
charge determines the value of n m and so determines the extent of inclusion of extra - framework
a cations ( i.e. , the value of m ) .
a visual
representation of our categorization is given in figure 4 , and it is by interpreting
the various trends that emerge from this diagram that we ultimately
aim to devise design strategies linking chemical composition and framework
structure .
organization of all known dicyanometallate frameworks according
to node coordination number and extent of incorporation of extra - framework
cations .
the frameworks shown are representative examples of isostructural
compounds ; the number of such compounds known is given in the corresponding
black circle .
the collection of frameworks contained within a single
hexagonal cell form a family with related composition ambxn .
the color of the
cell corresponds to the charge on the nodal cation ( inset ) .
what is immediately obvious is
that some families are considerably more populous than others .
the
largest and most topologically diverse family is that with composition
bx2 , consisting of a variety of structures based on four - connected
nets .
four - fold connectivity is well known to favor topological diversity hence the polymorphism of ,
e.g. , silica , zeolites , and imidazolate mofs . in the present case
, zn[au(cn)2]2 emerges as the most polymorphic of the bx2 systems , crystallizing
in at least four different forms that include analogues of cristobalite
and quartz .
in fact the parallel with
silica is a meaningful one : the combination of tetrahedral zn coordination geometry and aurophilic interactions capable
of bending the zn nc au cn zn linkers
resembles strongly the nonlinear si o si building units
of sio2 frameworks .
but one additional reason for the accessibility
of these bx2 structures will likely be the large number
of divalent cations with aqueous chemistry .
even transition metals
with octahedral coordination can yield four - connected frameworks if
spectator ligands are present in the synthesis mixture ( noting that
sometimes solvent itself may play this role ) .
indeed it is the doubly
ligated octahedral [ trans - bx4l2 ] coordination motif that yields the square grid topology
the
most frequently adopted network structure observed for this family . in principle , any such coordinated ligands may be susceptible to
exchange for dicyanometallate anions present in the reaction mixture ,
with concomitant incorporation of extra - framework cations : the equilibrium
position will depend on , among other factors , the ligand binding strength
and the stoichiometry of ax dicyanometallate salt in the reaction
mixture .
so , for example , while the cd / k[ag(cn)2 ] system forms the b(l2)x2 phase cd(py)2[ag(cn)2]2 when prepared in the presence
of pyridine , in the absence of pyridine the same pair of reagents
yields the abx3 phase kcd[ag(cn)2]3 .
likewise for the cd/[ppn][ag(cn)2 ] system
we obtain the five - coordinate a0.5b(l)x2.5 phase
[ ppn]0.5cd(etoh)[ag(cn)2]2.5 ( 3c ) as a first ( kinetic ) product ; subsequent exposure to additional
[ ppn][ag(cn)2 ] drives formation of the superperovskite
abx3 phase [ ppn]cd[ag(cn)2]33etohnh2o ( 4 ) .
this strategy of
varying b : ax stoichiometry seems an attractive method
of controlling network connectivity throughout the axbx2+x families .
the particular
topology adopted for the x 0 phases then
depends on the geometry of the a cation . for monatomic
a ( e.g. , k , na ) ,
the topology
is usually that of the corresponding a - deficient framework : this is
presumably a consequence of the much larger scale of the dicyanometallate
linker .
so , for example , both kfe[ag(cn)2]3 and
fe[ag(cn)2]3 share the same triply interpenetrating
-po network structure .
larger , molecular cations force topological
changes that reflect the cation shape . on the one hand ,
the cruciform
conformation of [ nbu4 ] helps drive the unusual
topologies of [ nbu4]0.5cd[ag(cn)2]2.5 and [ nbu4]ni[au(cn)2]3 . on the other hand , the more isotropic [ ppn ] cation
clearly favors the cubic - type nets of [ ppn]0.5cu[au(cn)2]2.5etoh and [ ppn]mn[au(cn)2]3 .
although there are far fewer examples based on monovalent
b cations the need for aqueous chemistry limiting
these systems to coinage metal salts
again we find that inclusion of monatomic extra - framework
cations appears to have essentially no effect on framework topology :
kcu[ag(cn)2]2 and cu(dmf)[au(cn)2]2 share the same basic square - grid
structures .
the tetrahedral [ net4 ] cation now
favors a diamondoid topology in [ net4]ag[ag(cn)2]2 ; and in [ cu2(2,2-bpy)2(cn)]0.5cu[cu(cn)2]1.5 the linear
nature of the [ cu2(2,2-bpy)2(cn ) ] cation drives a honeycomb structure with linear channels .
notable by its absence is the ( 10,3)-a or srs topology , a common topology for 3-connected mofs .
the apparent rarity of 3-connected structures is in large
part due to the propensity for cu to adopt alternative
binding modes , such as bifurcated linkages , though it may be possible to make a dicyanometallate framework with
( 10,3)-a topology using an octahedrally coordinated
metal with meridionally disposed capping ligands .
is likely responsible for the relative
paucity of structures known to contain b cations in the 3 + oxidation
state and the complete absence of higher - valent examples .
all known
trivalent dicyanometallate salts adopt one of just two dense framework
structures : either the triply interpenetrating -po net of fe[ag(cn)2]3 or the triply intepenetrating acs net of the lanthanide salts [ ln(oh2)3][au / ag(cn)2]3 .
the contrast with the structural diversity
of divalent bx2 systems could hardly be clearer , and will
reflect also the disparity of topological constraints associated with
six- and four - connected nodes . in principle , it may be possible to
access more open structure types through incorporation of extra - framework
cations .
naturally , this would require coordination numbers higher
than six which is certainly feasible for the lanthanide trications .
consequently , further exploration of
lanthanide dicyanometallate chemistry using molecular ax sources may yield as - yet unrealised systems with compositions
a0.5bx3.5 and/or abx4 .
in addition
to the general trends we discuss here , a number of interesting comparisons
can be drawn between neighboring pairs of structural families as shown
in figure 4 .
for example ,
there are clear diagonal relationships which connect the topologies
of higher- and lower - connected network structures . in this way
, the
ruddlesden popper - like structure of [ ppn]0.5cu[au(cn)2]2.5etoh ( an a0.5bx2.5 framework ) can be seen as a natural extension of the square - grid
structures of both cu[ag(cn)2]2 and kcu[ag(cn)2]2 ( bx2 and abx2 frameworks ,
respectively ) .
and then the superperovskite [ ppn]cd[au(cn)2]3 structure emerges as a further extension on this same
theme .
the triad of bx / a0.5bx1.5/abx2 systems cu[cu(cn)2]/[cu2(2,2-bpy)2(cn)]0.5cu[cu(cn)2]1.5/kcu[cu(cn)2]2 can be viewed in precisely the same terms .
we
anticipate there are many more such relationships , some of which may
become increasingly apparent only as new dicyanometallate frameworks
continue to be discovered .
while
it has long been clear that a large variety of dicyanometallate frameworks
can be prepared , what we have shown here is how the structures of
these frameworks might be influenced rationally through variation
in cation charge , coordination number , presence of coordinating ligands ,
and extra - framework cation size , shape and extent of incorporation .
the ability to controllably explore a variety of network structures
in this way reflects robson s original vision for coordination
polymer chemistry , and suggests that dicyanometalates might be considered
a model family for extended framework design . in principle
the diversity
of structures accessible to this family also suggests a similar diversity
of physical properties .
moreover , the structural flexibility imparted
by the dicyanometallate linker means that these properties may be
much more extreme in nature than in conventional ceramic frameworks as
is already known to be the case in a number of examples .
but what we see as particularly attractive about dicyanometalate
framework chemistry is the ability to include complex molecular cations
within a number of different framework topologies .
so much of the
rich structural chemistry and physics of conventional ceramic frameworks
has arisen from the interplay between lattice geometry , local degrees
of freedom , and nature of intersite interactions .
consequently the
extension of these same concepts to systems with new degrees of freedom ( e.g. , molecular cation orientations and conformations ) , new lattice geometries ( e.g. , the topology of compound 2 ) , and new types of interactions ( e.g. ,
cation and ) augurs well for
the discovery of new and interesting physical phenomena in this intriguing
model family of framework structures .
[ nbu4]ag(cn)]20.5h2o was prepared by
adding an aqueous solution of [ nbu4]cn ( 0.5510 g in 400
ml water ) to a suspension of agcn ( 0.3435 g in 100 ml water ) and stirring
for 1 h. the excess agcn was then removed by vacuum filtration and
the volume of the filtrate reduced by rotary evaporation .
the concentrated
filtrate was allowed to evaporate , and the resulting powder was dried
at 50 c for 2.5 h and then at 40 c for 18 h. [ ppn]au(cn)2 was prepared by the method outlined in ref ( 32 ) .
solutions of kau(cn)2 ( 2.583 g in 100 ml water ) and ppncl ( 5.40 g in 200 ml 50:50
water : ethanol ) were combined with immediate formation of a white precipitate .
the reaction mixture was covered and stirred for 1 h then collected
by vacuum filtration and washed with 300 ml cold water .
the solid
was left to air - dry overnight then dried in a vacuum oven at 40 c
for 3.5 h giving 6.80 g of product ( yield : 96.3% ) .
elemental analysis :
found ( calculated)% c 57.74 ( 57.95 ) ; h 3.80 ( 3.84 ) ; n 5.24 ( 5.34 ) .
solutions of kag(cn)2 ( 1.881 g in 100 ml water )
and ppncl ( 5.68 g in 200 ml 50:50 water : ethanol ) were combined with
immediate formation of a white precipitate .
the reaction mixture was
covered and stirred for 1 h then collected by vacuum filtration and
washed with 300 ml cold water .
the solid was left to air - dry overnight
then dried in a vacuum oven at 40 c for 1 week giving 5.87 g
of product ( yield : 90.0% ) .
elemental analysis : found ( calculated)%
c 65.05 ( 65.34 ) ; h 4.26 ( 4.33 ) ; n 5.88 ( 6.02 ) .
[ net4]ag[ag(cn)2]2 ( 1 ) single crystals were prepared by slow evaporation
of aqueous solutions of kag(cn)2 and [ net4]cl .
solutions of kag(cn)2 ( 0.2802 g , 1.411 mmol ) and [ n(c2h5)4]cl ( 0.2380 g , 1.436 mmol ) were
prepared with the minimum quantity of water and then mixed .
the resulting
colorless solution was left to evaporate slowly and crystals formed
within 3 weeks .
[ nbu4]0.5cd[ag(cn)2]2.5 ( 2 ) aqueous solutions of [ nbu4]ag(cn)20.5h2o and cd(no3)24h2o were prepared with the minimum
quantity of solvent , mixed , and left to evaporate from an open petri
dish .
[ ppn]0.5cu[au(cn)2]2.5 etoh ( 3a )
single crystals were prepared by slow - diffusion in an h - cell . into
one arm
was placed [ ppn]au(cn)2 ( 25 mg in 1 ml of ethanol )
and the other cucl22h2o ( 5.4 mg in 0.142
ml of ethanol ) .
[ ppn]0.5mn[ag(cn)2]2.5(etoh ) ( 3b ) was prepared by diffusion in a schott bottle .
cold solutions of [ ppn]ag(cn)2 ( 1.125 g in 20 ml of ethanol )
and mn(no3)24h2o ( 148 mg in
6 ml of ethanol ) were carefully layered with a buffer of 50 ml cold
ethanol separating the layers .
[ ppn]0.5cd[ag(cn)2]2.5etoh ( 3c ) single crystals were prepared by slow - diffusion in a vial .
cold
solutions of [ ppn]ag(cn)2 ( 101 mg in 2 ml of ethanol ) and
cd(no3)24h2o ( 19 mg in 0.2
ml of ethanol ) were carefully layered with a buffer of 5 ml of cold
ethanol separating the layers .
crystals formed overnight . for
[ ppn]cd[ag(cn)2]33etohnh2o ( 4 ) , a crystal was selected from a small
portion ( roughly 3 ml ) of a reaction mixture that initially yielded 3c .
solutions of [ ppn]ag(cn)2 ( 1.108 g in 25 ml
of ethanol ) and cd(no3)24h2o ( 183.7 mg in 7 ml of ethanol ) with a buffer of 50 ml of ethanol
separating the layers in a schott bottle .
colorless crystals formed
overnight and then a portion of these crystals , with mother liquor ,
were set aside in a sealed sample tube .
[ ppn]mn[au(cn)2]3 ( 5a ) was prepared by layering ethanolic
solutions of ppnau(cn)2 ( 50 mg in 2 ml of ethanol ) and
mn(clo4)2xh2o ( 14 mg in 0.2 ml of ethanol ) in a small sample tube .
colorless
crystals formed after 1 h. [ ppn]cd[au(cn)2]3 ( 5b ) was prepared by layering ethanolic solutions of
ppnau(cn)2 ( 50 mg in 2 ml of ethanol ) and cd(no3)24h2o ( 21 mg in 0.2 ml of ethanol ) in
a small sample tube .
colorless crystals formed after 1 h. single - crystal x - ray diffraction
data were collected using a nonius kappaccd diffractometer ( 2 and 5a ) or an oxford diffraction ( rigaku oxford
diffraction ) supernova diffractometer ( 1 , 3b , 3c , 4 , 5b ) fitted with an
oxford cryosystems cryostream 600 series/700 plus open - flow nitrogen
cooling device .
data for 3a were collected at diamond light source ( beamline i19 ) fitted with an oxford cryosystems helix open - flow
helium cryostat . denzo / scalepack , crysalispro , or
crystalclear were used for data collection and reduction as appropriate .
all structures were refined with full - matrix least - squares on f using crystals .
hydrogen atoms were , in some cases , visible in the difference fourier
map and treated in the usual manner . in the remaining cases , and particularly for disordered assemblies ,
hydrogen atoms were added geometrically .
full structural data are
included in the si , including cif files , and have been submitted to the ccdc
as numbers 14441341444141 .
these data can also be obtained
free of charge from the cambridge crystallographic data centre via http://www.ccdc.cam.ac.uk/data_request/cif . | we report the structures
of eight new dicyanometallate frameworks containing molecular extra - framework
cations .
these systems include a number of hybrid inorganic
organic
analogues of conventional ceramics , such as ruddlesden popper
phases and perovskites .
the structure types adopted are rationalized
in the broader context of all known dicyanometallate framework structures .
we show that the structural diversity of this family can be understood
in terms of ( i ) the charge and coordination preferences of the particular
metal cation acting as framework node , and ( ii ) the size , shape , and
extent of incorporation of extra - framework cations . in this way
, we
suggest that dicyanometallates form a particularly attractive model
family of extended frameworks in which to explore the interplay between
molecular degrees of freedom , framework topology , and supramolecular
interactions . |
cervical pain means that the collum or regio occipitalis , a muscle , or a muscle and a blood vessel inside the neck are pulled or that pain is felt such areas . in most cases ,
when a patient moves his or her neck towards a certain direction , pain appears , limiting in the neck s movement , and pressure pain appears on the collum .
cervical pain in oriental medicine is categorized as stiffness of the neck , headache , pain caused by stiffness , pain caused by stiffness of the neck , paresthesia , etc .
the causes are fatigue damage , stress , the aftereffect of wind and moiteur , congenital deformation , trauma , external contraction , etc . , which lead to the collateral meridian interruption and interruptions in the circulation of energy and blood .
cervical pain in western medicine is associated with a ruptured cervical disk , cervical spine osteoarthritis , cervical spondylosis , a spinal cord tumor , torticollis , damage to the cervical muscle and tendon , a cervical sprain , neurogenic muscle tone on a cervical area , etc . [ 3 , 4 ] .
pharmacopuncture therapy is a new form of therapy derived from the conjunction of meridian studies theory and herbal medicine theory .
meridian studies theory includes therapy to treat disease by controlling the functioning of the meridian system through acupuncture .
since pharmacopuncture therapy was first introduced to korea in 1960s , it has been frequently used clinically and is becoming a common therapy in oriental medicine . because pharmacopuncture therapy has been applied as a clinical treatment for some 40 years , studies on it have progressed in a variety of ways , and the results of many studies are becoming grounds for applying pharmacopuncture therapy as a clinical treatment [ 6 , 7 ] . recently , a number of studies about pharmacopuncture therapy have been done .
studies by han et al and kim et al dealt with bee venom pharmacopuncture , and studies by lee et al and cho et al dealt with hominis placenta acupuncture and scolopendra pharmacopuncture . thus , various studies according to the type of pharmacopuncture have been released , but analytic research on pharmacopuncture according to the disease is still not being actively pursued . also , pharmacopuncture is useful for patients with whiplash injuries and posterior neck pain patients , and its use has been on the rise .
accordingly , to explore the direction of future studies , the authors report the results of an analysis of theses released until now where pharmacopuncture was used for cervical spine disease . for that reason , we investigated until now the trend in research on pharmacopuncture used to treat cervical spine disease .
objects for search were original copies and abstracts of theses listed on a database or published until july 2014 .
the search was made on the oriental medicine advanced searching integrated system ( oasis ) , the national digital science library ( ndsl ) , and the korean traditional knowledge portal .
cervical pain , stiffness of the neck , posterior cervical pain , and neck disability , and those concerned with disease were cervical disk , whiplash injury ,
cervicalgia , herniated nucleus pulposus ( hnp) , and herniated intervertebral disc ( hivd) , ruptured cervical disk , and cervical disc disorder. these words were selected so that we could find theses involving patient groups undergoing korean medicine treatments for cervical pain .
the author selected these on research involving pharmacopuncture therapy for cervical spine disease from among theses released in korea .
after the authors had selected or excluded specific theses independently , the decision whether or not to include a thesis was made by the entire research staff .
such extraction was centered on the type of pharmacopuncture , the related disease and symptoms , the treatment s effectiveness , the publication year .
the number of reports in the literature that were searched by using the above method totaled 1594 . among them , 222 were selected ; 511 repeated reports and 861 theses that were not related to cervical disease were excluded .
finally , 25 clinical theses related to pharmacopuncture were selected ( table 1 ) chronologically , these were released as follows : 1 thesis in 2002 , 1 in 2004 , 3 in 2005 , 3 in 2006 , 4 in 2007 , 1 in 2009 , 1 in 2010 , 4 in 2011 , 4 in 2012 , and 2 in 2013 ( fig 1 ) . among the 25 selected theses , 13 used bee venom pharmacopuncture , among which a study by park et al involved a randomized controlled trial ( rct ) with hwangryun pharmacopuncture . in six theses ,
ouhyul pharmacopuncture was used , and among them , a study conducted by kim et al comparison of the effect of shinbaro pharmacopuncture to the effect of ouhyul pharmacopuncture .
in addition , three theses involved the use carthami - flos pharmacopuncture , and one each involved the use of scolopendra pharmacopuncture , soyeom pharmacopuncture , and hwangryunhaedok - tang pharmacopuncture ( fig 2 ) . among
the 25 selected theses , 11 came from the korean acupuncture & moxibustion society , 3 came from the journal of the pharmacopuncture institute , and 3 came from the journal of the korean academy of oriental rehabilitation medicine .
in addition , two each came from the institute of oriental medicine studies of daejeon university and the association of spine & joint korean medicine , and 1 each came from the korean journal of acupuncture , the korean association of oriental medicine information , the korea institute of oriental medicine , and the journal of the korean association of sports oriental medicine ( fig 3 ) .
the studies by lee et al , shin et al , and gwon et al showed a significant decrease when using then japanese orthopedic association score ( joa score ) as an assessment scale for cervical pain . among them , in the studies by lee et al and shin et al , bee venom pharmacopuncture was used ; in the study by gwon et al , scolopendra pharmacopuncture was used . among the selected theses , in the studies by park et al , lee et al , lee et al , woo et al , jang et al , and kim et al , the effect of pharmacopuncture for treating cervical pain
for example , in the studies by park et al and kim et al , the results achieved using hwangryun pharmacopuncture and bee venom pharmacopuncture were compared , as were the results achieved using shin- baro pharmacopuncture and ouhyul pharmacopuncture . in those two studies , bee venom pharmacopuncture and shinbaro pharmacopuncture were more effective . in a study by lee et al , chuna manual medicine and hwangryunhaedok- tang pharmacopuncture
for the first two weeks , both were effective , but after that , chuna manual medicine was more effective .
the study by lee et al included groups treated with chuna manual medicine , ouhyul pharmacopuncture , and chuna manual medicine combined with ouhyul pharmacopuncture . in that study , chuna manual medicine combined with ouhyul pharmacopuncture was a little more effective . in a study by woo et al ,
the improvements in the group that received acupuncture therapy combined with chuna manual medicine and in the group that received acupuncture therapy combined with ouhyul pharmacopuncture were compared .
significant improvements were seen in both groups , but difference in improvements between the two groups was not large . in the studies by an et al and kim et al , a group that received bee venom pharmacopuncture therapy and a group that received electroacupuncture were compared . here , the group that received bee venom pharmacopuncture therapy showed better effective . in the study by jang et al ,
the acupuncture group was compared with the group that received acupuncture combined with carthami - flos seeds and juglandis semen pharmacopuncture .
the result showed that acupuncture combined with pharmacopuncture was more effective . in a study of bee venom pharmacopuncture by gang et al , a group with bee venom pharmacopuncture therapy and a group with acupuncture
were compared after two treatments . in the group that received bee venom pharmacopuncture therapy ,
the visual analogue score ( vas ) ranged from 7.2 0.44 ( index ) to 1.5 0.36 ( index ) ; however , in the group that received acupuncture , the vas ranged from 6.4 0.46 ( index ) to 4.0 0.56 ( index ) . in a study by park et al ,
in the group that received bee venom pharmacopuncture therapy , the average response - to - treatment time was 12.3 days while in the acupuncture group , it was 15.6 days . in a study by song , after five treatments , vas changes were - 4.60 1.50 in the bee venom pharmacopuncture group and - 2.30 1.34 in the normal saline group , so bee venom pharmacopuncture was more effective . in a study by gang and song , the group that underwent bee venom pharmacopuncture therapy and the group that received acupuncture combined with bee venom pharmacopuncture were compared , and the levels of pain and the rates of improvement for the two groups were similar 5 days later . however , 10 days later , the group that received acupuncture combined with bee venom pharmacopuncture showed a better result than the group that received only the bee venom pharmacopuncture .
also , in the studies by gang et al , choi et al , lee et al , and kim et al , bee venom pharmacopuncture was reported to be effective for treating cervical pain . in the studies by yoon and seol et al ,
ouhyul pharmacopuncture and normal saline were used to treat posterior cervical patients . in both groups ,
the vas was significantly lower , but the ouhyul pharmacopuncture group had the better result .
in a study by lim et al , in the soyeom pharmacopuncture group five treatment later , the vas change was from 6.375 1.302 to 2.250 0.886 and the neck disability index ( ndi ) change was from 21.75 6.390 to 9.00 4.956 , both results being statistically significant . in a study by lim
et al , patients with posterior neck pain due to whiplash injuries caused by traffic accidents were treated with ouhyul pharmacopuncture and with soyeom pharmacopuncture , but no statistically significant results for the prepost difference index ( vas ndi short - form mcgill pain questionnaire ) were reported .
finally , in a study by park et al , the patients positive on the spurling test were treated with pharmacopuncture therapy for 2 weeks ; in 66.66% of the bee venom pharmacopuncture group , pain disappeared while it disappeared in only 40% of the hwangryun pharmacopuncture group . as a result of analyzing clinical theses involving the use of pharmacopuncture for treating diseases in cervical area according to the type of pharmacopuncture used by publication year , 9 out of the 13 clinical theses related to pharmacopuncture released before 2007 were theses involving the use of bee venom pharmacopuncture .
however , after 2007 , of the 12 clinical theses involving the use of pharmacopuncture to treat cervical pain , only 4 used bee venom pharmacopuncture .
this shows that the type of pharmacopuncture is changing from bee venom pharmacopuncture to ouhyul pharmacopuncture , carthami - flos pharmacopuncture , hwangryunhaedok - tang pharmacopuncture , etc .
these days , many people complain of cervical pain caused by excessive job , stress , etc .
thus , it has become necessary to actively study about the treatment of cervical pain in oriental medicine . in oriental medicine , many treatments are used for cervical pain : for example , acupuncture therapy , herbal medicine , pharmacopuncture , chuna manual medicine therapy , electroacupuncture . among them
, pharmacopuncture therapy combines existing meridian channel theory with the efficacy of herbal medicine . as efficacy of pharmacopuncture therapy
is excellent , its use is on the rise , and the types of pharmacopuncture are increasing . for those reasons ,
lee et al , cho et al , kim et al , and han et al performed studies according to the type of pharmacopuncture : for example , hominis placenta pharmacopuncture , scolopendra pharmacopuncture , bee venom pharmacopuncture .
for this reason , the authors investigated the use of pharmacopuncture therapy for treating cervical pain .
objects for search were original copies and abstracts of theses listed in a database or published until july 2014 .
the search was made on oasis the ndsl , and the korean traditional knowledge portal .
the number of reports in the literature that were searched by using the above method totaled 1594 .
finally , 25 clinical theses related to pharmacopuncture were selected . among the 25 selected theses , 13 ( 52% ) used bee venom pharmacopuncture .
thus , clinical studies using bee venom pharmacopuncture to treat cervical disease were in a slight majority . by academic journal in which the selected theses were published ,
11 came from the korean acupuncture & moxibustion society , which accounted for 44% of the total and ranked no . 1 , and 3 each came from the journal of the korean pharmacopuncture institute and the journal of the korean academy of oriental rehabilitation medicine , which accounted for 12% of the total and ranked no .
2 . also , among the 25 selected theses , 16 involved rcts . of these 16 ,
8 compared a pharmacopuncture group to an acupuncture combined with pharmacopuncture group , and the acupuncture combined with pharmacopuncture group showed a better result than the pharmacopuncture group . in the study by kim et al ,
shinbaro pharmacopuncture was more effective than ouhyul pharmacopuncture . in the study by park et al ,
bee venom pharmacopuncture was more effective than hwangryun pharmacopuncture , but in the study by lim et al , there was no difference between ouhyul pharmacopuncture and soyeom pharmacopuncture when treating patients with whiplash injuries caused by traffic accidents . in comparing chuna with pharmacopuncture ,
in a study by woo et al , there was no difference between the acupuncture combined with chuna group and the acupuncture combined with pharmacopuncture group . as a result of examining the recent trend in pharmacopuncture used to treat disease in the cervical area , we found that the main study theme before 2008 was bee venom pharmacopuncture .
however , from 2008 , the type of pharmacopuncture has been changing from bee venom pharmacopuncture to ouhyul pharmacopuncture , carthami- flos pharmacopuncture , hwangryunhaedok - tang pharmacopuncture , etc .
this means that clinical researchers are highly interested in various types of pharmacopuncture , but still there are not enough studies to confirm the effect pharmacopuncture .
thus , in the future , many studies are needed to identify the most effective pharmacopuncture treatments , along with their type , volume , procedure region , and number of treatments , and it is out hope that many such reports actually be published .
by analyzing theses related to cervical pain published in journals of academic institutes for korean medicine , we were able to draw the following conclusions : 1 .
pharmacopunctures used for cervical pain were pharmacopunctures used for cervical pain were bee venom pharmacopuncture , carthami - flos pharmacopuncture , scolopendra pharmacopuncture , ouhyul pharmacopuncturen , hwangryun pharmacopuncture , corpus pharmacopuncture , soyeom pharmacopuncture , hwangryunhaedoktang pharmacopuncture , shinbaro phamacopuncture .
2 . as a result of a rct study , a group treated with pharmacopuncture therapy combined with another method showed effective results .
, bee venom pharmacopuncture oriented studies were actively pursued , but the number of studies on various types of pharmacopuncture has been gradually increasing .
, the type of pharmacopuncture to be used should be selected based on the cause of the disease and the patient s condition . | objectives : the purpose of this study is to analyze trends in domestic studies on pharmacopuncture therapy for treating cervical disease.methods:this study was carried out on original copies and abstracts of theses listed in databases or published until july 2014 .
the search was made on the oriental medicine advanced searching integrated system ( oasis ) the national digital science library ( ndsl ) , and the korean traditional knowledge portal .
search words were
pain on cervical spine ,
cervical pain , ruptured cervical disk , cervical disc disorder , stiffness of the neck , cervical disk ,
whiplash injury ,
cervicalgia , posterior cervical pain , neck disability , herniated nucleus pulposus ( hnp) , and herniated intervertebral disc ( hivd).results : twenty - five clinical theses related to pharmacopuncture were selected and were analyzed by year according to the type of pharmacopuncture used , the academic journal in which the publication appeared , and the effect of pharmacopuncture therapy.conclusion:the significant conclusions are as follows : ( 1 ) pharmacopunctures used for cervical pain were bee venom pharmacopuncture , carthami - flos pharmacopuncture , scolopendra pharmacopuncture , ouhyul pharmacopuncturen , hwangryun pharmacopuncture , corpus pharmacopuncture , soyeom pharmacopuncture , hwangryunhaedoktang pharmacopuncture , shinbaro phamacopuncture .
( 2 ) randomized controlled trials showed that pharmacopuncture therapy combined with other methods was more effective .
( 3 ) in the past , studies oriented toward bee venom pharmacopuncture were actively pursued , but the number of studies on various other types of pharmacopuncture gradually began to increase .
( 4 ) for treating a patient with cervical pain , the type of pharmacopuncture to be used should be selected based on the cause of the disease and the patient s condition . |
, usa . tricine , 2-mercaptoethanol , hynic were purchased from sigma - aldrich , usa , and stannous sulfate was purchased from merck .
five hundred milligram of higg was dissolved in 10 ml distilled water and this solution was drawn onto slide - a - lyzer dialysis cell and dialyzed overnight at 4c using normal saline .
the contents of dialysis cell were drawn through millipore filter ( 0.22 micron ) and 0.8 mg of hynic solution was added in 100 l dmso .
the ph was adjusted to 6.4 with 1.5 m sodium phosphate buffer and dialyzed for overnight at 4c in a sterile dialysis cell using 1.5 m sodium phosphate buffer .
finally , aliquots were dispensed into 15 ml vials , frozen overnight at -20c , lyophilized , sealed under vacuum and stored in a refrigerator until use .
hundred milligram of tricine was dissolved in 8 ml of distilled water and acidified with 0.5 ml of 2.0 m hcl .
0.5 ml of stannous sulphate solution ( 20 mg / ml ) was added and ph adjusted to 5.5 using 2 m sodium hydroxide solution .
the contents were filtered through 0.22 millipore filter and stored overnight at -20c , lyophilized sealed under vacuum and stored in a refrigerator until use .
tc - hynic - higg was synthesized according to procedures described previously , with slight modification . in brief , vial containing higg - hynic was thawed at room temperature and 60 l of freshly prepared trycine kit was added to it followed by addition of 20 - 30 mci tc - perthecnetate .
the contents were dissolved by swirling the vial gently and incubated at room temperature for 20 min to complete the labeling reaction .
strips ( 112 cm ) of itlc - sg , ( gelman , usa ) were used as stationary phase and acetone was used as the mobile phase to separate free pertechnetate and tc - hynic - higg , which moved with the solvent .
citrate buffer ( ph 5.5 ) was used as the mobile phase to separate tc - hynic - higg , and tc - hynic .
one hundred microliters of the radiolabeled antibody was incubated in 1.9 ml of saline or human serum at 37c .
small aliquots were withdrawn at different time intervals up to 24 h and radiolabeling efficiency was evaluated by itlc using citrate buffer as mobile phase .
aliquots of 0.5 ml of tc - hynic - higg ( 100 l ) were challenged over a range of cysteine and dtpa concentrations ( 25100 mmol / l ) at 37c for 1 h. the percentage of tc displaced by cysteine and dtpa was determined by itlc - sg using normal saline as mobile phase .
( labeled - hynic - hynic , rf = 0.00.15 , free tc , rf = 0.751.0 and cystine / dtpa- tc , rf = 0.60.7 ) .
hundred microliters of the radiolabeled antibody was mixed with 2.0 ml of dichloromethane and 1.9 ml of normal saline .
the mixture was kept at room temperature for 1 h and radioactive counts of the two separated phases were taken using counter ( capintec , usa ) to determine per cent lipophilicity of the radiolabeled product .
one hundred microliters of tc - hynic - higg , was mixed with 1.9 ml of fresh serum and the contents were kept at 37c for 1 h. 2 ml of 10% trichloric acetic acid was added to the mixture and the contents were centrifuged for 5 min at 3500 rpm . per cent radioactive counts in supernatant and precipitate fractions were noted using g - counter ( capintec , usa ) to determine extent of protein binding .
all animal experiments were conducted in accordance with our institutional guidelines , and the experimental procedure was approved by the institutional animal ethical committee duly constituted for the purpose .
biodistribution experiments were performed by intravenous administration of 100 l of tc- hynic - higg ( 1 ci ) to strain - a mice ( 2540 g ; n = 10 ) .
organs of interest , namely , heart , liver , lungs , spleen , kidneys , stomach , intestine and muscle were removed , washed with saline and weighed , and respective radioactivity counts were determined with the help of a g - counter ( capintec , usa ) .
200 l of tc - hynic - higg ( 100 ci ) was intravenously administered to rabbits ( n = 5 ) through the ear vein .
radioactivity per unit volume was calculated by counting a known volume from the sample at each time point .
whole blood was considered as 7% of the body weight to calculate the percentage of administered radioactivity present in total body blood .
a group of three male new zealand rabbits , weighing 23 kg were used in the immunogenicity studies .
infection was induced by inoculation of e. coli ( atcc 22923 ) into the left thigh muscle .
after 45 days , when gross swelling was apparent in the infected thigh , 3 mci of tc - hynic - higg was injected intravenously via ear vein .
the anesthetized animal was placed in the supine position and scanned under the gamma camera .
anterior whole body image and spot views of the lower extremities were obtained hourly for the first 2 h and subsequently at 24 h. acute aseptic inflammation was induced in three other new zealand white rabbits , weighing 23 kg with turpentine .
commercial grade turpentine liquid was sterilized by ultrafiltration using a 0.45 nm bio - filter .
each animal received 0.1 ml of the sterile turpentine liquid by intramuscular injection into the right thigh muscle .
the rabbits were then intravenously administered with 3.0 mci of tc - hynic - igg via ear vein .
the anesthetized animal was placed in the supine position and scintigraphy was done under the gamma camera .
normal biodistribution study was carried out using tc- hynic - higg scintigraphy in human volunteers .
briefly , 10.0 mci tc - hynic - higg ( equivalent of 2.0 mg higg ) was administered in normal male volunteers ( n = 2 ) intravenously for the acquisition of imaging data .
anterior and posterior whole - body images were obtained immediately at 6 h and 24 h post - injection using a dual head hawkeye gamma camera system ( ge , milwaukee , usa ) using the inbuilt software entegra version-2 .
regions of interest ( roi ) were constructed at each time point for the whole body and all organs visualized .
the geometric mean of activity in these regions was calculated and corrected for radioactive decay .
organ radioactivity , as a fraction of the administered dose , was calculated by using the immediate whole body as 100% of the injected dose . from the imaging results , the residence time for tc - hynic - higg in the major source organs ( heart chambers , kidneys , liver , lungs , spleen , and the remainder whole body )
results of biodistribution experiments were statistically analyzed using a one - way anova followed by the dunnett post - hoc test .
, usa . tricine , 2-mercaptoethanol , hynic were purchased from sigma - aldrich , usa , and stannous sulfate was purchased from merck .
five hundred milligram of higg was dissolved in 10 ml distilled water and this solution was drawn onto slide - a - lyzer dialysis cell and dialyzed overnight at 4c using normal saline .
the contents of dialysis cell were drawn through millipore filter ( 0.22 micron ) and 0.8 mg of hynic solution was added in 100 l dmso .
the ph was adjusted to 6.4 with 1.5 m sodium phosphate buffer and dialyzed for overnight at 4c in a sterile dialysis cell using 1.5 m sodium phosphate buffer .
finally , aliquots were dispensed into 15 ml vials , frozen overnight at -20c , lyophilized , sealed under vacuum and stored in a refrigerator until use .
hundred milligram of tricine was dissolved in 8 ml of distilled water and acidified with 0.5 ml of 2.0 m hcl .
0.5 ml of stannous sulphate solution ( 20 mg / ml ) was added and ph adjusted to 5.5 using 2 m sodium hydroxide solution .
the contents were filtered through 0.22 millipore filter and stored overnight at -20c , lyophilized sealed under vacuum and stored in a refrigerator until use .
tc - hynic - higg was synthesized according to procedures described previously , with slight modification . in brief , vial containing higg - hynic was thawed at room temperature and 60 l of freshly prepared trycine kit was added to it followed by addition of 20 - 30 mci tc - perthecnetate .
the contents were dissolved by swirling the vial gently and incubated at room temperature for 20 min to complete the labeling reaction .
strips ( 112 cm ) of itlc - sg , ( gelman , usa ) were used as stationary phase and acetone was used as the mobile phase to separate free pertechnetate and tc - hynic - higg , which moved with the solvent .
citrate buffer ( ph 5.5 ) was used as the mobile phase to separate tc - hynic - higg , and tc - hynic .
one hundred microliters of the radiolabeled antibody was incubated in 1.9 ml of saline or human serum at 37c .
small aliquots were withdrawn at different time intervals up to 24 h and radiolabeling efficiency was evaluated by itlc using citrate buffer as mobile phase .
aliquots of 0.5 ml of tc - hynic - higg ( 100 l ) were challenged over a range of cysteine and dtpa concentrations ( 25100 mmol / l ) at 37c for 1 h. the percentage of tc displaced by cysteine and dtpa was determined by itlc - sg using normal saline as mobile phase .
( labeled - hynic - hynic , rf = 0.00.15 , free tc , rf = 0.751.0 and cystine / dtpa- tc , rf = 0.60.7 ) .
hundred microliters of the radiolabeled antibody was mixed with 2.0 ml of dichloromethane and 1.9 ml of normal saline .
the mixture was kept at room temperature for 1 h and radioactive counts of the two separated phases were taken using counter ( capintec , usa ) to determine per cent lipophilicity of the radiolabeled product .
one hundred microliters of tc - hynic - higg , was mixed with 1.9 ml of fresh serum and the contents were kept at 37c for 1 h. 2 ml of 10% trichloric acetic acid was added to the mixture and the contents were centrifuged for 5 min at 3500 rpm . per cent radioactive counts in supernatant and precipitate fractions were noted using g - counter ( capintec , usa ) to determine extent of protein binding .
all animal experiments were conducted in accordance with our institutional guidelines , and the experimental procedure was approved by the institutional animal ethical committee duly constituted for the purpose .
biodistribution experiments were performed by intravenous administration of 100 l of tc- hynic - higg ( 1 ci ) to strain - a mice ( 2540 g ; n = 10 ) .
organs of interest , namely , heart , liver , lungs , spleen , kidneys , stomach , intestine and muscle were removed , washed with saline and weighed , and respective radioactivity counts were determined with the help of a g - counter ( capintec , usa ) .
200 l of tc - hynic - higg ( 100 ci ) was intravenously administered to rabbits ( n = 5 ) through the ear vein .
radioactivity per unit volume was calculated by counting a known volume from the sample at each time point .
whole blood was considered as 7% of the body weight to calculate the percentage of administered radioactivity present in total body blood .
a group of three male new zealand rabbits , weighing 23 kg were used in the immunogenicity studies .
infection was induced by inoculation of e. coli ( atcc 22923 ) into the left thigh muscle .
after 45 days , when gross swelling was apparent in the infected thigh , 3 mci of tc - hynic - higg was injected intravenously via ear vein .
the anesthetized animal was placed in the supine position and scanned under the gamma camera .
anterior whole body image and spot views of the lower extremities were obtained hourly for the first 2 h and subsequently at 24 h. acute aseptic inflammation was induced in three other new zealand white rabbits , weighing 23 kg with turpentine .
commercial grade turpentine liquid was sterilized by ultrafiltration using a 0.45 nm bio - filter .
each animal received 0.1 ml of the sterile turpentine liquid by intramuscular injection into the right thigh muscle .
the rabbits were then intravenously administered with 3.0 mci of tc - hynic - igg via ear vein .
the anesthetized animal was placed in the supine position and scintigraphy was done under the gamma camera .
normal biodistribution study was carried out using tc- hynic - higg scintigraphy in human volunteers . briefly
, 10.0 mci tc - hynic - higg ( equivalent of 2.0 mg higg ) was administered in normal male volunteers ( n = 2 ) intravenously for the acquisition of imaging data .
anterior and posterior whole - body images were obtained immediately at 6 h and 24 h post - injection using a dual head hawkeye gamma camera system ( ge , milwaukee , usa ) using the inbuilt software entegra version-2 .
regions of interest ( roi ) were constructed at each time point for the whole body and all organs visualized .
the geometric mean of activity in these regions was calculated and corrected for radioactive decay .
organ radioactivity , as a fraction of the administered dose , was calculated by using the immediate whole body as 100% of the injected dose . from the imaging results , the residence time for tc - hynic - higg in the major source organs ( heart chambers , kidneys , liver , lungs , spleen , and the remainder whole body )
results of biodistribution experiments were statistically analyzed using a one - way anova followed by the dunnett post - hoc test .
the potential value of radiolabeled higg as an imaging agent for detecting focal infection has been previously reported .
an indirect in - labelling via chelating group attached to the antibody has been exploited successfully ; this labeling approach is not preferred due to the variable binding affinity of in to the antibody 's amino groups , which may partly be located in the hypervariable region of the antibody molecule , thereby altering its immunoreactivity .
the radiolabeling procedure being reported here for labeling higg with tc-99 m using hynic gives consistently high ( > 96% ) radiolabeling efficiency with respect to the radiolabeled product [ table 1 ] .
stability of higg kit ( shelf life ) was also evaluated by storing the kit for different time periods .
results indicate that the kit is stable up to three months with only 1.6% reduction in radiochemical purity of the tc-99 m labeled product [ table 2 ] . in vitro stability study of radiolabeled higg
was studied in saline at room temperature and in human serum at 37c till 24 h [ table 3 ] , up to 4 h , no free pertechnetate was detected irrespective of the storage condition , indicating no leaching of tc-99 m from the product . even at 24 h the free pertechnetate content were only 5.1% and 9.5% in case of serum and saline , respectively .
high binding affinity of tc - higg was ascertained by allowing the tagged protein to incubate with dtpa and cysteine at different molar ratios [ table 4 ] .
dtpa at a molar concentration of 25 and 50 times that of higg decreased labeled higg by only 3.1% and 7.2% respectively . however , when dtpa was increased 100 times , the labeled percentage of higg was reduced by 11.83% . in case of transchelation with cysteine the reduction of the labeled higg was more pronounced . even at a molar ratio of 25 times
the decrease was about 5.8% , which increased to 23.7% at 100 times cysteine concentration .
the above observations suggested a high binding affinity of higg sh group for tc-99 m .
lipophilicity study of the labeled product showed good lipophilic character of the molecule [ table 5 ] .
optimization of radiolabeling efficiency of tc - higg with different concentration of reducing agent using itlc method of quality control stability of higg kit after storing these kits for different time period stability of 99m - higg when incubated at room temperature in saline and at 37c in human serum , respectively , using itlc method of quality control different molar concentration of dtpa and cysteine were mixed with tc - higg .
each value is the mean of three separate experiments of the same preparation lipophilicity profile of the 99mtc - hynic - higg biodistribution of 99mtc - hynic - higg in mice at 4 h and 24 h after intravenous administration is shown in figure 1 . among
the various organs studied , significant accumulation of the radiotracer was found in liver ( 4.9% ) , kidney ( 3.7% ) and intestine ( 2.1% ) at 4 h interval . by 24 h interval
the remaining organs ( heart , lungs , spleen , stomach and muscles ) had very low accumulation of the radiotracer .
low activity in the stomach , suggest an insignificant amount of free pertechnetate in the tc - higg preparation , further confirming the in vivo stability of the radiolabeled higg molecule .
significant amount of radioactivity in liver , intestine and kidneys suggest hepatobiliary as well as renal route of clearance for tc - hynic - higg .
blood clearance data in rabbits after intravenously administering 2 - 3 mci of tc - hynic - higg exhibited slow and biphasic clearance [ figure 2 ] . at 1
h , 15.1% of the injected radioactivity was present in blood , which reduced to 4.93% by 24 h. biodistribution of tc - hynic - higg
. the animal were intravenously administered with 1 uci tracer and the radioactivity in various organ was measured at 4 and 24 h. each value is the mean of 6 mice expressed in % dose administered per organ blood clearance of tc - hynic - higg in rabbit after administering it intravenously .
each value is the mean of three independent experiments anatomical delineation of the extent of focal inflammation is critical to clinical management of infectious processes , both for diagnosis and for monitoring the response to therapy . in our experiments ,
acute inflammatory lesion was developed within 3 days following the injection of sterile turpentine liquid .
an opaque creamy whitish lesion developed at the site of injection surrounded by inflammatory tissue .
no pus was found in the inflamed tissue during autopsy and the entire inflamed thigh muscle tissue measured about 3 cm in size .
the site of inflammation appeared as an area of diffuse uptake of tc- hynic - higg during scintigraphy . in animals that were injected with e. coli to generate an infection model , abscess formation occurred within 78 days post inoculation of e. coli .
a small firm mass measuring less than 1 cm in size was found in the infected thigh with noticeable gross swelling .
the anterior whole body and spot scintigraphy images at 2 h and 24 h showed increased uptake of tc - hynic - higg , with the area seen as a focal hot spot , indicating good localization of the radiolabeled higg at the site of infection [ figure 3 ] .
the image of an inflammatory lesion of patient [ figure 4 ] obtained using a spect camera after administering tc - hynic - higg indicates not only the quality of the kit and radiopharmaceutical preparation , but also clinical usefulness .
( a ) 2 h and ( b ) 24 h anterior and posterior view showing 99mtc - higg accumulation in rabbit .
right thigh shows the acute infection developed by sterile turpentine while right thigh of rabbit shows the site of abscess formation by e. coli tc - hynic - higg distribution in human volunteer at 24 h the present method of radiolabeling higg with hynic is a simple , easy to perform radiolabeling procedure that gives high radiolabeling yield with minimum colloid formation .
our experimental observations indicate good accumulation of tc - hynic - higg at the infection and/ or inflammation site .
tc - hynic - higg imaging data indicated higher lesion to background ratio in the infected foci as compared to the inflammatory foci the technique may therefore have a good diagnostic utility in the differentiation of infection from inflammation in clinical situations . | background : radiolabeled human immunoglobulin - g ( higg ) has demonstrated its utility in inflammation and infection imaging .
however , the present method of radiolabeling higg is time - consuming and complex.objective:to develop a simplified method of radiolabeling higg with technetium-99 m ( 99mtc ) via a nicotinyl hydrazine derivative ( 99mtc - hynic - higg ) and its biological evaluation.results:in vitro and in vivo studies showed that 99mtc - higg prepared by this method was fairly stable in physiological saline and human serum till 24 h. only 4.3% degradation of the radiolabeled drug was seen till 24 h. blood clearance pattern of the radiopharmaceutical exhibited biphasic exponential pattern .
biodistribution of 99mtc - hynic - higg in mice was observed up to 24 h. significant accumulation of the radiotracer was found in liver ( 4.93 % ) , kidney ( 3.67% ) and intestine ( 2.12 % ) at 4 h interval by 24 h interval , it was reduced to 1.99% , 2.18% and 1.93 % respectively .
significant amount of radioactivity in liver , kidney and intestine suggest hepatobilliary as well as renal route of clearance for 99mtc - hynic - higg .
the anterior whole body and spot scintigraphy images showed increased uptake of 99mtc - hynic - higg , with the area seen as a focal hot spot , indicating good localization of the radiolabeled higg at the site of infection.conclusion:the present findings indicate that 99mtc - hynic - higg holds great potential for the scintigraphy localization of inflammation .
the shelf life of the developed kit , when stored at ( ) 20c was found to be at least 3 months . |
weight loss surgery is an effective and durable treatment for clinically severe obesity and its associated conditions .
bariatric surgery not only promotes weight loss but also significantly reduces cardiac - related death , myocardial infarction , and stroke.1 approximately 220,000 americans had some form of bariatric surgery in 2008 , with the roux - en - y gastric bypass ( rygb ) procedure accounting for the vast majority.2 postoperative anemia has been reported in approximately one - fourth to one - half of the patients within 6 months to 4 years following surgery.39 despite its proven benefits , several nutritional deficiencies may be seen after gastric bypass .
these include iron , calcium , the fat - soluble vitamins a , d , e , and k , vitamin c , copper , selenium , and zinc.1016 however , by far the most common of these is iron deficiency .
women , especially those who are menstruating , appear to be at higher risk for developing iron deficiency,5,8,17 and oral iron supplementation is inconsistent in preventing anemia.7,1319 moreover , data beyond 45 years are sparce , and the long - term need for intravenous ( iv ) iron replacement has not been well characterized . since iron deficiency anemia may take years to develop , we sought to determine the long - term incidence of anemia , iron deficiency , and the prevalence of iv iron infusions five or more years after rygb surgery .
in accordance with the principles of the declaration of helsinki , and following the approval of the penn state college of medicine institutional review board , 904 subjects were identified from our prospective bariatric surgery database as having undergone rygb surgery at hershey medical center from 1999 to 2006 .
electronic medical records ( emrs ) were assessed on all subjects for iron supplementation , pre- and postoperative complete blood counts ( cbcs ) , ferritin levels , menstruation status at time of surgery , and length of the roux limb .
women were considered to be menopausal if their age was > 50 years at the time of surgery .
subjects were considered eligible for inclusion if a preoperative cbc and at least one postoperative cbc at > 6 months from the time of surgery were available .
anemia was defined as a hemoglobin < 12 g / dl for women and < 14 g / dl for men .
iv iron therapy was given at the discretion of the hematology consultant for patients with symptomatic iron deficiency anemia who were intolerant or refractory to oral iron supplements .
the conventional roux limb was between 100 and 150 cm and the pancreatobiliary limb between 40 and 50 cm .
all patients were instructed to take one of the more absorbable forms of oral iron , such as funarate / gluconate , 28 mg at bedtime , preferably with vitamin c 500 mg , and not within 2 hours of calcium supplements , in addition to calcium , vitamin d , a multivitamin containing vitamin a , and vitamin b12 supplements .
follow - up visits were routinely scheduled at 1 month with the dietician , at 3 months with the surgeon and dietician , at 9 months with the dietician , and at 12 months and then annually for 5 years with the surgeon . at 1 month , patients were given an information sheet to be given to their primary physician requesting cbcs along with routine laboratory monitoring quarterly for the first year and annually thereafter .
univariate analyses using chi - squared test and student s t - test were performed between the outcomes ( incidence of anemia and iron deficiency anemia ) and variables ( sex , age at time of surgery , length of roux limb , oral iron supplementation , menstruation status , postoperative complications , and presence of preoperative anemia ) . since the follow - up time varied across subjects ,
particularly , kaplan meier curves were constructed to estimate the cumulative event rate of anemia over a 7-year time period .
the log - rank test was used to determine statistical significance of various patient characteristics as risk factors for developing anemia , and 95% confidence bands were constructed .
multivariable cox proportional hazards regression analysis was used to jointly model the effects of the characteristics identified by the log - rank test .
in accordance with the principles of the declaration of helsinki , and following the approval of the penn state college of medicine institutional review board , 904 subjects were identified from our prospective bariatric surgery database as having undergone rygb surgery at hershey medical center from 1999 to 2006 .
electronic medical records ( emrs ) were assessed on all subjects for iron supplementation , pre- and postoperative complete blood counts ( cbcs ) , ferritin levels , menstruation status at time of surgery , and length of the roux limb .
women were considered to be menopausal if their age was > 50 years at the time of surgery .
subjects were considered eligible for inclusion if a preoperative cbc and at least one postoperative cbc at > 6 months from the time of surgery were available .
anemia was defined as a hemoglobin < 12 g / dl for women and < 14 g / dl for men .
iv iron therapy was given at the discretion of the hematology consultant for patients with symptomatic iron deficiency anemia who were intolerant or refractory to oral iron supplements .
the conventional roux limb was between 100 and 150 cm and the pancreatobiliary limb between 40 and 50 cm .
all patients were instructed to take one of the more absorbable forms of oral iron , such as funarate / gluconate , 28 mg at bedtime , preferably with vitamin c 500 mg , and not within 2 hours of calcium supplements , in addition to calcium , vitamin d , a multivitamin containing vitamin a , and vitamin b12 supplements .
follow - up visits were routinely scheduled at 1 month with the dietician , at 3 months with the surgeon and dietician , at 9 months with the dietician , and at 12 months and then annually for 5 years with the surgeon . at 1 month
, patients were given an information sheet to be given to their primary physician requesting cbcs along with routine laboratory monitoring quarterly for the first year and annually thereafter .
univariate analyses using chi - squared test and student s t - test were performed between the outcomes ( incidence of anemia and iron deficiency anemia ) and variables ( sex , age at time of surgery , length of roux limb , oral iron supplementation , menstruation status , postoperative complications , and presence of preoperative anemia ) . since the follow - up time varied across subjects ,
particularly , kaplan meier curves were constructed to estimate the cumulative event rate of anemia over a 7-year time period .
the log - rank test was used to determine statistical significance of various patient characteristics as risk factors for developing anemia , and 95% confidence bands were constructed .
multivariable cox proportional hazards regression analysis was used to jointly model the effects of the characteristics identified by the log - rank test .
three - hundred and nineteen subjects met the inclusion criteria of a preoperative cbc and at least one postoperative cbc > 6 months from the time of surgery recorded in the emr .
two - hundred and sixty - nine ( 84% ) were women and 50 ( 16% ) were men .
one - hundred and forty - eight ( 55% ) of the women were menstruating at the time of surgery .
the median follow - up time of the 319 subjects was 56 months with an interquartile range 3171 months ( minimum 5.9 months , maximum 90.4 months ) .
subjects who did not meet the inclusion criteria had similar distributions in age , sex , weight , and body mass index ( bmi ) , except for a higher ( 73% ) proportion of women menstruating at the time of surgery .
preoperative hemoglobin levels were observed for 62% of the subjects who did not meet the inclusion criteria , with a mean ( standard deviation ) of 13.6 ( 1.3 ) g / dl compared to 13.4 ( 1.2 ) for the subjects who met the inclusion criteria .
twenty - two ( 61% ) of the 36 subjects tested , representing 18% of the 122 subjects with anemia , were found to be iron deficient using a standard cut - off of 10 ng / ml .
fourteen ( 64% ) of the 22 subjects with iron deficiency anemia were women of menstrual age . for patients who developed anemia ,
the average postoperative hemoglobin concentration measured at > 6 months postoperatively was 12.6 ( 1.1 ) g / dl in men and 10.7 ( 1.1 ) g / dl in women .
time to event analysis showed that the risk of developing anemia by 7 years post procedure was 58% ( fig .
the risk of postoperative anemia was significantly higher in the women who menstruated at the time of surgery compared to those who did not ( p = 0.002 ) .
suprisingly , the rate of development of anemia in men was similar to that of menstruating women ( fig .
one - hundred and seventy - five ( 55% ) subjects received supplemental oral iron after surgery .
however , postoperative iron replacement did not reduce the overall rate of anemia ( fig . 5 ) .
longer roux limbs seemed to be associated with the development of anemia at years 14 , but the overall difference in the whole study period did not reach statistical significance ( fig .
twenty - seven subjects representing 8.5% of our study cohort and 22% of our anemic subjects with intolerance or refractoriness to oral iron received iv iron sucrose or low molecular weight iron dextran for iron deficiency anemia with a hemoglobin of < 10 g / dl 1111 months postoperatively ( mean 51 , median 52 months ) ( fig .
the usual doses for each event were 1 g of iron sucrose given over three infusions , or a single infusion of 1 g of iron dextran .
twenty - four were women , of whom 18 ( 75% ) were under 51 years of age .
ten of the 27 also received red cell transfusions more than 1 year postoperatively ( mean 59 months , median 50 months ) , and two additional women received red cell transufsions but no iv iron 2 and 3 years postoperatively ( data not shown ) .
thirteen of the 27 subjects had hemoglobin values recorded before and after receiving intravenous iron ( table 1 ) .
all but one had a hemoglobin rise of > 2 g / dl within 46 weeks following the infusion .
the average pre - infusion hemoglobin was 9.1 g / dl and the average post - infusion hemoglobin for the those not receiving red cell transfusions was 12.4 g / dl .
menstruation status and presence of preoperative anemia were predictive of postoperative anemia in the univariate analysis ( table 2 , p = 0.005 and < 0.0001 , respectively ) . in the multivariable cox regression , menstruation status and the presence of preoperative anemia were also significant ( p = 0.0014 and 0.044 , respectively ) ( table 2 ) .
baseline characteristics , such as age and bmi , were not significant and thus were removed from the final model .
mammalian iron absorption requires the transfer of iron across both the apical and basolateral membranes of enterocytes .
this takes place preferentially in the duodenum and occurs in both nonheme and heme forms .
nonheme iron enters the cell through the divalent metal transporter dmt-1 and is transferred across the basolateral membrane to the circulation by ferroportin .
heme binds to its receptor hcp-1 and is absorbed directly into the duodenal enterocyte by a process that is not well understood.20,21 however , there is a gradient for nonheme iron absorption throughout the small intestine , at least in animals.2224 rygb is a procedure in which the distal stomach , duodenum , and proximal jejunum are bypassed , precluding exposure of the food bolus to these sites in the postoperative biliopancreatic limb .
food bolus exposure to the normal gastric acid gradient along the foregut is also altered .
therefore , exclusion of the duodenum from alimentary flow and ph alterations along mucosa with absorptive potential are both implicated in the development of iron deficiency anemia after rygb .
perioperative blood loss is unlikely to contribute significantly because the reported rate of bleeding requiring transfusion during or immediately after rygp is on the order of only 1%4%.25 our study is in agreement with previous reports that iron deficiency and iron deficiency anemia are significant complications following rygb .
reported rates of iron deficiency range from 15% to 60% and of iron deficiency anemia from 10% to 52% in retrospective studies.4,6,8,9,1115 however , only three of these studies have follow - ups of 4 years or more,4,9,11 and the true incidence of iron deficiency and iron deficiency anemia is difficult to ascertain . in a prospective study by ruz et al of 51 individuals ,
the percentage of anemic subjects changed from 1.5% at the beginning of the study to 38.8% at 18 months.3 in a larger prospective study by avinoah et al of a random group of 200 patients undergoing bariatric surgery , mean values of hemoglobin and iron showed a gradual and significant decrease 68 years after surgery;26 however , the actual values were not reported .
our study of 319 subjects showed a cumulative incidence of anemia of 58% by 7 years post procedure .
this is one of only three studies8,9,17 to systematically report long - term results beyond 5 years .
the main findings were that the rate of development of anemia in individual subjects continued to increase over at least 7 years , and that 8.5% of all 319 subjects and 22% of 122 anemic subjects received iv iron replacement with good response at an average of 4 years postoperatively .
eighteen percent of our anemic subjects were confirmed to have iron deficiency , consistent with published literature .
however , this is likely a gross underestimate , since only one - third had ferritin levels available for review , and of these , 61% had iron deficiency .
furthermore , serum ferritin alone may not be the optimal way to define iron deficiency , as it is an acute phase reactant that is generally higher in obese people , presumably secondary to underlying chronic inflammation.27 in this setting , our threshold of 10 g / l may have been too low .
previous studies have assessed serum iron and total iron binding capacity ( tibc ) , but these also are poor markers for iron deficiency because serum iron is influenced by iron absorption from meals , infection , inflammation , and diurnal variation , and tibc is affected by malnutrition , inflammation , chronic infection , and cancer
. a ferritin level of < 10 ng / ml is very specific for iron deficiency , but in older patients or those with inflammatory states one can not rule out iron deficiency until the ferritin level is > 100 ng / ml.28 guyatt et al found that the likelihood ratio for iron deficiency was positive up to a ferritin level of 40 ng / ml in the absence of inflammation and up to 70 ng / ml in the presence of inflammation.29 had we raised the serum ferritin threshold to 40 ng / ml , we would have increased our percentage of anemic patients with iron deficiency from 18% to 21% .
had we raised it to 70 ng / ml , 29 ( 24% ) of our anemic patients would have been classified as being iron deficient .
although raising the ferritin cutoff may improve the rate of detection of iron deficiency , an accurate assessment of iron deficiency in the setting of chronic inflammation awaits the development of a reliable test to measure hepcidin levels .
rates of anemia and iron deficiency were highest in menstruating women , 62% of whom became anemic by 7 years postoperatively .
this is greatly increased from the 12% incidence of iron deficiency in women of childbearing age in the national health and nutrition examination survey,30 and the 13% incidence of anemia in women of reproductive age in the who survey.31 iron supplementation did not appear to correct the development of anemia over time .
suprisingly , the rate of anemia in men was similar to that in menstruating women .
this may have been affected by the small sample size or the higher rate of preoperative anemia in the men .
the length of the roux limb was associated with development of anemia at post - surgical yearly intervals 14 , but lost significance by year 5 and was not significant in the multivariate analysis .
mechanistically , this early association could be due to greater malabsorption of nutrients in patients with longer limbs , with subsequent adaption of enterocytes facilitating iron absorption over time .
a gradient for nonheme iron absorption throughout the small intestine has been demonstrated in animals.2224 such an adaptive mechanism could explain why some patients absorb enough food and supplemental iron post gastric bypass surgery to prevent the development of anemia in the absence of other causes such as menstrual blood loss , occult gastrointestinal bleeding , or a deficiency of other micronutrients , which were not examined in this study .
the significant association of preoperative anemia with postoperative anemia is not unexpected , given that preexisting conditions other than the role of adipose tissue leading to low hemoglobin concentrations preoperatively would be unlikely to change with gastric bypass .
the fact that oral iron after surgery did not correct the anemia is not surprising , since oral iron is excluded from the biliopancreatic limb encompassing the excluded stomach , duodenum , and proximal jejunum .
a recent report has shown that iron absorption from a single oral dose of ferrous sulfate is reduced by more than 60% after rygb.32 published data identifying patients receiving iv iron supplementation following rygb are limited . in one study ,
anemia was corrected in most of 23 patients who received iron dextran post rygb for refractory iron deficiency anemia , with an 85% probability of remaining in an iron - replete state at 1 year.33 in another study , intravenous iron was required in 6.7%.17 in an earlier retrospective analysis of 42 bariatric surgery patients who received iv iron , premenopausal women required earlier replacement.34 this represented approximately 4% of the patients who underwent all forms of bariatric surgery at the parent institution during the study period .
clearly , there is a range of iron deficiency that is moderate and does not require iv iron .
however , a seminal observation in our study was that 27 subjects , representing 8.5% of our study cohort and 22% of anemic subjects , received iv iron infusions a mean of 44 months and a median of 32 months after surgery .
the vast majority of these ( 90% ) were women , of whom 75% were premenopausal .
increases of > 2 g / dl of hemoglobin within 6 months following iv iron infusions were observed in a subset of all but 1 of 13 subjects with data pre and post infusion . for this group
, the average hemoglobin rose from 9.1 to 12.4 g / dl , confirming the efficacy of iv iron replacement in subjects previously unresponsive or intolerant to oral iron supplements .
although currently approved iv iron preparations have been shown to have an excellent safety profile in multiple studies comprising thousands of subjects with iron deficiency anemia , all iron products can cause hypersensitivity or other reactions , some of which are severe.35 this small but potentially avoidable risk , plus the inconvenience and cost of repeated iron infusions , can have a major impact on quality of life .
only 35% met our inclusion criteria on review of the emr because the others had no follow - up cbc recorded 6 months or longer after surgery .
this low response rate may indicate a self - selection bias , with subjects more likely to return for follow - up if they were symptomatic from anemia or otherwise ill , thereby increasing the prevalence of anemia in our study population .
however , those who were excluded had similar distributions of age , gender , weight , and bmi , except for a higher proportion of menstruating women , which would argue strongly against a self - selection bias . additionally , only 30% of anemic subjects had ferritin levels recorded .
however , our overall rates of anemia , iron deficiency , and iron deficiency anemia were consistent with the published literature .
other limitations include lack of data regarding compliance and the coexistence of other micronutrient deficiencies such as b12 and folate deficiency , which have been shown to be substantial following rygb . despite a clearly articulated schedule requesting follow - up visits and cbcs at least annually for 5 years following rygb
, the lack of data for review in this single - institution study highlights the fragmented care received by this patient population .
the reported 5-year follow up rate of 1359 eligible patients in the international bariatric surgery registry was only 5.1%,36 and the 5-year follow - up at major bariatric centers is as low as 14% ( oral communication from matthew hutter , md , massachusetts general hospital ) .
when taken in this context , our 35% follow - up rate at 7 years is commendable .
clearly , a better system for long - term follow - up is needed to detect anemia from deficiencies of iron and other nutrients , which may take years to develop .
credentialing of bariatric centers of excellence includes recommended rates of annual follow - up that are difficult to achieve . traditionally , credentialed bariatric centers of excellence
have been required to demonstrate a plan for at least 5-year follow - up of patients undergoing weight loss surgery . while a plan may in fact be in place , follow - up of patients , particularly in countries without national health insurance , is generally poor .
patient mobility , changes in providers , loss of health insurance , cost of care , cost of supplements , lack of uniformity of electronic health records , and innumerable other factors limit patient follow - up .
more definitive guidelines , closer collaboration with primary care providers , and improved methods of tracking are needed to increase our understanding of the long - term consequences and to improve outcomes following rygb .
this is particularly true given the rise in popularity of bariatric surgery for the control of obesity and obesity - related comorbid conditions . in summary , we have shown a 58% cumulative incidence of anemia by 7 years post rygb .
iron deficiency anemia was demonstrated in 18% of a subset with ferritin levels available for review .
importantly , 8.5% , two - thirds of whom were women of menstrual age 50 years , and 22% of all subjects with anemia required iv iron supplementation a mean of 47 months postoperatively because of unresponsiveness or intolerance to oral iron .
well - designed prospective studies are needed to determine the magnitude of this under recognized longterm risk for the development of iron deficiency anemia after rygb and to better define those at increased risk for needing iv iron infusions .
additionally , translational studies to elucidate factors controlling iron uptake by the remaining alimentary surface are needed to identify adaptive mechanisms that may reduce the risk for development of iron deficiency following rygb surgery . | the frequency of anemia , iron deficiency , and the long - term need for iv iron following roux - en - y gastric bypass ( rygb ) surgery has not been well characterized .
three - hundred and nineteen out of 904 consecutive subjects who underwent rygb at penn state hershey medical center from 1999 to 2006 met the inclusion criteria for a preoperative complete blood count ( cbc ) and at least one cbc > 6 months following surgery .
cumulative incidence of anemia 7 years post procedure was 58% .
menstruation status and presence of preoperative anemia were predictive of anemia by univariate analysis and multivariable cox regression ( p = 0.0014 and 0.044 , respectively ) .
twenty - seven subjects , primarily premenopausal women , representing 8.5% of the cohort and 22% of the 122 anemic subjects , needed intravenous ( iv ) iron a mean of 51 months postoperatively for anemia unresponsive or refractory to oral iron .
the risk for development of anemia necessitating iv iron therapy following rygb is highest in menstruating women and continues to increase for many years , even in post - menopausal women .
well - designed prospective studies are needed to identify the incidence of iron deficiency anemia and the patient populations at increased risk for requiring iv iron replacement after rygb surgery . |
most of the bacteria existing in the oral cavity are present as complex aggregates known as biofilms on the surfaces of the teeth and these biofilms are termed dental plaques [ 12 ] .
streptococci are said to be the first bacteria which colonize in oral surfaces and may consist 70% of the cultivable bacteria existing in the human dental plaque and streptococcus mutans as the primry odontopathogen presents in the supragingival plaque and results in one kind of oral disease known as dental caries [ 34 ] . since both dental caries and periodontal disease start initially by plaque accumulation on the oral soft and hard tissues , conventional mechanical debridement and good oral hygiene may accomplish a temporary decrease of microorganisms in dental plaques . to overcome these problems , it is essential to develop new antimicrobial therapic approaches .
recently , vaccines for dental caries and periodontal diseases have been produced and applied on patients [ 67].one alternative method is photodynamic therapy ( pdt ) , showing great potential for the treatment of neoplastic and non - neoplastic diseases which was firstly demonstrated by jodlbauer and von tappeiner in 1904 [ 89 ] . in this method ,
a photoactive dye , termed a photosensitizer [ ps ] is taken up into the cells and is then irradiated with light of an appropriate wavelength .
generally , in photosensitization processes , the laser or ps alone are not toxic and only cells that contain the photosensitizer and also receive laser are affected by the treatment finally .
thus , the use of this method provides an opportunity to achieve selectivity and to target specific sites of the mouth or the plaque .
antibacterial photosensitizers currently under investigation for use in the mouth include tbo and chlorin e6 [ 1314 ] .
these agents show great promise , but will necessarily be subjected to lengthy experimental and clinical assessments .
one such photosensitizer is radachlorin which is a chlorophyll a derivative , including mainly sodium chlorin e6 , having been successfully applied in diagnosing tumors and treating surface tumors .
there have been only a few studies on the antimicrobial photodynamic therapic ( apdt ) effects of radachlorin , although there have been several studies on chlorin e6 , which is a major component of radachlorin . on the other hand ,
tbo is a widely known photosensitizer that has been in use for many years and is efficient in producing singlet oxygen under the maximum absorption wavelength of 630 nm .
tbo has also been reported as an effective dye for inactivation of yeasts , gram positive and gram negative bacteria in assistance with laser irradiation [ 1719 ] .
therefore , this prompted us to carry out an in vitro study on the subject of the antimicrobial photodynamic effect of radachlorin in comparison with tbo on the viability of streptococcus mutans to enhance pdt application in plaque - related disease treatment .
the standard strain of streptococcus mutans ( atcc 35668 , ptcc 1683 ) was purchased from the iranian science organization of science and technology ( irost ) in tehran , iran .
the bacterium was subcultured on mitis salivarious agar ( quelab , canada ) and then incubated at 37c in the presence of 10% co2 for 24 hours .
overnight cultures were prepared in trypticase soy broth ( merk , germany ) by transferring a few colonies grown on mitis salivarious agar .
the bacterial suspensions were then diluted in broth to an optical density of mcfarland no : 0.5 ( approximate numbers 1.510 bacteria ml ) .
radachlorin gel ( 0.1% , 25 g ) was obtained from rada - farma ltd , russia and stored at 08 c in the dark ( fig 1 ) .
toluidine blue powder was taken from micromedia chemicals - hungry , dissolved in sterile saline firstly to reach the final concentration of 0.1% and then subsequently kept in the dark ( fig 2 ) .
the laser sources used for each photosensitizer were a diode laser ( milon - lahta , russia ) ( fig 3 ) with a fiber optic diameter of 800 micrometer , a maximum output of 2.5 w and a predominant wavelength of 662 nm for radachlorin and the irradiation of a diode laser ( mustang 2000 , moscow , russia ) ( fig 4 ) with a hand held probe of klo4 and a maximum power output of 30 mw at a wavelength of 633 nm for tbo .
the distance between the laser tips and the illuminated area was adjusted to create a spot light of 1 cm in diameter with a fixed power density for each photosensitizer .
photodynamic therapy : the laser parameters used in this study for bacterial suspension were 100 mw / cm ( power density ) and 12 j / cm ( energy density ) and continuous mode for radachlorin and 30 mw / cm , 3 j / cm and continuous mode for toluidine blue o. the concentration of 0.2 ml of each photosensitizer was applied on 0.2 ml of the bacterial suspensions .
the following groups were used : ( i ) l ps ( no laser , no photosensitizer ) , ( ii ) l ps ( treated only with ps ) , ( iii ) l ps ( treated only with laser ) and ( iv ) l ps ( treated with laser and ps : photodynamic therapy group ) .
group i and ii were kept in the incubator at 37 c in the presence of 10% co2 .
bacterial suspensions in group iii and especially group iv which were incubated with ps for 10 minutes in the dark at room temperature , were exposed to 662 and 633 nm laser from above for 120 and 150 seconds in the dark at room temperature and subsequently transferred to the incubator .
after overnight incubation of all groups , they were cultured on mitis salivarious agar and viable microorganisms grown on the plates were counted in the next day . in order to access the differences between the groups , the variable bacterium reduction promoted by each treatment was analyzed by kruskal - wallis and mann - whitney u test .
statistical significance was accepted at p<0.05 . the statistical package for social sciences ( spss)16 for windows , (
the standard strain of streptococcus mutans ( atcc 35668 , ptcc 1683 ) was purchased from the iranian science organization of science and technology ( irost ) in tehran , iran .
the bacterium was subcultured on mitis salivarious agar ( quelab , canada ) and then incubated at 37c in the presence of 10% co2 for 24 hours .
overnight cultures were prepared in trypticase soy broth ( merk , germany ) by transferring a few colonies grown on mitis salivarious agar .
the bacterial suspensions were then diluted in broth to an optical density of mcfarland no : 0.5 ( approximate numbers 1.510 bacteria ml ) .
radachlorin gel ( 0.1% , 25 g ) was obtained from rada - farma ltd , russia and stored at 08 c in the dark ( fig 1 ) .
toluidine blue powder was taken from micromedia chemicals - hungry , dissolved in sterile saline firstly to reach the final concentration of 0.1% and then subsequently kept in the dark ( fig 2 ) .
the laser sources used for each photosensitizer were a diode laser ( milon - lahta , russia ) ( fig 3 ) with a fiber optic diameter of 800 micrometer , a maximum output of 2.5 w and a predominant wavelength of 662 nm for radachlorin and the irradiation of a diode laser ( mustang 2000 , moscow , russia ) ( fig 4 ) with a hand held probe of klo4 and a maximum power output of 30 mw at a wavelength of 633 nm for tbo .
the distance between the laser tips and the illuminated area was adjusted to create a spot light of 1 cm in diameter with a fixed power density for each photosensitizer .
photodynamic therapy : the laser parameters used in this study for bacterial suspension were 100 mw / cm ( power density ) and 12 j / cm ( energy density ) and continuous mode for radachlorin and 30 mw / cm , 3 j / cm and continuous mode for toluidine blue o. the concentration of 0.2 ml of each photosensitizer was applied on 0.2 ml of the bacterial suspensions .
the following groups were used : ( i ) l ps ( no laser , no photosensitizer ) , ( ii ) l ps ( treated only with ps ) , ( iii ) l ps ( treated only with laser ) and ( iv ) l ps ( treated with laser and ps : photodynamic therapy group ) .
group i and ii were kept in the incubator at 37 c in the presence of 10% co2 .
bacterial suspensions in group iii and especially group iv which were incubated with ps for 10 minutes in the dark at room temperature , were exposed to 662 and 633 nm laser from above for 120 and 150 seconds in the dark at room temperature and subsequently transferred to the incubator .
after overnight incubation of all groups , they were cultured on mitis salivarious agar and viable microorganisms grown on the plates were counted in the next day .
in order to access the differences between the groups , the variable bacterium reduction promoted by each treatment was analyzed by kruskal - wallis and mann - whitney u test .
statistical significance was accepted at p<0.05 . the statistical package for social sciences ( spss)16 for windows , ( spss inc , chicago , il , usa )
the growing bacterial resistance in spite of antibiotic drugs , conventional mechanical debridement and chemical agents has questioned the efficiency of these therapies . to overcome these problems ,
photodynamic therapy has become a possible alternative antibacterial therapy for plaque - related diseases such as dental caries .
the advantages of pdt over conventional antimicrobial agents are non - invasive nature , ease of reaching deeply situated areas , repeatability , high selectivity , no resistance to drugs , rapid killing of target microorganisms in a few minutes depending mainly on the energy densities delivered ( on the contrary , in conventional antimicrobial agents hours or even days are necessary ) and finally that antimicrobial effects may be limited to the site of the lesion by careful topical application of photosensitizers and the site of irradiation may even be restricted further by using an optical fiber [ 2122 ] .
in fact , in the human oral cavity , there are very large numbers of bacterial species , which comprise a complex ecosystem .
thus , the response of the bacterial community to photodynamic treatment may differ greatly from that of their in vitro cultured isolates in many aspects , such as growth rate , metabolic activity and gene expression [ 2324 ] .
wainwright also demonstrated that photodynamic inactivation ( pdi ) of microorganisms depends on the chemical structure of ps and the incubation time of the drug with the bacterial cells .
damage to the bacterial cell wall , increased permeability of cytoplasmic membrane and nucleic acid strand breakage may be resulted following with pdi .
based on these advantages , several studies were carried out using pdt approving that oral bacteria are susceptible to pdt [ 2627 ] .
photosensitizers are vital elements in pdt ; several studies have demonstrated the efficacy of a range of photosensitizers in the elimination or reduction of oral bacteria [ 11,2829 ] .
tbo is an attractive option because of its affordable cost and intense absorption wavelength in the red light spectrum ( > 600 nm ) , while radachlorin is a chlorophyll a derivative , including mainly sodium chlorin e6 , which has already passed complete pre - clinical assessments .
these clinical trials have clarified significant advantages ; such as very low toxicity in the dark , high contrast of tumor accumulation , much more rapid body evacuation ( only two days ) , intensive absorption band at relatively large wavelengths where tissues are more transparent and finally the high phototoxicity .
soukos , rovaldi and pfitzner determined the antimicrobial activity of chlorin e6 derivatives like poly - l - lysine chlorin e6 conjugates and new photosensitizer blc1010 , blc 1014 on anaerobic bacteria compared with pure chlorin e6 .
risovannaia also reported that radachlorin-mediated photodynamic therapy could eliminate streptococcus pyogens in the animals infected tissues .
the results obtained in this study demonstrated that tbo - mediated photodynamic therapy was more successful compared to radachlorin in effective bacterial reduction .
the most effective combination is 0.1% tbo with 3 j / cm laser at 30 mw .
our findings regarding tbo are in accordance with those previous studies which have shown that it is possible to kill periodontal bacteria by using low concentration of toluidine blue and low energy densities [ 3537 ] .
although the bacterial count reduction in 3 j / cm laser irradiation alone was more than our expectation , it may be explained by attenuation in bacterial growth as fastidious microorganism .
one explanation for the decreased photodynamic effect of radachlorin may be that extra radachlorin molecules which could not bond to the bacterium excessively spend the limited oxygen molecules dissolved in the suspension and would reduce available oxygen for the photosensitizer molecules inside or close to the bacterium . another explanation may be defined according to wainwright s study which was carried out on fotolon as a major component of radachlorin indicating that high power density over a short time period may give different antimicrobial effects in comparison with lower power density over a longer time even with the same energy density in both cases .
he also declared that the reduced photobleaching rates for higher chlorin e6 concentrations may be explained by the self - shielding effect . in a higher concentration of the dye , the distance traveled by the excitation light may be reduced due to its loss in intensity . in such a case , superficial layers of the dye absorb the laser very efficiently , but they prevent its penetration into deeper layers .
finally , the photobleached superficial layers become transparent while deeper layers still strongly absorb the laser .
this is why radiant exposures for a highly concentrated photosensitizer may be underrated , leading to reduced pdi efficiency . during the antibacterial experiments , the energy density of 15 j / cm under 60 sec illumination could not completely photobleach chlorin e6 solution and in fact , a higher dose ( such as a dose higher than 30 j / cm )
in conclusion , our results demonstrate that the association of tbo with a diode laser and the energy density of 3 j / cm may be more effective in reducing the viability of streptococcus mutans pure cultures compared with radachlorin-mediated 12 j / cm laser irradiation . | objectives : dental caries and periodontal diseases are caused by infection of teeth and supporting tissues due to complex aggregate of bacteria known as biofilm , firstly colonized by streptococci .
the main purpose of this in vitro study was to evaluate the antimicrobial effects of toluidine blue o ( tbo ) and radachlorin in combination with a diode laser on the viability of streptococcus mutans.materials and methods : bacterial suspensions of streptococcus mutans were exposed to either 0.1% tbo associated with ( 20 mw , 633 nm diode laser , continuous mode , 150 s ) or 0.1% radachlorin and laser irradiation ( 100 mw , 662 nm diode laser , continuous mode , 120 s ) .
those in control groups were subjected to laser irradiation alone or tbo
/ radachlorin alone or received neither tbo / radachlorin nor laser exposure .
the suspensions were then spread over specific agar plates and incubated aerobically at 37c .
finally , the bactericidal effects were evaluated based on colony formation.results:potential bacterial cell killing was only observed following photosensitization with tbo and 3 j / cm2 laser exposure ( p<0.05 ) , whereas radachlorin showed significant reduction in dark condition compared to laser exposure ( p<0.05).conclusion : tbo - mediated photodynamic therapy seems to be more efficient than radachlorin in significantly reducing the viability of streptococcus mutans in vitro . |
to prevent medical accidents , users must be informed of the cautions written on the package inserts of medicines . these are exclusive legal documents that describe detailed information for each drug , such as the composition , efficacy , dosage , and cautions .
however , since the descriptions in package inserts are excessive and there are over 20,000 medicines in japan , it is not easy for healthcare workers to read all documents . as a countermeasure ,
the utilization of information technology has been focused on in recent years . to develop an information system that alerts healthcare workers at the user interface of an ordering system and an electronic health record system , we need to implement a drug information database as a source of information alerts . in order to utilize the same in information systems , the pharmaceutical and medical devices agency (
pmda)1 has provided standard general markup language ( sgml ) formatted data of package insert information since the year 1999.2 some medical institutions have developed drug databases based on the sgml data to retrieve drug information.3 however , this is not easy since the descriptions in package inserts are too complex and their information poorly structured.48 in this sgml data , most information is directly stored as statements and terms have not been standardized or encoded . since computer systems can not interpret drug information , existing systems can not check prescriptions and administration based on a drug information database . in order to develop such systems ,
it is necessary to develop a drug information database which has a structure suitable for the purpose . in this paper
the reason for analyzing precautions for application data is that it contains information concerning the usage situation that must be known at the time of prescription .
we obtained the sgml data from the pmda web site , utilizing the yj code as a key , which is a drug identification code and included in the standard drug master ( 9/30/2007 ) provided by the medical information system development center ( medis - dc).9 there were 11,685 sgml files .
in our target sgml data , since precautionary statements are described in the precautionsforapplication field ( as shown in figure 1 ) , we extracted text data from the item and detail fields in the same .
after this extraction , we split each sentence and converted 1 byte characters to 2 bytes . in this study , we removed the sentences described in parentheses , since these can be regarded as additional information . as a result
additionally , we applied dependency analysis to the extracted statements via software named cabocha ( v0.53 ; nara institute of science and technology , nara , japan).10 this technique extracts words , parts of speech , segments , and dependency relations between each segment by probabilistic calculation based on defined grammar and dictionary data . in this study , we investigated them by using information such as morphemes , segments , dependency relations , and parts of speech provided by the software .
since predicate words are often described at the end of the sentence in japanese , we extracted only headwords in the final segment from the results of dependency analysis .
( must ) was the most commonly occurring word , with a frequency of 83% .
it emerges with meanings of order , if we place it after verbs , eg ,
( must ) , although there are other order expressions that are weaker , eg ,
( desirable ) and that of possibility
( can ) . to extract information concerning the order description , we collected verbs modifying
( must ) in the previous subsection and obtained 415 kinds of headwords in table 2 . in this table , we can see that there are verbs expressing direct orders such as
( use ) , causative orders such as
( make someone use ) , direct interdictions such as
( do not use ) , and causative interdictions such as
( do not make user use ) . since the descriptions of order and interdiction coexist in precautionary statements ,
therefore , it is necessary to make order / interdiction flags in the database and to provide medical staff with the flags separately by information systems .
furthermore , since direct and causative statements coexist , targets to be ordered are not clear .
although humans can read them and understand targets , it is difficult to make computers interpret targets . in order to alert to appropriate targets , it is necessary to identify the appropriate targets to be alerted .
focusing on these points , we categorized the statements into four categories : direct orders , causative orders , direct interdictions and causative interdictions . in order to summarize information for each category ,
we tried to employ the word - linking method.11 however , we can not obtain relations between verbs and subjects / objects using this technique because the method extracts only nouns , verbs , and adjectives . to extract such relations , we focused on the case markers ( particles ) appearing at the end of segments that modify the verbs .
these are words that indicate the dependency relationship of a certain word to a predicate verb .
although we obtained three types of case markers using cabocha : case markers general ,
case markers collocation , and case markers citation , we used only case markers general , since it accounted for 90.65% and there were fewer case markers in that type than others . in this study , we focus on the case markers ,
( wo ) ,
( ni ) ,
( ga ) ,
( kara ) ,
( de ) ,
we aggregated ternaries , a verb , a case marker , and the verb modifier , which also appears in the segment containing the case marker .
after aggregation , we visualized the ternaries as nodes of a network to find major information in the order statement concerning the four cases , which are direct order , interdiction , causative order , and causative interdiction .
in the network , the nodes are collected verbs , case markers , and keywords , and the edges express the connections among them , while their frequencies are expressed as their width .
the threshold of appearance frequencies in the data limits the nodes and edges in figures 25 .
( use ) ,
( be careful ) , and
( stop ) ; we can also see statements such as
( use for eye drops ) ,
though it is an order to avoid , it can be interpreted as an interdiction , since the avoidance is implied in the concept of interdiction .
figure 3 shows that the major expressions concern the interdiction of usage . in particular , we can see an interdiction to apply medicines to the affected areas , such as
( do not apply to inflammatory part )
moreover , we obtained statements that indicate an interdiction of mixing drugs , eg ,
the first is for medical experts and patients , and another type is for medicines .
the former gives examples such as
( make patients take the medicine without chewing ) ,
( make patients take the drug after a meal ) , and
( make patients take the medicine with water ) .
the latter gives examples such as
( neutralize ) and
( dry ) .
they express methods for the preparation and storage of medicines . as causative order statements already mentioned , the subjects of the former include both humans ( pharmacists , nurses and patients ) and medicines . in figure 5 ,
statements such as
( do not make patients avoid water ) and
( do not regurgitate from syringe ) can be found . based on these results , we found two points concerning precautionary statements that should be considered .
since this may lead to the misreading of these statements , they should be described separately .
the second point is that we should distinguish two kinds of subjects of statements , namely patients and medical experts .
this is important for system checking because the system must know the subjects to whom warnings should be given .
since predicate words are often described at the end of the sentence in japanese , we extracted only headwords in the final segment from the results of dependency analysis .
( must ) was the most commonly occurring word , with a frequency of 83% .
it emerges with meanings of order , if we place it after verbs , eg ,
( must ) , although there are other order expressions that are weaker , eg ,
( must ) in the previous subsection and obtained 415 kinds of headwords in table 2 . in this table
, we can see that there are verbs expressing direct orders such as
( use ) , causative orders such as
( make someone use ) , direct interdictions such as
( do not use ) , and causative interdictions such as
( do not make user use ) . since the descriptions of order and interdiction coexist in precautionary statements ,
therefore , it is necessary to make order / interdiction flags in the database and to provide medical staff with the flags separately by information systems .
furthermore , since direct and causative statements coexist , targets to be ordered are not clear .
although humans can read them and understand targets , it is difficult to make computers interpret targets . in order to alert to appropriate targets , it is necessary to identify the appropriate targets to be alerted .
focusing on these points , we categorized the statements into four categories : direct orders , causative orders , direct interdictions and causative interdictions . in order to summarize information for each category
, we tried to employ the word - linking method.11 however , we can not obtain relations between verbs and subjects / objects using this technique because the method extracts only nouns , verbs , and adjectives . to extract such relations , we focused on the case markers ( particles ) appearing at the end of segments that modify the verbs .
these are words that indicate the dependency relationship of a certain word to a predicate verb .
although we obtained three types of case markers using cabocha : case markers general ,
we used only case markers general , since it accounted for 90.65% and there were fewer case markers in that type than others . in this study , we focus on the case markers ,
we aggregated ternaries , a verb , a case marker , and the verb modifier , which also appears in the segment containing the case marker .
after aggregation , we visualized the ternaries as nodes of a network to find major information in the order statement concerning the four cases , which are direct order , interdiction , causative order , and causative interdiction .
in the network , the nodes are collected verbs , case markers , and keywords , and the edges express the connections among them , while their frequencies are expressed as their width .
the threshold of appearance frequencies in the data limits the nodes and edges in figures 25 .
( use ) ,
( be careful ) , and
( stop ) ; we can also see statements such as
( use for eye drops ) ,
( use after washing ) . it should be noted that the statement
though it is an order to avoid , it can be interpreted as an interdiction , since the avoidance is implied in the concept of interdiction .
figure 3 shows that the major expressions concern the interdiction of usage . in particular , we can see an interdiction to apply medicines to the affected areas , such as
( do not apply to inflammatory part )
moreover , we obtained statements that indicate an interdiction of mixing drugs , eg ,
( do not mix with drugs ) . in figure 4 , we see two types of causative verbs .
the first is for medical experts and patients , and another type is for medicines .
the former gives examples such as
( make patients take the medicine without chewing ) ,
( make patients take the drug after a meal ) , and
the latter gives examples such as
( neutralize ) and
( dry ) .
they express methods for the preparation and storage of medicines . as causative order statements already mentioned , the subjects of the former include both humans ( pharmacists , nurses and patients ) and medicines . in figure 5 ,
statements such as
( do not make patients avoid water ) and
( do not regurgitate from syringe ) can be found .
based on these results , we found two points concerning precautionary statements that should be considered .
since this may lead to the misreading of these statements , they should be described separately .
the second point is that we should distinguish two kinds of subjects of statements , namely patients and medical experts .
this is important for system checking because the system must know the subjects to whom warnings should be given .
( use ) ,
( be careful ) , and
( stop ) ; we can also see statements such as
( use for eye drops ) ,
though it is an order to avoid , it can be interpreted as an interdiction , since the avoidance is implied in the concept of interdiction .
figure 3 shows that the major expressions concern the interdiction of usage . in particular , we can see an interdiction to apply medicines to the affected areas , such as
( do not apply to inflammatory part )
moreover , we obtained statements that indicate an interdiction of mixing drugs , eg ,
the first is for medical experts and patients , and another type is for medicines .
the former gives examples such as
( make patients take the medicine without chewing ) ,
( make patients take the drug after a meal ) , and
( make patients take the medicine with water ) .
the latter gives examples such as
( neutralize ) and
( dry ) .
as causative order statements already mentioned , the subjects of the former include both humans ( pharmacists , nurses and patients ) and medicines . in figure 5 ,
statements such as
( do not make patients avoid water ) and
( do not regurgitate from syringe ) can be found . based on these results , we found two points concerning precautionary statements that should be considered .
since this may lead to the misreading of these statements , they should be described separately .
the second point is that we should distinguish two kinds of subjects of statements , namely patients and medical experts .
this is important for system checking because the system must know the subjects to whom warnings should be given .
in the previous section , we found words that express the time to perform an action eg ,
orders should be provided to medical staff and patients at the appropriate time . in order to clarify correspondences between timing and orders , we extracted words representing timing from the target data and classified them based on procedures to use drugs .
initially , we compiled keywords where the part of speech is an adverbial noun ( table 3 ) .
( when ) as feature words that express the concept of timing , since most segments expressing timings contain these words .
the precautionary statements contain mainly two types of descriptions , one of which is that the order is described after the timing expressions in statements eg ,
( be careful at the time of use ) , and another of which where the timing expression appears singly eg ,
( delivery time of medicines ) in the
< item > field of sgml data . in the former case , we extracted keywords that modify the verb expressing the order and whose segments contain the feature words defined above . in the latter case , we extracted keywords that are at the tail of sentences and have feature words .
the resultant keywords contained those with similar meanings . to reduce the variety of expressions , we identified such synonymous expressions by hands .
we investigated the top 50 extracted words and found six groups : at the time of delivery , at the time of preparation , in use ,
we found that the caution related to in use was the largest group . utilizing this classification
, we obtained characteristic words that express an order and appear only at specific instances , eg ,
( dispose of ) after use and
( store under light interception ) at times of storage .
the obtained timing for alerts are vital to prevent accidents in order for the system to specify the time at which to send an alert .
in this study , we analyzed precautionary statements in the package inserts of medicines using a text - mining method to develop a drug information database . consequently , we found the major statements used to express orders and interdictions in use .
moreover , we obtained points of consideration concerning the targets of orders in the statements and timing of their execution . in this study , although we applied several methods to text data written in japanese , our methods can be employed for any language if a dependency analysis is applied to them .
we can also use prepositions in english or other languages instead of particles in japanese language .
this suggests that our method is applicable to languages other than japanese . utilizing the knowledge obtained
it should contain information such as the actions described in the statement , the flag to express an order or interdiction , the subject to be ordered , and the timing . in the future , this database will be accessed from various systems such as ordering systems and portable information terminals for nurses and patients , and these systems will provide orders and cautions to the appropriate targets at the appropriate time .
namely , the systems will provide orders of administration methods to patients and/or nurses just before administration . in future work
, we will analyze other information in the package inserts and develop a drug information database based on the results . | backgroundto prevent medical accidents , users must be informed of the cautions written in medical package inserts . to realize countermeasures by utilizing information systems
, we must also implement a drug information database . however , this is not easy to develop , since the descriptions in package inserts are too complex and their information poorly structured .
it is necessary to analyze package insert information and propose a data structure.methodswe analyzed the descriptions of
precautions for application in package inserts via text mining methods . in order to summarize statements , we applied dependency analysis to statements and visualized their relations between predicate words and other words .
furthermore , we extracted words representing timing to execute the order.resultswe found that there are four types of statements : direct orders such as
( use ) , causative orders such as
( make someone use ) , direct interdictions such as
( do not use ) , and causative interdictions such as
( do not make user use ) . as for words representing timing , we extracted six groups : at the time of delivery , at the time of preparation , in use ,
after use , and at the time of storage .
from these results , we obtained points of consideration concerning the subjects of orders in the statements and timing of their execution.conclusionfrom the obtained knowledge , we can define the information structure used to describe the precautionary statement .
it should contain information such as the actions described in the statement , the flag to express an order or interdiction , the subject to be ordered , and the timing . |
cholangiograms through this route are frequently performed to assess the biliary tree prior to removing the t - tube and discharging the patients from hospital . however ,
utilization of t - tube for percutaneous cholangioplasty has been described in a few case reports only .
the pain was mild - to - moderate in severity , intermittent , with occasional vomiting .
there was no radiation of pain , and the patient had little relief with analgesics .
the patient had decreased urine output over the past 24 h. he was a chronic smoker .
there was mild elevation of liver enzymes [ aspartate transaminase ( ast ) 87 iu / l , alanine transaminase ( alt ) 95
abdominal usg ( iu22 matrix , philips , best , netherlands ) revealed echogenic contents within gallbladder ( gb ) lumen .
there was no intrahepatic biliary radical ( ihbr ) dilatation and the pancreas was normal . a magnetic resonance cholangiography ( magnetom aera , siemens , erlangen , germany ) was performed that revealed mild dilatation of cbd and intrahepatic biliary radicles [ figure 1 ] .
there was worsening of the abdominal pain over the ensuing few days associated with progressive elevation of conjugated bilirubin ( 7.5 mg / dl ) .
a naso - biliary drainage ( nbd ) tube was placed which resulted in decline of conjugated bilirubin to 4.4 mg / dl .
cholangiogram was performed through the nbd after 1 week , which revealed filling defects within the gb and cbd suggestive of blood clots .
thick slab magnetic resonance cholangiopancreatography ( mrcp ) image reveals mild dilatation of the entire common bile duct ( cbd , arrow ) with smooth distal tapering ( short arrow ) .
no filling defects are seen within the cbd the main pancreatic duct is of normal calibre ( arrowhead ) patient continued to deteriorate with a falling hemoglobin suggesting ongoing hemobilia .
a vascular cause [ pseudoaneurysm or arteriovenous ( av ) malformation ] was suspected and computed tomography ( ct ) angiography ( somatom definition flash , siemens , erlangen , germany ) and digital subtraction angiography ( allura xper fd 10 , best , netherlands ) was performed .
however , 4 weeks later , there was mild rise in the serum bilirubin as well as alkaline phosphatase .
there were filling defects within the proximal cbd with no opacification of ihbr suggestive of an inflammatory stricture at or just beyond the confluence [ figure 2 ] .
a 0.035 guidewire was passed into the left ductal system across the stricture at the confluence .
after removal of the t - tube , a 7f 35 cm sheath was inserted over the wire and advanced into the biliary tree .
balloon dilatation was performed using a 10 mm diameter by 40 mm long angioplasty balloon ( advance , atb pta dilatation catheter ; cook medical , bloomington , in , usa ) , inflated for 10 min .
we also did cholangioplasty of the lower end of cbd as filling defects were seen in the proximal cbd on the t - tube cholangiogram suggesting distal stasis .
repeat balloon dilatation of the hilar stricture was done after 3 weeks . a temporary external drainage tube [ 12f percutaneous transhepatic biliary drainage catheter ( ptbd catheter ) ]
was placed with its tip just across the stricture , after each session of cholangioplasty .
cholangiography via the biliary catheter following the procedure revealed good opacification of the entire biliary tree [ figure 3 ] .
the external biliary drainage catheter was finally removed 3 weeks after the second session of cholangioplasty .
patient was followed with serial evaluation of serum bilirubin , alkaline phosphatase , and ultrasound examinations . at the last follow - up ,
t - tube cholangiogram reveals lack of contrast opacification of the common hepatic duct and intrahepatic biliary radicles ( arrowheads ) .
note the opacified cbd ( arrow ) and t - tube in situ ( short arrow ) cholangiogram performed through percutaneous transhepatic biliary drainage catheter ( ptbd catheter , arrow ) following balloon dilatation of stricture reveals good opacification of the entire biliary tree
various treatment options for benign biliary strictures include endoscopic sphincterotomy , percutaneous cholangioplasty / stenting , and surgical biliary enteric diversion .
surgical diversion is best reserved for patients with anastomotic , extrahepatic , extra - pancreatic biliary stricture .
thus , in majority of patients , interventional radiology techniques in the form of percutaneous cholangioplasty / stenting are employed . however , a successful and long - term favorable outcome involves consideration of several important issues .
traumatic , post - transplant , post - infective , etc . ) , site of stricture ( viz .
hilar , proximal or distal cbd , peri - ampullary ) , presence of any pre - existing access ( viz .
t - tube , ptbd , hutson russell loop ) , and length of stricture are all expected to have a bearing on the results of cholangioplasty .
cholangioplasty involves dilatation of the stricture using a transluminal angioplasty balloon ( balloon cholangioplasty / biliary balloon dilatation ) .
it involves access to the intrahepatic biliary radicles through percutaneous puncture , either under ultrasound guidance ( when the ducts are dilated ) or fluoroscopic guidance after obtaining a cholangiographic picture following a central puncture ( when there is minimal ductal dilatation ) .
two options follow : ( a ) placing an internal - external drain catheter and upgrading the catheter every 3 weeks to a larger caliber to dilate the tract or ( 2 ) balloon dilatation of the stenotic segment every 3 weeks , upsizing the balloon size every session .
former approach leads to reduced quality of life and the external catheter is prone to infections .
alternative access routes have been rarely described . when there is indication of repeated dilatations for biliary stenoses and endoscopic access is not available , a transjejunal approach has been described . in these cases ,
the dilatation is performed through the fixed limb ( either afferent or efferent limb ) of a roux - en - y hepaticojejunostomy . in this context , it is helpful to remember that the efferent limb of the roux loop is attached to the peritoneum in the right anterior location and the afferent limb is fixed anteriorly in the central upper abdomen . under fluoroscopic or ct guidance , the selected loop is punctured using a thin needle .
subsequently , the biliary tree is catheterized in a retrograde fashion and bilioplasty is performed .
fontein , et al . described their experience in 494 patients who were planned for cholangioplasty through the roux loop . in 86% of the interventions ,
another potentially useful , yet a rarely described route for balloon dilatation , is the t - tube .
t - tube is left in place following surgery for benign or malignant disease . in cholelithiasis with suspected choledocholithiasis , following cbd exploration , the t - tube is left in place to facilitate endoscopic treatment through the mature tract .
t - tube placement is also performed in a difficult biliary - enteric anastomosis and extensive invasion of the distal bile duct .
kim , et al . reported three cases of biliary stent placement through the t - tube .
the cause of biliary obstruction in these cases was malignant disease process in the cbd or peri - ampullary region .
the procedure was successful in all three cases and follow - up did not show recurrence of jaundice .
in conclusion , unconventional access routes for cholangioplasty constitute a viable treatment option for benign biliary strictures . | percutaneous cholangioplasty is a commonly performed procedure for both benign and malignant diseases .
the most common route for accessing the biliary tree is transhepatic , following ultrasound or fluoroscopic - guided percutaneous puncture .
there are situations when alternative routes can be utilized to access the common bile duct ( cbd ) .
we accessed the cbd via t - tube placed surgically in a 57-year - old man who had obstructive jaundice of obscure etiology which was likely inflammatory . |
there is growing belief that administering emergency medical care is a key skill for physicians .
but as early as 1981 , an article in the lancet reported a lack of skills among house - officers being tested on cardiopulmonary resuscitation.1 similar results were found in studies at the addenbrooke s hospital of cambridge ( uk ) in 19842 and the wellington school of medicine ( new zealand ) in 1989.3 a study from 1989 among family physicians and pediatricians in chicago ( usa ) reported that they were not trained in basic life support at all.4 possible inadequate competence of physicians relating to aspects of emergency care has later been confirmed by studies from australia ( 1999),5 the united states ( 2001),6 norway ( 2001),7 denmark ( 2002),8 south africa ( 2005)9 and the netherlands ( 2006),10 indicating that little progress has been made since . in belgium , no similar studies have been performed , but our own research in 2008 among medical students and junior graduates in their postgraduate training to become a family physician suggest at least a low confidence in their own skills and knowledge regarding emergency medical care .
a number of publications express the need for better education in medical emergency skills , and better and more accessible training modules for physicians.11,12,13 also , the need for repetitive and continuous re - education has been illustrated by numerous studies,1417 suggesting that 6 months is the optimal time interval with which skills should be recapped.1820 based on these scientific results , initiatives to improve education in clinical skills and emergency care were set up in several countries .
these initiatives involve , for example , reviewing of university curricula in medicine,21 supporting rural family physicians in acquiring and retaining urgency skills,22,23 formulating clearer final qualifications for medicine graduates,24,25 evaluating clinical skills using objective structured clinical examinations11 and incorporating clinical skills assessments in medical license exams.25 even though the study groups and assessed skills were rather heterogeneous among the different publications , we can safely say that practical knowledge of emergency medical care among physicians seems to be insufficient worldwide . however , almost no data is available concerning physicians in belgium.11 it is essential for belgian family physicians to be able to manage emergency conditions .
basic emergency care is done by family physicians within their own practice or during on - call hours . especially outside of the cities ,
people usually do nt seek care in emergency departments without first seeing their own family physician .
belgium is said to have one of the highest health care standards in the world , which implies that its physicians are probably confident and skilled .
the aim of this study was to ascertain how confident dutch - speaking family physicians in belgium feel about their ability to give adequate emergency medical care and to examine their assessment of knowledge of relevant medical conditions .
all local family physician organizations of dutch - speaking physicians in belgium were asked to invite their members to take part in our study in september 2009 .
a convenience sample consisting of 974 family practitioners was contacted through email and asked to complete a web - based questionnaire .
physicians in their postgraduate training to become a family physician were also included , since they work independently as qualified physicians and are an integral part of the medical corps in belgium .
we opted for this subgroup of belgian physicians because they consist of a homogenous group .
the two main language groups in belgium ( french and dutch ) have a different organization of medical studies .
curricula are different , and contrary to their french - speaking counterparts , flemish physicians have been trained in an interuniversity setting for more than 25 years . because the vast majority of dutch - speaking physicians work in flanders and brussels , we performed our study only in these two parts of the country .
we opted for an online questionnaire , hosted on server space of the vrije universiteit brussel .
cookies as well as ip address analysis were used to avoid single users filling in the questionnaire multiple times .
for each skill , the participant was asked for a self - assessment , using a likert scale with the following possibilities : 0 , has no experience or knowledge whatsoever ; 1 , has theoretical knowledge ; 2 , has had a demonstration of the skill or has observed it ; 3 , is able to perform the skill independently ; and 4 , has proficiency in the respective skill . the second part consisted of 31 questions ascertaining the participant s knowledge of conditions relevant to urgent medical care .
again , a likert scale was used as follows : 0 , has never heard of the disease or condition ; 1 , has heard of the disease / condition but is unable to manage it ; 2 , can manage the disease / condition ; 3 , can diagnose the disease / condition ; and 4 , can independently treat the disease / condition .
our questionnaire was based on a list used by hekkert in a similar study in the netherlands.21 a test study has been performed on medical students , using the same methodology and questionnaire.11 using the information gained from that study , changes were made to scales , descriptions , questions , and questionnaire setup .
however , we limited these changes to the essential ones , in order to maintain our possibility to compare .
it is unclear in what way the original instrument was validated by the composers , but since hekkert used the validated criteria set from the dutch raamplan 200124 and based on consistency within and comparison with other studies using the same questionnaire , we estimated the questionnaire to be valid and reliable .
the dutch raamplan is also used in belgium , as a basis for the final qualifications in medical studies .
the participants were divided into subgroups : based on sex , age ( five groups ) , geographical location ( rural , urban , metropolitan ) , size of the family practice ( four groups ) , and on the organization of the practice ( five groups ) .
they were also asked if and when they had taken any additional training in emergency care .
the context of this training was not further defined , meaning that every initiative being regarded as additional training by the participant was included . for each participant , a mean score for skills and
although a decimal score on a likert scale has no meaning as such , we assumed that means would provide more information than the raw score itself .
questionnaires in which a participant indicated the same score on each question , or where the pattern of answers showed a consequent algorithm , were assumed to be intentionally misleading and were excluded .
the raamplan states that at the end of the medical studies , a physician should be able to perform skills in an appropriate manner , not always by acting independently but also by referring out , if needed.24 in our opinion , having had a demonstration of a skill ( likert score 2 ) does not comply with this criterion .
therefore , we set the minimum criterion to meet our standards at likert score 3 , meaning the participant was able to perform the skill independently . even though it could be theoretically assumed that a licensed physician meets this criterion in all mentioned skills , this assumption is highly unrealistic .
hence , we assumed that a participant met our standards in a reasonable manner when scoring 3 on at least half of the questioned skills .
for assessing knowledge of relevant conditions , we used the appendix to the raamplan , which differentiates between the conditions .
we assumed that a participant fulfilled the criteria in a reasonable way if he or she met the set criterion on at least half of the questioned pathologies . for statistical interpretation , we used spss statistics for windows , version 19.0 ( ibm corporation , armonk , ny , usa ) . due to the small subgroups and the presence of some extreme results
, we could not assume a normal distribution of our data , as was indicated by a shapiro
however , we decided to include extremes in our statistics , in line with growing statistical belief.26 in most cases , we used a kruskal wallis test to compare subgroups .
we used a mann whitney u - test to compare men and women , and to compare holders of an ecg or spirometry certificate to those without . in order to compare the number of physicians in the subgroups meeting our set standards , we used a chi - square test
however , we kept the nonparametric results , with their stronger stratification and bigger distribution independence , since this did not differentiate any additional significance .
all local family physician organizations of dutch - speaking physicians in belgium were asked to invite their members to take part in our study in september 2009 .
a convenience sample consisting of 974 family practitioners was contacted through email and asked to complete a web - based questionnaire .
physicians in their postgraduate training to become a family physician were also included , since they work independently as qualified physicians and are an integral part of the medical corps in belgium .
we opted for this subgroup of belgian physicians because they consist of a homogenous group .
the two main language groups in belgium ( french and dutch ) have a different organization of medical studies .
curricula are different , and contrary to their french - speaking counterparts , flemish physicians have been trained in an interuniversity setting for more than 25 years . because the vast majority of dutch - speaking physicians work in flanders and brussels , we performed our study only in these two parts of the country .
we opted for an online questionnaire , hosted on server space of the vrije universiteit brussel .
cookies as well as ip address analysis were used to avoid single users filling in the questionnaire multiple times .
for each skill , the participant was asked for a self - assessment , using a likert scale with the following possibilities : 0 , has no experience or knowledge whatsoever ; 1 , has theoretical knowledge ; 2 , has had a demonstration of the skill or has observed it ; 3 , is able to perform the skill independently ; and 4 , has proficiency in the respective skill . the second part consisted of 31 questions ascertaining the participant s knowledge of conditions relevant to urgent medical care .
again , a likert scale was used as follows : 0 , has never heard of the disease or condition ; 1 , has heard of the disease / condition but is unable to manage it ; 2 , can manage the disease / condition ; 3 , can diagnose the disease / condition ; and 4 , can independently treat the disease / condition .
our questionnaire was based on a list used by hekkert in a similar study in the netherlands.21 a test study has been performed on medical students , using the same methodology and questionnaire.11 using the information gained from that study , changes were made to scales , descriptions , questions , and questionnaire setup .
however , we limited these changes to the essential ones , in order to maintain our possibility to compare .
it is unclear in what way the original instrument was validated by the composers , but since hekkert used the validated criteria set from the dutch raamplan 200124 and based on consistency within and comparison with other studies using the same questionnaire , we estimated the questionnaire to be valid and reliable . the dutch raamplan is also used in belgium , as a basis for the final qualifications in medical studies .
the participants were divided into subgroups : based on sex , age ( five groups ) , geographical location ( rural , urban , metropolitan ) , size of the family practice ( four groups ) , and on the organization of the practice ( five groups ) .
they were also asked if and when they had taken any additional training in emergency care .
the context of this training was not further defined , meaning that every initiative being regarded as additional training by the participant was included . for each participant , a mean score for skills and
although a decimal score on a likert scale has no meaning as such , we assumed that means would provide more information than the raw score itself .
questionnaires in which a participant indicated the same score on each question , or where the pattern of answers showed a consequent algorithm , were assumed to be intentionally misleading and were excluded .
the raamplan states that at the end of the medical studies , a physician should be able to perform skills in an appropriate manner , not always by acting independently but also by referring out , if needed.24 in our opinion , having had a demonstration of a skill ( likert score 2 ) does not comply with this criterion .
therefore , we set the minimum criterion to meet our standards at likert score 3 , meaning the participant was able to perform the skill independently . even though it could be theoretically assumed that a licensed physician meets this criterion in all mentioned skills , this assumption is highly unrealistic .
hence , we assumed that a participant met our standards in a reasonable manner when scoring 3 on at least half of the questioned skills .
for assessing knowledge of relevant conditions , we used the appendix to the raamplan , which differentiates between the conditions .
we assumed that a participant fulfilled the criteria in a reasonable way if he or she met the set criterion on at least half of the questioned pathologies .
for statistical interpretation , we used spss statistics for windows , version 19.0 ( ibm corporation , armonk , ny , usa ) . due to the small subgroups and the presence of some extreme results , we could not assume a normal distribution of our data , as was indicated by a shapiro
however , we decided to include extremes in our statistics , in line with growing statistical belief.26 in most cases , we used a kruskal wallis test to compare subgroups .
we used a mann whitney u - test to compare men and women , and to compare holders of an ecg or spirometry certificate to those without . in order to compare the number of physicians in the subgroups meeting our set standards , we used a chi - square test
however , we kept the nonparametric results , with their stronger stratification and bigger distribution independence , since this did not differentiate any additional significance .
the recruitment rate to our email invitation was approximately 22% ( n = 210 ) .
after exclusion of incomplete questionnaires ( n = 51 ) or those with obviously intentional misleading answers ( n = 1 ) , our study group consisted of 158 family physicians , meaning a completion rate of 75% .
this sample size was adequate , and by comparing its composition to the demographic composition of belgian family physicians , it seemed to be highly representative .
additional training in emergency medical care had been completed by 39% of participants , an ecg certificate was held by 92% , and 61% held a spirometry certificate .
holders of a spirometry certificate scored higher than their colleagues without such a certificate ( 2.94 and 2.72 respectively ; p = 0.035 ) . also , family physicians with an additional training in emergency care had clearly higher scores than those without ( p < 0.001 ; means , 3.07 and 2.72 respectively ) . any influence of time ( delay ) pertaining to additional training
no other significant differences between subgroups were found . the mean likert score on skills was 2.50 . based on the questionnaire results ,
the least - known skills were : tilting to recovery position ( mean , 1.30 ) , use of an external defibrillator ( 1.79 ) , and resuscitation of a child ( 1.93 ) .
the best - known skills were : removal of a foreign body ( 3.53 ) , applying bandages ( 3.09 ) , and external heart massage ( 3.08 ) .
our minimum criteria were met by 64% of participants . in 14 of the 22 skill items ,
the skills criteria met by the highest number of participants were : removal of a foreign body ( 92% ) , external heart massage ( 87% ) , and applying bandages ( 80% ) .
the skills criteria met by the lowest number of participants were : tilting to recovery position ( 19% ) , use of the external defibrillator ( 28% ) , and safe transport of an accident victim ( 29% ) ( table 1 ) .
holders of a spirometry certificate scored higher than their colleagues without such a certificate ( 2.61 and 2.34 respectively ; p = 0.035 ) .
also , family physicians with an additional training in emergency care had clearly higher scores than those without ( p < 0.001 ; means , 2.81 and 2.31 respectively ) , with more meeting our criteria ( 77% versus 56% ; p = 0.006 ) .
the least - known diseases or conditions were : a fall from great height ( mean , 2.09 ) , skull base fracture ( 2.28 ) , and electrocution ( 2.28 ) . the best - known diseases or conditions were : superficial wounds ( 3.91 ) , insect bites ( 3.86 ) , and hyperventilation ( 3.85 ) .
our minimum criteria were met by 55% of participants . on 24 of the 31 diseases / conditions , at least half of the physicians met our criteria .
the skills criteria met by the highest number of participants were : superficial wounds ( 99% ) , cerebrovascular abnormalities ( 99% ) , and epilepsy ( 97% ) .
the skills criteria met by the lowest number of participants were : electrocution ( 11% ) , poisoning ( 11% ) , and a fall from great height ( 13% ) ( table 3 ) .
more holders of a spirometry certificate met our criteria than their colleagues without this certificate ( 63% versus 43% ; p = 0.013 ) . also , family physicians with additional training in emergency care had clearly higher scores than those without ( p = 0.003 ; means , 3.33 and 2.14 respectively ) , with more meeting our criteria ( 70% versus 45% ; p = 0.002 ) .
the mean score of physicians working in rural areas was higher than that of those working in urban or metropolitan areas ( p = 0.033 ; means , 3.25 and 3.15 respectively ) .
more physicians from rural areas met our criteria compared to those from urban or metropolitan areas ( 61% versus 45% ; p = 0.047 ) ( table 2 ) .
no significant differences between men and women , age groups , or practice size could be found .
whether or not the physician was the holder of an ecg certificate did not have a significant influence on the results .
the recruitment rate to our email invitation was approximately 22% ( n = 210 ) .
after exclusion of incomplete questionnaires ( n = 51 ) or those with obviously intentional misleading answers ( n = 1 ) , our study group consisted of 158 family physicians , meaning a completion rate of 75% .
this sample size was adequate , and by comparing its composition to the demographic composition of belgian family physicians , it seemed to be highly representative .
additional training in emergency medical care had been completed by 39% of participants , an ecg certificate was held by 92% , and 61% held a spirometry certificate .
holders of a spirometry certificate scored higher than their colleagues without such a certificate ( 2.94 and 2.72 respectively ; p = 0.035 ) .
also , family physicians with an additional training in emergency care had clearly higher scores than those without ( p < 0.001 ; means , 3.07 and 2.72 respectively ) . any influence of time ( delay ) pertaining to additional training could not be proven from our results ( p = 0.091 ) .
no other significant differences between subgroups were found . the mean likert score on skills was 2.50 . based on the questionnaire results ,
the least - known skills were : tilting to recovery position ( mean , 1.30 ) , use of an external defibrillator ( 1.79 ) , and resuscitation of a child ( 1.93 ) .
the best - known skills were : removal of a foreign body ( 3.53 ) , applying bandages ( 3.09 ) , and external heart massage ( 3.08 ) .
our minimum criteria were met by 64% of participants . in 14 of the 22 skill items , at least half of the physicians met our criteria .
the skills criteria met by the highest number of participants were : removal of a foreign body ( 92% ) , external heart massage ( 87% ) , and applying bandages ( 80% ) .
the skills criteria met by the lowest number of participants were : tilting to recovery position ( 19% ) , use of the external defibrillator ( 28% ) , and safe transport of an accident victim ( 29% ) ( table 1 ) .
holders of a spirometry certificate scored higher than their colleagues without such a certificate ( 2.61 and 2.34 respectively ; p = 0.035 ) .
also , family physicians with an additional training in emergency care had clearly higher scores than those without ( p < 0.001 ; means , 2.81 and 2.31 respectively ) , with more meeting our criteria ( 77% versus 56% ; p = 0.006 ) .
the least - known diseases or conditions were : a fall from great height ( mean , 2.09 ) , skull base fracture ( 2.28 ) , and electrocution ( 2.28 ) . the best - known diseases or conditions were : superficial wounds ( 3.91 ) , insect bites ( 3.86 ) , and hyperventilation ( 3.85 ) .
our minimum criteria were met by 55% of participants . on 24 of the 31 diseases / conditions , at least half of the physicians met our criteria .
the skills criteria met by the highest number of participants were : superficial wounds ( 99% ) , cerebrovascular abnormalities ( 99% ) , and epilepsy ( 97% ) .
the skills criteria met by the lowest number of participants were : electrocution ( 11% ) , poisoning ( 11% ) , and a fall from great height ( 13% ) ( table 3 ) .
more holders of a spirometry certificate met our criteria than their colleagues without this certificate ( 63% versus 43% ; p = 0.013 ) .
also , family physicians with additional training in emergency care had clearly higher scores than those without ( p = 0.003 ; means , 3.33 and 2.14 respectively ) , with more meeting our criteria ( 70% versus 45% ; p = 0.002 ) .
the mean score of physicians working in rural areas was higher than that of those working in urban or metropolitan areas ( p = 0.033 ; means , 3.25 and 3.15 respectively ) .
more physicians from rural areas met our criteria compared to those from urban or metropolitan areas ( 61% versus 45% ; p = 0.047 ) ( table 2 ) .
no significant differences between men and women , age groups , or practice size could be found . whether or not the physician was the holder of an ecg certificate
based on the results of our study , we can confidently state that dutch - speaking family physicians in belgium do not feel able to adequately administer most aspects of emergency care . as described in the introduction ,
this is in line with studies in many other countries.110 our results were also highly comparable with those obtained in our similar study from 2008 by junior doctors in their postgraduate training to become family physicians.11 factors of influence on skills and knowledge relating to urgent medical care seem limited . having taken an additional training in emergency medicine is the most influencing positive factor on both skills and knowledge .
this sounds logical , but could be a result of the profile of the participant rather than of the training itself .
holding a spirometry certificate proves to be of positive influence on the physician s self - assessed skills and knowledge .
holding an ecg certificate , however , had no influence on the physician s skills and knowledge in emergency medical care , probably because the certified training is incorporated in the basic curriculum for most medicine students and is therefore independent of the student s profile . in most cases ,
obtaining an ecg certification is independent of the physician s personal interests and preparedness to take additional training , whereas obtaining a spirometry certificate is highly influenced by these factors .
it is plausible that a doctor seeking additional training has an overall more proactive professional approach .
therefore , the found link between the certified emergency , ecg or spirometry training , and the assessed confidence could be a result of the physician s profile rather than of the certified training itself .
this seems likely , because spirometry is of little practical use in emergency medical care .
according to our results , family physicians in rural areas are more confident about relevant knowledge than their urban or metropolitan counterparts .
this could be explained by the proximity of hospital emergency services for urban patients , lowering the threshold and thus surpassing the family physician .
even though the participants indicated a lack of confidence in skills and knowledge relating to emergency care , this needs to be seen in perspective .
most physicians felt quite confident in topics they had experience with or that were common in daily practice .
the items on which they indicated low confidence were generally less common and mostly treated by specialist care anyway .
this may limit the practical implications of our results . also , we need to stress that it was not our aim to test individual skills , but to assess the overall confidence of the physician .
however , we did include a mention of individual skills , since this provides a good idea of the specific areas in which the physician lacks confidence .
specific confidence or skill in the different aspects of individual emergencies needs to be the subject of further research .
. these may be the organization of education in emergency medicine in our universities , the lack of additional training and recapping , a noticeable inconsistency for some guidelines , or the frequency with which these guidelines change.2729 finally , it is important to underline that this study only assessed physician s confidence in a very specific area .
our study does show , however , that dutch - speaking belgian family physicians do nt feel very confident in the area of emergency care , regardless of the quality of the care they provide . throughout our study ,
we have guarded the validity and reliability of the instruments used , thus ensuring the quality and relevance of our results .
we used an existing questionnaire11,21 based on final qualifications described in the raamplan.24 the relevancy of our results obviously depends on the relevancy of the criteria set in the raamplan .
but these qualifications were established after a long and careful process , likely ensuring their relevancy .
we tried to avoid as many confounders as possible and limit expectation and confirmation bias by carefully designing and respecting the study protocol .
we are aware of the fact that our method held a risk of selection bias , but by randomly choosing the potential participants , we attempted to limit this as well .
self - assessment is not the most adequate instrument to evaluate skills.9,30 an objective structured clinical examination would have been more appropriate,18 but was not feasible within the context of this study . also , the tested items are only surrogate markers .
it is plausible that with lesser confidence , training , or knowledge a good outcome is still possible with a proper attitude and awareness of the physician s own limitations .
it is regrettable that only a small sample of family physicians were prepared to take part in our study .
it is possible that due to the small study group , we were not able to prove the influence of all relating factors .
however , in comparison to the official rapport of the belgian medical corps,31 we are confident that our study group is representative of the entire population of family physicians ( table 4 ) .
many epidemiological studies have been performed with a representative sample of only 1%2%.32 even with the limitations of our study in mind , we feel secure in stating that the scientific value of our study is assured .
throughout our study , we have guarded the validity and reliability of the instruments used , thus ensuring the quality and relevance of our results .
we used an existing questionnaire11,21 based on final qualifications described in the raamplan.24 the relevancy of our results obviously depends on the relevancy of the criteria set in the raamplan .
but these qualifications were established after a long and careful process , likely ensuring their relevancy .
we tried to avoid as many confounders as possible and limit expectation and confirmation bias by carefully designing and respecting the study protocol .
we are aware of the fact that our method held a risk of selection bias , but by randomly choosing the potential participants , we attempted to limit this as well .
self - assessment is not the most adequate instrument to evaluate skills.9,30 an objective structured clinical examination would have been more appropriate,18 but was not feasible within the context of this study .
also , the tested items are only surrogate markers . the real clinical relevance of emergency care is determined by patient outcome .
it is plausible that with lesser confidence , training , or knowledge a good outcome is still possible with a proper attitude and awareness of the physician s own limitations .
it is regrettable that only a small sample of family physicians were prepared to take part in our study .
it is possible that due to the small study group , we were not able to prove the influence of all relating factors .
however , in comparison to the official rapport of the belgian medical corps,31 we are confident that our study group is representative of the entire population of family physicians ( table 4 ) .
many epidemiological studies have been performed with a representative sample of only 1%2%.32 even with the limitations of our study in mind , we feel secure in stating that the scientific value of our study is assured .
almost half of the family physicians in the dutch - speaking area of belgium feel insufficiently competent to offer most aspects of emergency medical care .
it is not clear if this lack of confidence has any consequences for the quality and outcome of patient care .
it does prove , however , that family physicians know the limits of their own medical skills . | backgroundpractical knowledge of emergency medical care among physicians seems to be insufficient worldwide .
research specifically aimed at family physicians is rather scarce .
additionally , in belgium there are no data on this subject.purposesour aim was to ascertain how confident belgian family physicians feel about their ability to give adequate emergency care and to examine their assessment of their knowledge of relevant medical conditions.methodswe used a web - based questionnaire for which a convenience sample of 974 dutch - speaking family practitioners was invited through email .
the survey assessed how these physicians perceived their own emergency skills and their knowledge of relevant medical conditions.resultsthe survey had a recruitment rate of 22% ( n = 210 ) , with a 75% completion rate .
the minimum criteria formulated pertaining to skills and knowledge were met by 64% and 55% of the participants , respectively .
the mean cumulative scores on skills and knowledge were 2.5 and 3.2 , respectively ( on a scale from 0 to 4 ) .
physicians with additional training in emergency care ( 3.07 versus 2.72 ) , or with a spirometry certificate ( 2.94 versus 2.72 ) scored better than those without . practitioners from rural areas felt more confident than those from urbanized regions ( 3.25 versus 3.15 ) .
physicians felt more competent in aspects of emergency care where they had experience.conclusionalmost half of the dutch - speaking family physicians in belgium felt insufficiently competent to offer emergency medical care . |
diarrhea , caused by acute infectious or chronic diseases , may be considered as an imbalance between the approximately 7 l of fluids ingested or secreted into the gastrointestinal tract each day , and the reabsorption of almost an equivalent volume .
infectious organisms such as escherichia coli or vibrio cholerae produce toxins that stimulate normal secretory pathways in the intestine , resulting in potentially life - threatening secretory diarrhea and dehydration .
the gold standard of treatment is rehydration with glucose - electrolyte solution but , regrettably , there is still a high mortality from these conditions in developing countries ; hence , more effective treatments are needed .
for example , v. cholerae infection induces enteroendocrine cells to secrete 5-hydroxytryptamine ( 5-ht or serotonin ) , which stimulates secretion of water and electrolytes from small intestinal epithelial cells or enterocytes .
it has been shown that secretions from goblet cells include the peptides guanylin and uroguanylin , which have similar amino acid sequences to the heat - stable enterotoxin of e. coli .
these enterotoxins and endogenous peptides bind to guanylate cyclase c ( gc - c ) and stimulate the production of increased levels of intracellular cyclic guanosine monophosphate ( cgmp ) . increased levels of cgmp ultimately activate the secretion of chloride ions through the cystic fibrosis transmembrane conductance regulator ( cftr ) .
when chloride reaches the intestinal lumen , its osmotic force pulls water out of the interstitial fluid through the spaces between enterocytes and this ultimately leads to dehydration .
it has been demonstrated that enterotoxigenic e. coli requires the gc - c receptor to induce intestinal secretion .
in addition to its presence in goblet cells , guanylin is also located in enteroendocrine cells , as well as in neuroendocrine tumors such as carcinoids .
in fact , enhanced local secretion of guanylin may contribute to secretory diarrhea in these patients .
the relationship of enterocytes , goblet cells and enteroendocrine cells in the lining of the intestine and the role of secretomotor mechanisms has been reviewed previously . although some of the signaling pathways involved in abnormal intestinal secretion have been identified , targeted approaches to reverse secretion are needed to complement rehydration therapy .
this article highlights a recent study that identifies the molecular genetic basis of a familial diarrhea ( fd ) , illustrating a mechanism that can be pharmacologically targeted to enhance secretion in constipation or to inhibit secretion in diarrheal diseases .
fiskerstrand et al . identified a norwegian family with a rare , early onset form of chronic or intermittent fd .
their investigation started with careful clinical observations of an 88-year - old admitted to hospital for acute diarrhea and dehydration . it was noted that affected members of the family started to develop symptoms in infancy , had chronic , relatively mild diarrhea , and the symptoms tended to subside by middle age in some members of the family .
the fd was a dominantly inherited , fully penetrant disease in 32 ( 18 male and 14 female ) members of the family .
fourteen unaffected members of the family were also evaluated . in a series of elegant studies ,
the authors used whole genome linkage analysis based on arrays with snps to identify a candidate region on chromosome 12 , and then sequenced gucy2c , encoding gc - c .
linkage analysis was used to determine the candidate region for the mutation in samples from 11 affected members and 14 unaffected members .
this analysis revealed only one shared region in the affected members , located on the short arm of chromosome 12 ( 12p ) with a maximum lod ( logarithm ( base 10 ) of odds ) score of 5.1 .
a haplotype spanning approximately 2.9 megabases showed complete segregation with the disease , and the region contained 28 putative protein coding genes , including gucy2c .
exome sequencing of the entire candidate region from three affected family members excluded mutations in genes other than gucy2c as possible causes of or contributors to susceptibility to the disease .
in addition , fiskerstrand et al . conducted functional studies of mutant gc - c in hek293 t cells .
exposure of the mutant receptor to its ligands ( heat - stable enterotoxin , uroguanylin and guanylin ) resulted in markedly increased ( up to eightfold ) production of cgmp .
these functional assays demonstrate that the mutation is capable of inducing the intracellular production of the second messenger cgmp , which results in intestinal secretion through cftr .
the authors appropriately describe this familial variation as a form of ' genetic knock - in ' of the mechanism that is stimulated by enterotoxigenic e. coli , and this represents an intriguing confluence of the effects of bugs and genes .
the fiskerstrand et al . study , which identified the causative gucy2c mutation , has important implications from mechanistic , diagnostic and therapeutic perspectives .
first , identification of this gene mutation in fd provides evidence that the pathway is an important mechanism of chronic diarrhea in addition to infectious enterotoxic diarrhea .
second , mutation in gucy2c should be considered in the differential diagnosis of chronic diarrhea , especially when there is a positive history of fd .
third , understanding the pathway provides a target for the development of a new drug class to treat constipation by inducing intestinal secretion .
since analogs of guanylin and uroguanylin ( linaclotide and plecanatide ) already exist , clinical translation of the science to develop secretagogue drugs for relief of constipation and associated symptoms ( bloating and abdominal pain ) had preceded the intriguing molecular genetic discovery of fiskerstrand et al . .
targeting the receptor represents an alternative therapeutic approach , in lieu of targeting the gene itself . however , there are some loose ends in the story .
the susceptibility of the norwegian family to inflammatory bowel disease ( ibd ) , small bowel obstruction and esophagitis in the absence of hiatal hernia can not be easily attributed to the effects of the gucy2c mutation on gc - c , based on current knowledge .
in addition , there are no reports of any association of the gucy2c mutation and ibd , even with genome - wide association studies .
nevertheless , the gc - c agonists plecanatide and sp-333 ( analogs of uroguanylin ) have been shown to ameliorate inflammation in murine colitis models via downregulation of certain pro - inflammatory cytokines , such as il-6 , il-17 and il-23 , and they may also have therapeutic potential in ibd and , possibly , carcinogenesis . this remarkable story may eventually have a happy ending for patients with this specific fd . the report should stimulate the development of blockers of the gc - c and cftr intestinal secretory pathways , and this will have potential for wide application to life - threatening infectious diarrheas , in addition to the chronic , mild , non - fatal diarrhea afflicting this family ; these diarrheas could then be specifically targeted with personalized or precision medicine .
cgmp : cyclic guanosine monophosphate ; cftr : cystic fibrosis transmembrane conductance regulator ; fd : familial diarrhea ; gc - c : guanylate cyclase c ; ibd : inflammatory bowel disease ; il : interleukin ; snp : single - nucleotide polymorphism .
| acute infectious and chronic diarrheal diseases are important public health problems .
a recent study by fiskerstrand and colleagues identified a family with a rare early onset familial diarrhea . by linkage analysis and exon sequencing
, the authors identified a heterozygous missense mutation in gucy2c , encoding the guanylate cyclase c receptor , which is involved in intestinal secretion .
this newly identified gene in the etiology of a familial diarrhea provides a candidate target for the development not only of new treatments for diarrhea , but also of a new drug class to treat constipation . |
sexuality is an integrant and essential part of quality of life ( qol ) , and patient affected
by neurological disability , including stroke , should also be investigated and treated for
sexual disorders1 .
sexual dysfunction is
common after stroke , but is frequently not addressed by healthcare providers2 .
the latter is defined as a decline in libido in both genders , erectile
dysfunction and poor ejaculation in men and diminished lubrication and orgasm in women3 , 4 .
post - stroke patients reported a marked decline in all the measured sexual functions , ie ,
libido , coital frequency , erectile and orgastic ability , and vaginal lubrication , as well as
in their sexual satisfaction .
the spouses also reported a significant decline in their
libido , sexual activity , and sexual satisfaction as a consequence of stroke5 .
sexual dysfunctions after stroke reported
to be due to multiple etiologies , including both physical and psychosocial causes1 , 2 .
sexual function relies on a complex network of peripheral and central pathways involving
the participation of autonomic and somatic nerves and the integration of numerous spinal and
supraspinal sites in the central nervous system , with the hypothalamic and limbic regions
playing a pivotal role6 , 7 .
many studies have shown significant relationships between symptoms of depression and
functional outcome in patients from 3 months to 2 years after stroke . also , reduced quality
of life after stroke appears to be related not only to the physical disability but also to
psychologic factors8,9,10 .
depression and fear of a
recurrent stroke are examples of psychological factors influencing sexual function and , in
particular , sexual desire3 .
the role of
psychological factors in poststroke sexual dysfunctions ( sd ) is further confirmed by the
observation that sexual disorders are reported not only by the patients , but also by their
spouses or partners8 , 11 , 12 .
nevertheless , most of
the patients received little information about sexuality following stroke on sexual behavior
and attitudes of the stroke patients spouses , although they are very important in terms of
stroke survivors well - being .
the decreased sexual activities of the stroke patients in korea mainly result from
psychological burden and physical disability caused by a disorder , but most of them tend to
avoid consultation with experts13 .
additionally , avoiding sexual intercourse and conversation is another important factor of
decreasing sexual life due to a concern about aggravating a spouse s condition14 .
random forest is a bagging algorithm which reduces variance of a large number of complex
models with low bias15 . while boosting ,
which uses a set of week classifiers and combine them to derive a strong classifier , reduces
bias of a large number of small models with low variance16 .
we compared the result of some machine learning algorithms , among
them random forest and boosting algorithm are the best for our purpose .
many studies on changed psychological and physical health regarding rehabilitation after
stroke have been conducted , but there are not enough comparative studies on the general
characteristics of the patients and sexual index .
the purpose of this study was to examine
patient preferences for counseling related to sexuality of post - stroke patients in korea by
using the random forest , boosting algorithm and manova .
studies about stroke patients
sexual life which were unofficially discussed could be an important factor to improve the
quality of patients life and family life including a spouse as well .
the survey was conducted by 147 patients who were diagnosed with stroke from university
hospitals located in seoul / gyeonggi , south korea .
the survey procedure was sufficiently
explained beforehand to the subjects who voluntarily participated by signing the survey
consent form .
we firstly revised and used brief sexual functioning questionnaire for men ( bsfq ) and brief
index of sexual functioning for woman ( bisf - w ) .
the revised bsfq , bisf - w are used to
diagnose a disorder of sexual behavior and function , according to the objective of this
study17 , 18 .
the revised questionnaire was then reviewed by an expert group
consisting of three professors in the department of physical therapy to examine content
validity and suitability of the questions .
the questionnaire was composed of 27 questions
such as 8 independent variables related general characteristics of the patients , 7 dependent
variables in conjunction with sexual intercourse and changed muscle tone , 6 questions
regarding a changed sexual function , and 6 questions about a changed motor and a sensory
function after stroke ( table 1table 1.classification of group and definition
for each variablesgroupvariablesdefinitiongroupvariablesdefinitionindependent1duration of illnessdependent 315changes of
muscle tone after strokedependent 12communication about sexual activities16balance ability after stroke3necessity of study about sexual
activities17fine movement
after stroke4have a sex partner18changes of tactile sense after stroke5try to sexual intercourse after stroke19changes of smell sense after
stroke6frequency
of sexual intercourse / month20changes of taste sense after stroke7changes of muscle tone during sexual
intercourseindependent21gender8level of
muscle tone during sexual intercourse22agedependent 29changes of frequency of sexual intercourse after stroke23weight10frequency of kissing and
romantic touching after stroke24marriage11thinking about sex with interest or desire after
stroke25occupation12ability to become sexually aroused after stroke26education level13overall level of sexual
satisfaction after stroke27income level / month14obstacles during sexual intercoursedependent 1 : sexual intercourse and changed
muscle tone ; dependent 2 : changed sexual function ; dependent 3 : changed motor &
sensory function ) .
the alpha value of cronbach was
0.688 which was used to indicate internal consistency of the dependent variables .
data for
this study had been collected for 98 days : feasibility study for 8 days and main survey 90
days .
a physical therapist at the hospital fully explained the objectives and procedures of
this study to the patients , and the questionnaires were filled by voluntary patients .
total
156 questionnaires out of 200 were collected , and 147 questionnaires except 9 unsuitable
questionnaires were used for final analysis .
dependent 1 : sexual intercourse and changed
muscle tone ; dependent 2 : changed sexual function ; dependent 3 : changed motor &
sensory function the collected questionnaires were analyzed by r version 3.3.1 . to figure out an
interrelationship between the general characteristics of the patients and the variables of
the sexual function , random forest , boosting algorithm and manova
the general characteristics of 147 subjects are age , duration of illness , weight , gender ,
marital status , education level , income level and occupation .
the mean age of our study
population ( 97 men ; 65.99% and 50 women ; 34.00% ) was 53.56 years .
the forty - six patients ( 31.30% ) have more than 1 year but
no longer than 3 years of a duration of illness , and it is examined the mean duration of
illness is 3.18 years . for the weight factor ,
the most number of the patients are fifty - four
( 36.73% ) whose weight is between 60 kg and 70 kg , and its mean is 67.05 kg . for the martial
status factor , one hundred thirty - seven patients ( 93.20% )
the factor of education level , sixty - seven patients
( 45.58% ) are graduated in high school . for the income level factor ,
fifty - six ( 38.10% ) of
patient s income is over 3 million won per month . in occupation factor ,
self - employed
patients are twenty - eight ( 19.05% ) , company employee twenty - two ( 14.97% ) , housewife eighteen
( 12.24% ) .
the random forest is the best method to find variable importance , we applied this method
for each variable group , in variable group 1 , var1 is the most important , and then var22 ,
var23 , var26 , var27 , var25 , var21 and var 24 respectively .
the most important variable in
variable group 2 is var22 , and then var26 , var23 , var25 , var1 , var27 , var21 and var 24 .
finally , for variable group 3 , var1 has the most important percentage , and we have the order
var26 , var23 , var27 , var22 , var25 , var21 and var 24 among the rest of variables ( table 2table
2.interrelationship between the general characteristics of the
patients and the variables of the sexual functionindependent
variablesdependent
variablesgroup 1group 2group
3%incmse%incmse%incmse130.6***19.3***29.5**2115.5**14.419.52226.5***28.7***24.72325.2***25.2 * 26.7**2410.7***10.1 * 4.82519.619.6**22.12623.3***25.4***27.32723.1**19.3 * 25.5%incmse : % increase in mse ( mean
squared error)variable importance in random forest*p0.05 ; * * p0.01 ;
* * * p0.001 ) .
% incmse : % increase in mse ( mean
squared error ) variable importance in random forest * p0.05 ; * * p0.01 ;
* * * p0.001 the result of variable importance in boosting algorithm is somehow the same as that of
random forest .
the purpose of this study was to examine patient preferences for counseling related to
sexuality of post - stroke patients in korea by using the random forest , boosting algorithm
and manova .
random forest is an excellent method to determine variable importance , as it fits a number
of decision tree classifiers on various sub - samples of the dataset .
it uses averaging to
improve the predictive accuracy and control over - fitting , so there is no need for
cross - validation to get better ( or unbiased ) estimation , since according what we said it is
already done internally .
however , it is difficult to interpret the result with multiple
outcome variables in random forest , for this purpose using multivariate tree boosting that
is a method for non - parametric regression is useful . in this study
we are extending these
three methods along with linear regression for one group of independent variable and three
different groups of dependent variables to analyze the dataset and find the importance of
group of independent variables to predict the group of dependent variables as well as
illustrate the correlation between groups of dependent variables .
this study is to realize
the effect of sexual functions in stroke patients in computer science point of view .
moreover , we applied 10-fold cross - validation for getting better result in multiple linear
regressions19 and compared the result
of these three methods on our data that we conclude the best result from random forest and
boosting algorithm . as a result of questionnaire analysis , duration of illness , age , and education level among
8 independent variables strongly affect by other dependent variables which are sexual
intercourse and changed muscle , changed sexual function , and changed motor & sensory
function . according to analysis the quality of life targeted at stroke patients who have less than
1 year duration of illness , the quantitative and qualitative measures of most patients
related to sexual life were decreased12 .
patients who have more than 1 year duration of illness had the same result14 .
this is due to the fact that most stroke
patients did not enjoy traveling and an engaging in outdoor activities so that the quality
of life was reduced12 .
the sexual
function of most stroke patients in this study was also reduced because patients and their
spouses focused on rehabilitation for the first year after an illness occurred , and there
was absence of sex education after one year21 . forsbergwrleby
et al.12 pointed out the necessity of sex education for stroke patients and
their spouses in terms of the quality of life .
appropriate
sexual life and sex education according to the duration of illness are also considered
important factors to improve the quality of life , and it needs to examine a detailed sexual
life and an education based on the seriousness of a stroke in the future .
approximately 60% of the stroke patients between 60s and 70s showed reduction of the sexual
function with the erectile dysfunction , and it reported the high possibility of the erectile
dysfunction as getting old21 . in this
study ,
the sexual activity of the patients between 40s and 50s was reduced after a stroke
occurred compared to patients between 60s and 70s . since this referred to the physiological
age - related characteristic and they had active sexual life compared to other age groups ,
variables related to a sexual function were proportionally lower than they were before
illness occurred .
an education level is categorized according to graduation : before graduating
elementary school , graduated from middle school , graduated from high school , and after
graduating university .
as an education level goes up , it shows that patients who graduated
university tend to often have a conversation about the sexual topic , and this group also
showed the high statistical result regarding the necessity of a sexual study , sexual
intercourse status , and the number of sexual intercourse .
this was due to the possibility
that they might have more sexual education opportunities so that they could have more
chances of sexual intercourse14 , 20 .
patients between 60s and 70s did not respond to some
questionnaire items due to passive attitude and repulsion .
this limitation caused the low
response rate so that we had a difficulty to generalize the research result .
yet , we found
out that sexual education for disabled koreans needed to be developed urgently and hopefully
this study would be a good guideline for developing such sexual program . in conclusion , this study found out that duration of illness , age , and education level were
important factors that affected patients sexual life and function .
it is required to make
more specific descriptions of the questionnaire items and needs to complement a
questionnaire for the future work . | [ purpose ] the purpose of this study is to examine patient preferences for counseling
related to sexuality post - stroke in korea .
[ subjects and methods ] a survey was conducted
on 200 stroke patients . among the 200 submitted questionnaires , 156 responded but 147
cases are available .
the questionnaire is composed of 27 questions such as 8 independent
variables related to the general characteristics of the patients , 7 dependent variables in
conjunction with sexual intercourse and changed muscle tone , 6 questions regarding to
changed sexual function , and 6 questions about a changed motor and a sensory function
after stroke . to analyze the factors related to a sexual function after a stroke , we used
the random forest , boosting algorithm and manova .
[ results ] the most important variable in
variable group 1 is var1 , and then var22 , var23 , var26 , var27 , var25 , var21 and var 24
respectively .
the most important variable in variable group 2 is var22 , and then var26 ,
var23 , var25 , var1 , var27 , var21 and var 24 .
finally , for variable group 3 , var1 has the
most important percentage , and we have the order as var26 , var23 , var27 , var22 , var25 ,
var21 and var 24 among the rest of variables .
the result of variable importance in
boosting algorithm is somehow the same as that of random forest .
[ conclusion ] as a result
of our analysis , we figured out that duration of illness , age , and education level are
important factors of sexual functions for korean stroke patients . |
during visual exploration of the natural world , humans make saccades , vergence , and combined saccade - vergence movements .
the saccades are the fast eye movements used to change the direction of fixation rapidly ; they are conjugate movements , that is , the eyes move equally and in the same direction .
vergence eye movements are necessary to change fixation from a far to a close target , or vice versa ; these movements are disconjugate ( in opposite direction in the two eyes ) .
combined movements are the movements most frequently made when looking at objects in the space needing a shift of gaze both in direction and in depth . to our knowledge , a fine exploration of the speed and accuracy characteristics of all these eye movements used during the everyday life has never been done in dyslexic readers .
indeed , the majority of the studies dealing with eye movements and dyslexia are limited to saccades made during reading or made towards isolated single target ( tracking condition ) .
pavlidis reported frequent regressive saccades and unstable fixation in dyslexia during reading and during sequential tracking of leds .
shorter saccade amplitude and longer fixation durations during reading have been reported [ 3 , 4 ] .
in contrast , olson et al . found no eye movement abnormalities in dyslexic readers when reading pseudowords and hutzler et al . also reported no eye movement differences between dyslexic and non - dyslexic reader subjects on a string - processing task or while reading pseudowords .
specifically , there were no differences in the number of fixations , or in the duration of the first fixation , or in the total reading time .
recently our group [ 7 , 8 ] reported in dyslexic readers poor binocular coordination of saccades and of the postsaccadic fixation period not only during reading single words and during tracking condition but also during free exploration of artwork , suggesting that such problems occur independently from the reading process and might reflect immaturity of normal oculomotor learning mechanisms .
another group of studies has examined the latency of saccades under various oculomotor conditions and at different viewing distances .
dossetor and papaioannou and pirozzolo observed longer saccade latency in dyslexic readers , while other authors [ 11 , 12 ] did not find latency difference between dyslexic readers and non - dyslexic readers .
fischer and weber also examined saccade latency distribution in dyslexic readers and they reported longer mean latencies and large standard deviation in these children ; moreover , other researchers observed a high number of express saccades with the gap and with the overlap paradigms in dyslexic readers .
all these findings suggested abnormal function of the fixation system in dyslexia , and abnormalities in saccade initiation and accuracy .
our group recently extended studies in dyslexic readers examining several types of eye movements in the natural far and near space ; we reported that the latency of saccades at far distance and of saccades combined with convergence was significantly longer in dyslexic readers ; moreover , we found higher rates of express latency in dyslexic readers that occur not only for saccades but for other types of eye movements , particularly for divergence .
most likely , problems of both voluntary and reflexive controls of attention during eye movement preparation could explain the findings observed in dyslexic readers .
latency as well as fixation stability are known to be controlled by central and cortical structures ; in contrast , speed and accuracy of eye movements are controlled by both cortical and premotor structures at the brainstem level where the motor command is prepared .
more precisely , the speed and the amplitude of the saccades depend on the properties of the saccade burst generator located for the horizontal direction at the paramedian pontine reticular formation ( pprf ) in the brain stem .
the pprf receives inputs from several cortical structures ( frontal eye field , parietal cortex ) , the superior colliculus , and the cerebellum .
the neural organisation of the vergence brainstem generators looks like that of the saccade system .
indeed , neurons involved specifically in the control of vergence movements have been found in the mesencephalic reticular formation of monkeys [ 1719 ] .
convergence and divergence burst neurons were found to discharge in relation to vergence velocity ; moreover , [ 20 , 21 ] provided evidence that the activity of neurons in the prearcuate area , close to the frontal eye field of monkeys was related to vergence dynamic .
some authors [ 22 , 23 ] proposed a model in which combined eye movements could be produced by a coactivation of the two distinct oculomotor systems , that of the saccade and that of the vergence .
the omnipause neurons ( opn ) could gate the activity of a specific pool of neurons , the so - called saccade - related - vergence bursters responsible for generating both saccade and vergence commands .
chaturvedi and van gisbergen showed that the superior colliculus via the opn controls the saccade and the vergence responses during combined movements . indeed , when the target called for combined movement , electrical stimulation of the rostral region of the superior colliculus of monkeys inactivated both the saccadic and the vergence systems .
behavioural oculomotor studies carried out in children and in adults give us some information on the development state of such premotor structures .
for instance , cohen and henn showed that speed of saccades does not change significantly with age , suggesting that the pprf is completed developed by the age of 4 years .
subsequent studies confirmed this results reporting that saccade speed in children of 4 years was similar to that observed in adults [ 26 , 27 ] and that saccade amplitude was constant from the age of 12 years .
yang and kapoula explored eye movements in natural space in children from the age of 4.5 years ; they showed that saccades , vergence , and combined movements are similarly accurate as for adults ( close to 90% of the amplitude required by the target ) in children from the age of 4.5 years .
moreover , these authors did not found any significant difference in the speed of all types of eye movements between children and adults .
these observations indicate early maturation of all distinct brainstem structures controlling eye movements ( saccades and vergence ) and their interaction . the goal of the present study was to explore further the speed and the accuracy characteristics of natural eye movements in 3d space in dyslexic children in order to find out whether some kind of dysfunction in the brainstem oculomotor circuits could be present in this population of children .
data from dyslexic readers were compared to those from an age - matched group of non - dyslexic reader children .
furthermore , two paradigms ( gap and simultaneous ) were used to elicit eye movements in natural space .
it is well known that the gap paradigm decreases the latency of eye movements ; and our prior study showed in dyslexic reader children this effect for the latency of several types of eye movements ( saccades , vergence , and combined movements ) . here
we want to examine whether speed and accuracy characteristics could also be affected by the gap paradigm and eventually to find out differences in the two groups of children ( dyslexic and non - dyslexic readers ) .
our general objective was to contribute to enlarge knowledge on oculomotor behaviour in dyslexic reader children .
dyslexic children were recruited from the pediatric hospital where they are referred for a complete evaluation of their dyslexia state with an extensive examination including neurological / psychological and phonological capabilities . for each child , the time of reading a text , its comprehension , and the capacity of reading word / pseudowords hve been evaluated by using the l2ma battery .
this is the standard test developed by the applied psychology centre of paris , and is used everywhere in france .
inclusion criteria were scores on this test beyond 2 standard deviations and a normal mean intelligence quotient ( iq , evaluated with wisc iii ) , that is , between 85 and 115 .
the mean age of the dyslexic children was 12 0.08 years , the mean iq was 100 6 , and the mean reading age was 9 1 years . a carefully selected age - matched control group ( mean age : 12 1 years ) of thirteen non - dyslexic children was selected .
these children had to satisfy the following criteria : no known neurological or psychiatric abnormalities , no history of reading difficulty , and no visual stress or any difficulties with near vision .
iq and reading measurements were not available for these children , but they were selected by the director of the school on the basis of their school performances ; their score on french ( reading , understanding , and orthography ) , mathematic , and foreigner languages were all beyond the mean score of the class .
recruitment for controls , based on school performance alone has been used by others [ 3133 ] .
both non - dyslexic and dyslexic reader children underwent an ophthalmologic examination accompanied by orthoptic evaluation of their visual function ( median values showed in table 1 ) .
all children had normal binocular vision ( 60 seconds of arc or better ) , that was evaluated with the tno random dot test .
visual acuity was normal ( 20/25 ) for all children , dyslexic and non - dyslexic readers .
the near point of convergence was abnormal ( > 8 cm ) in 30% of dyslexic children while it was normal ( 7 cm ) for all non - dyslexic children .
moreover , an orthoptic evaluation of vergence fusion capability using prisms and maddox rod was done at far and at near distances .
the divergence amplitude was limited in 27% and 76% of the cases for dyslexic readers , respectively , at far and at near distances ; while for non - dyslexic children , limited divergence was observed only at near distance in 46% of the cases .
( at far and near at distance , respectively ) and in the normal range for all but one non - dyslexic child at far distance .
phoria ( i.e. , latent deviation of one eye when the other eye is covered , using the cover - uncover test ) was abnormal in 18% of dyslexic reader children at far as well as at near distances ; while for non - dyslexic children , phoria was abnormal in 15% and in 7% at far and at near distance , respectively .
in sum , orthoptic evaluation showed a tendency of poor divergence amplitude particularly at near distance in dyslexic reader children in line with another study on a larger population of dyslexic and non - dyslexic reader children .
the investigation adhered to the principles of the declaration of helsinki and was approved by our institutional human experimentation committee .
informed consent was obtained from the children 's parents after the procedure for the experiment was explained .
note that the majority of children , dyslexic and non - dyslexic readers , participated already to our previous study in which only eye movement latencies have been analyzed .
the spatial and the temporal arrangement are the same to that used in our previous study .
briefly , eight leds were embedded in two isovergence circles at different distance ( 20 and 150 cm ) on an horizontal table ( see figure 1(a ) ) .
five leds were placed 150 cm from the subjects ' eyes , one at the center , two at 10 , and two at 20. the required mean angle of vergence for fixating these diodes was 2.3. the other three leds were placed at a distance of 20 cm , one at the center and two at 20 ; the mean angle of vergence was 17.1. three types of movements were elicited : pure saccades , pure vergence , and combined movements .
the fixation point was either the central led at the distance of 20 cm or the central led at the distance of 150 cm .
pure saccades to the left or to the right were elicited either at a close distance of 20 cm or at a far distance of 150 cm .
pure vergence was either convergence or divergence between the two leds placed on the median plane at 20 cm and 150 cm .
the required vergence movement was always 14.8 ( 17.1 2.3 ) for both pure vergence along the median plane and combined eye movements .
two temporal paradigms were used ( gap and simultaneous , see figure 1(b ) ) .
the gap paradigm was used to elicit short - latency eye movements . for each trial , the central led ( at a distance of either 20 cm or 150 cm ) was switched on for a period of 2.5-second .
then it was switched off , and a target led appeared 200 ms later ( gap period ) .
after a 2.5 seconds fixation period , the central led was switched off , and simultaneously the target led was switched on for 1.5 seconds .
the instruction given to the children was to look at the target led as accurately and as rapidly as possible .
children were in a dark room and faced the horizontal table ; viewing was binocular .
child performed 4 blocks of 36 trials each , two with the gap and two with the simultaneous paradigm ; each block was separated by a few minutes of rest . in each block
calibration was done before and after each block to allow accurate evaluation of the amplitude of the saccades .
calibration factors for each eye were extracted from the eye positions during the calibration procedure ; a polynomial function with five parameters was used to fit the calibration data .
a standard saccade paradigm was used to elicit visually guided saccades to target - leds presented at 0 , 10 , and 20 at the far isovergence surface .
the child fixated the central led for 2 seconds ; the central led disappeared and another led appeared for 2 seconds at an eccentric position to the left or to the right at 10 or 20. child was instructed to fixate the led as accurately as possible ; the led presentation was sufficiently long to allow accurate fixation .
data collection was controlled by rex , a software developed for real - time experiments and run on a pc .
horizontal eye movements from both eyes were recorded simultaneously with a photoelectric device ( oculometer , bouis , karlsruhe , germany ) .
this system has a resolution of 2 minutes of arc and a linear range of 20. there is no obstruction of the visual field with this recording system .
eye - position signals were lowpass filtered with a cutoff frequency of 200 hz and digitized with a 12-bit analogue - to - digital converter ; each channel was sampled at 500 hz . a polynomial function with five parameters was used to calibrate individual eye position signal . from these two signals , we derived the conjugate saccadic signal [ ( left eye + right eye)/2 ] and the vergence disconjugate signal ( left eye right eye ) .
off - line computer algorithms based on standard velocity and acceleration criteria were used to determine the saccade and vergence onset and offset .
the onset of the conjugate saccadic component ( for pure saccades and for the saccade component of combined movements ) was defined as the time when the eye velocity reached 5% of the saccadic peak velocity ; the offset of this signal was defined as the time when the eye velocity dropped below 10/s .
the onset and the offset of the vergence signals ( for pure vergence movements and for the vergence component of combined movements ) were defined as the time point when the eye velocity exceeded or dropped 5/s .
these criteria are standard and similar to those used by other authors . for all types of eye movements and for both saccade and vergence components , we measured the gain , which is the ratio of the amplitude of the total movement over the target excursion amplitude and the mean velocity ( amplitude of the movement over duration ) .
statistical analysis was performed by the two - way anovas as with between subject factor of the two groups of children ( dyslexic readers and non - dyslexic readers ) and as within subject factor of the individual means of the mean velocity and of the gain for each type of eye movements for the two paradigms used ( gap and simultaneous ) .
figure 2 shows the mean velocity of all eye movements tested in the gap and in the simultaneous paradigm for the two groups of children ( dyslexic and non - dyslexic ) ; in figures 2(a ) and 2(b ) , movements with high mean velocity , saccades in pure and combined form are shown , and in figures 2(c ) and 2(d ) , movements with slow mean velocity ( vergence pure and combined with saccades ) are shown .
table 2 shows the minimum and the maximum mean velocity values for dyslexic and non - dyslexic reader children for each type of eye movements examined in the two paradigms .
the mean velocity value for each type of eye movements examined in dyslexic readers is similar to that found in non - dyslexic readers . for each type of eye movements
, the anova does not show any significant difference between the two groups of children ( f(1,28 ) = 1.43 , p = .24 ) ; there is no significant effect of the paradigms and there is no significant interaction between the groups of children and the paradigms . in sum ,
figure 3 shows the mean gain ( amplitude of the movement / target excursion ) of all types of eye movements tested ( saccades at far and at near distance , convergence , divergence , and both components of combined saccade - vergence movements ) in the gap and in the simultaneous paradigms for dyslexic and non - dyslexic reader children .
table 3 shows the minimum and the maximum gain values for each group of children for each type of eye movements examined in the two paradigms tested . for
each type of eye movements examined , the mean gain in dyslexic readers is similar to that found in non - dyslexic readers . for each type of eye movements
, the anova does not show any significant difference between the two groups of children ( f(1,28 ) = 1.74 , p = .19 ) ; there is no significant effect of the paradigms and there is no significant interaction between the groups of children and the paradigms . in conclusion ,
this study shows that the mean velocity and the accuracy of eye movements in natural space ( saccades , vergence , and combined eye movements ) in dyslexic readers are as good as in non - dyslexic children of similar age .
these findings are new and contribute to know further the oculomotor capabilities in a population of dyslexic readers . indeed ,
while studying latency of eye movements provides information about cortical function , speed - accuracy parameters are important measures to understand the functioning of the premotor and central circuits involved in the triggering of eye movements .
our previous study conducted on dyslexic reader children showed abnormally longer latency for some types of eye movements suggesting that such population of children has some difficulties on both voluntary and reflexive controls of attention .
in contrast , the present study shows similar speed - accuracy characteristics in dyslexic and non - dyslexic reader children .
based on all these findings , we can advance the hypothesis that dyslexic reader children have problems in the central and cortical processes involved in the preparation of eye movements while the premotor and central areas responsible of the triggering of such eye movements are working as well as in non - dyslexic readers .
this idea is also in line with our findings on binocular control of saccades and fixation in dyslexic readers [ 7 , 8 ] .
indeed , in these studies we reported that dyslexic children showed poor quality of binocular coordination of saccades and fixation , independently of the task used , suggesting an intrinsic ocular motor deficit .
such a deficiency could be related to immaturity of the normal ocular motor learning mechanisms via which ocular motor coordination and stable fixation are achieved .
the cerebellum and cortical areas of the magnocellular stream such as the parietal cortex could be the sites of ocular motor learning . consequently , we suggest that dyslexic reader children may have immaturity and/or deficiency in this network . in the future ,
studies examining further adaptive cortical learning mechanisms alone and combined with visual attention training activities in dyslexic and non - dyslexic children are needed to improve the knowledge on dyslexia .
finally , this study also shows that temporal manipulation of the stimulus presentation ( by using gap and simultaneous paradigms ) does not influence the speed and the accuracy characteristics of eye movements for the two populations of children here tested ( dyslexic readers as well as non - dyslexic readers ) .
the majority of studies dealing with gap effect reported only data on latency of eye movements , showing the decrease of latency by the gap paradigm ; the work of pratt examined both the latency and the kinematic features as saccadic amplitude , duration , average velocity , peak velocity , and peak acceleration in young adults .
results showed greater peak velocities for saccades elicited by the gap paradigm with respect to the overlap paradigm .
such result is in contrast with the data presented here ; however , as also suggested by the author , these differences could be due to the specific experimental set up employed in the study ( e.g. , presence of a warning tone on every trial , limited number of subjects tested , overlap and gap paradigm compared ) that was different to those used here . in conclusion ,
our data found out from a larger population of children ( both dyslexic and non - dyslexic reader ) suggest that speed and accuracy characteristics of eye movements are predominantly determined by brainstem structures and they are not influenced by temporal manipulation of the stimulus presentation ; while the gap paradigm acts on physiological cortical mechanisms subtending attention and motor preparation leading to change in eye movement 's latency . | objective .
latency of eye movements depends on
cortical structures while speed of execution and accuracy depends
mostly on subcortical brainstem structures .
prior studies reported
in dyslexic reader children abnormalities of latencies of saccades
( isolated and combined with vergence ) ; such abnormalities were
attributed to deficits of fixation control and of visual
attention . in this study
we examine speed and accuracy
characteristics of horizontal eye movements in natural space
( saccades , vergence and combined movements ) in dyslexic reader
children .
methods .
two paradigms are tested : gap
paradigm ( fixation offset 200 ms prior to target onset ) ,
producing shorter latencies , in both non - dyslexic reader and
dyslexic reader children and simultaneous paradigm .
seventeen
dyslexic reader children ( mean age :
12 0.08
years ) and thirteen non - dyslexic reader children ( mean age :
12 1
years ) were tested .
horizontal eye movements from both eyes were
recorded simultaneously by a photoelectric device ( oculometer , dr .
bouis ) . results . for all movements tested
( saccades , vergence , isolated or combined ) and for both paradigms ,
the mean velocity and accuracy were similar in dyslexic readers
and non - dyslexic readers ; no significant difference was found .
conclusion .
this negative but important result ,
suggests no dysfunction of brainstem ocular motor circuits in
dyslexic readers .
it contrasts results on latencies related to
visual attention dysfunction at cortical level . |
genome - wide association studies ( gwas ) have successfully discovered nearly 1500 genetic loci associated with over 200 common disease and quantitative traits ( http://www.genome.gov/gwastudies/ ) .
gene interactions ( epistasis ) that could be a potential source of missing heritability evidenced in these studies ( 1,2 ) .
a recent study demonstrated that epistasis could exist widely in biological pathways and create substantial phantom heritability undetectable via conventional gwas ( 3 ) .
indeed , additional tests of interactions involving gwas loci with significant marginal effects have successfully discovered epistasis in several studies ( 47 ) . however , despite enormous effort , so far studying epistasis in gwas has been far more challenging and less fruitful than conventional gwas focussing on single locus effects ( 3,8 ) .
one major challenge in studying epistasis in gwas ( typically with hundreds of thousands of snp markers ) is the need to scrutinize billions of pairwise snp combinations in order to consider all possible interactions .
the challenge is increasing as more sequencing data ( and consequently more snps ) become available in future gwas .
these methods are confined to either binary disease or quantitative traits and several are designed specifically for computers equipped with particular graphical processing units .
substantial computing time is still required in some of these methods , especially when widely available computer systems are used ( 11,13 ) .
there is therefore great demand for high - throughput tools that can run on widely used computer platforms to analyse both quantitative and disease traits , making the analysis of epistasis routine in gwas and ultimately improving our understanding of the role of epistasis in complex traits .
another major challenge is that many current gwas populations may have only limited power to detect and replicate significant epistasis signals due to their relatively small sample sizes ( 8,14 ) .
the approach of meta - analysis of multiple gwas ( 15 ) may enhance the power of detection of epistasis but this is not yet applicable due to the lack of powerful computational tools to support epistasis analysis in such data sets using imputed ( not categorical ) genotype data .
pathway - based approaches may narrow the search space and also enhance power , for example , by seeking pathway pathway interactions ( 3 ) or by identifying common pathways enriched in epistatic genes with modest interaction signals ( i.e. strong but not necessarily genome - wide significant ) detected from multiple gwas populations ( 16 ) . in either case , fast screening of pairwise interactions in individual gwas populations appears to be critical in order to provide information for pathway - based analyses .
we have developed the biforce toolbox to address the challenges of high - throughput detection of epistasis .
biforce toolbox is programmed in java to support large scale analysis of pairwise epistasis in quantitative and disease traits on commonly used computer systems ( e.g. running either windows , osx or linux operating system ) via a graphical user interface ( gui ) or the command line .
it integrates available algorithms and advanced computing technologies such as bitwise computing technologies first adopted in boost ( 12 ) and fastepistasis ( 11 ) and multi - threaded parallelization into one software package to offer rapid and comprehensive pairwise genome scans .
biforce toolbox is built on concepts and algorithms we previously developed to maximize the power of detection of different forms of epistasis while controlling false positive rates ( 9,17,18 ) .
it has been rigorously tested and successfully identified interesting interaction signals and pathways in multiple studies using large gwas data sets ( 7,16 ) . here
, we describe the main features and functionality of the biforce toolbox using the body mass index ( bmi ) trait in the northern finland birth cohort 1966 ( nfbc1966 ) ( 16,19 ) as an example .
the nfbc1966 gwas data were provided by the database of genotype and phenotype ( dbgap ; http://www.ncbi.nlm.nih.gov/gap ) via specific data use certification .
biforce toolbox is named to reflect its main features : fast screening of pairwise interactions in gwas of complex disease and quantitative traits , using the brute force computational power of bitwise computing and multi - threaded parallelization .
it is implemented in java to enable its use on most commonly used computer systems , allowing local secure analysis of gwas data sets and comprehensive fast genome - wide scans for epistasis .
it has been designed to be user friendly and benefits from an intuitive gui as well as command line access for automated submission of jobs .
biforce toolbox uses a combined search algorithm ( 17,18 ) that integrates a full pairwise genome scans with specific tests of interactions involving snps with marginal effects that are genome - wide significant ( marginal - snps ) to increase the power of detection .
biforce toolbox is free and the binary files compiled for the three operating systems ( mac osx , windows and linux ) can be downloaded from http://bioinfo.utu.fi / biforcetoolbox/. biforce toolbox takes ordinary gwas data ( in genotype and phenotype files ) as input , where snp genotypes need to pass normal quality control procedures and phenotypes are recommended to be adjusted for covariates and relatedness for quantitative traits ( 7,16 ) or post biforce analysis for disease traits . after data input , it converts the snp genotype data into boolean bit values and the data are stored in memory - efficient java bitset arrays that allow missing snp genotypes to be handled easily and boolean bitwise operations ( e.g. logical and ) to be applied to the arrays of bit values , which makes the association tests ( see below ) extremely fast .
further , biforce toolbox partitions the whole pairwise genome scan automatically into smaller tasks and feed them evenly to available processing threads to compute in parallel and store results appropriately .
the combined search algorithm implemented in biforce toolbox includes two consecutive genome scans : single snp - based genome - wide association tests and pairwise epistatic interaction tests of all snp combinations .
marginal - snps are identified in the first scan and used to test interactions involving them . by default
the 5% genome - wide significance thresholds are derived based on the bonferroni correction for the total number of tests performed . given n to be the total number of snps in a gwas with k ( k > 0 ) marginal
- snps being identified , the thresholds are : p = 0.05/n for marginal - snps , p = 0.05/[(n 1 ) * k ] for marginal - snp interactions and p = 0.05/[n * ( n 1)/2 ] for a pairwise genome scan . alternatively , user specified significance thresholds can be specified in the analysis .
association tests are based on linear regression models , where the genotypes of each snp ( i.e. homozygote of the minor allele , homozygote of the major allele and heterozygote ) are fitted as fixed factors .
pairwise snp interactions are assessed using contingency tables which makes biforce toolbox applicable to both quantitative and binary disease traits .
briefly , a saturated model ( fitting two snps and interactions ) is tested against a reduced model ( fitting the two snps without interactions ) with four degrees of freedom and then the f ratio for quantitative traits or log - likelihood ratio for binary traits is calculated .
p values are then derived according to specific test statistic distributions and appropriate degrees of freedom ( assuming fixed four degrees of freedom in interaction tests for disease traits ) . for disease traits ,
biforce toolbox adopts the approximation filtering algorithm developed in boost ( 12 ) as a default option to accelerate the exhaustive pairwise genome scan which can be dismissed when necessary ( i.e. using only log - likelihood ratio tests in the scan ) . on completion of the search process
, biforce toolbox can be used to generate summary information and examine the identified snp pairs ( e.g. allele frequencies and contingency tables ) after reloading the input data files and the retained results ( figure 1 ) .
furthermore , it can perform a generic analysis of pathways enriched within groups of genes showing interaction signals to provide an initial view of the potential biology underlying these signals .
such an analysis involves two steps : annotating snps ( with reference snp i d numbers ) to the nearest genes and then performing overrepresentation analysis based on hypergeometric tests against a variety of gene - centred functional data such as gene ontology ( go ) and kegg pathway annotation ( 20 ) .
further details of such an analysis can be found in the manual of biforce toolbox .
gwas data are generated by microarray or next - generation sequencing platforms ( a ) .
snapshot of the biforce toolbox gui after loading and conversion of the bmi data of the nfbc1966 cohort ( note the multi - tab options such as working with subsets of snps or snp - pairs and performing single snp - based genome - wide association ) ( d ) .
snapshot of the biforce toolbox gui running the pairwise genome scan of the bmi data with progress reporting at the bottom ( note snp information , genotype counts , contingency table are showed for a retained pair of snps ; the retained results of the pairwise genome scan can be exported as tab - separated text file or excel compatible spreadsheet for further analyses by users ) ( e ) . a graphic view of positions of interaction signals in bmi of the nfbc1966 cohort generated by a third party tool circos , where chromosome ideograms are shown around the outer ring and are oriented pter
snapshot of the biforce toolbox gui running pathway enrichment analysis after the pairwise genome scan using epistatic genes with interaction log10
p values greater than 7.3 ( i.e. p < 5.0e ; note the member epistatic genes and associated snps are displayed for a pathway selected ) ( g ) .
gwas data are generated by microarray or next - generation sequencing platforms ( a ) .
snapshot of the biforce toolbox gui after loading and conversion of the bmi data of the nfbc1966 cohort ( note the multi - tab options such as working with subsets of snps or snp - pairs and performing single snp - based genome - wide association ) ( d ) .
snapshot of the biforce toolbox gui running the pairwise genome scan of the bmi data with progress reporting at the bottom ( note snp information , genotype counts , contingency table are showed for a retained pair of snps ; the retained results of the pairwise genome scan can be exported as tab - separated text file or excel compatible spreadsheet for further analyses by users ) ( e ) .
a graphic view of positions of interaction signals in bmi of the nfbc1966 cohort generated by a third party tool circos , where chromosome ideograms are shown around the outer ring and are oriented pter
snapshot of the biforce toolbox gui running pathway enrichment analysis after the pairwise genome scan using epistatic genes with interaction log10
p values greater than 7.3 ( i.e. p < 5.0e ; note the member epistatic genes and associated snps are displayed for a pathway selected ) ( g ) .
we use bmi of the nfbc1966 cohort to illustrate a typical analysis procedure using biforce toolbox .
the genotype and phenotype data were pre - processed following the instructions given in the original gwas ( 19 ) .
bmi was further corrected for covariates and relatedness and normalized to prevent spurious associations as detailed elsewhere ( 16 ) . in total 323 697 autosomal snps and
the remaining analysis is summarized in figure 1 , with an example list of the retained results of the pairwise genome scan ( none is genome - wide significant ) shown in table 1 .
table 1.a short list of the results of the pairwise genome scan of bmi in nfbc1966 cohortsnp1snp2anova_pairanova_intpair_log10pint_log10pgenoclassnors9873966rs9506047.70414.8509.58711.3419rs9873966rs44063737.64614.7859.49611.2879rs2310173rs96914727.61514.3119.44810.8939rs7536830rs111690637.64413.9289.49210.5769rs1453405rs22890257.23713.7448.86210.4239rs2962896rs68906736.81013.4668.20310.1939rs983936rs10746517.38013.4659.08310.1929rs983936rs15416947.32413.4478.99710.1779rs10490096rs178160027.30516.7898.01810.1248rs3980965rs117639727.26013.0018.8979.8089snp1 ( snp2 ) : the first ( second ) snp ; anova_pair ( anova_int ) : f ratio of a whole pair of snps with interaction ( interaction between a pair of snps ) ; pair_log10p ( int_log10p ) : log10
p value of a whole pair of snps with interaction ( interaction between a pair of snps ) ; genoclassno : the number of joint genotype classes ( 9 in total ) with samples . a short list of the results of the pairwise genome scan of bmi in nfbc1966 cohort snp1 ( snp2 ) : the first ( second ) snp ; anova_pair ( anova_int ) : f ratio of a whole pair of snps with interaction ( interaction between a pair of snps ) ; pair_log10p ( int_log10p ) : log10
p value of a whole pair of snps with interaction ( interaction between a pair of snps ) ; genoclassno : the number of joint genotype classes ( 9 in total ) with samples .
we measured the biforce toolbox performance in analysing disease ( 50% cases and 50% controls ) and quantitative traits of gwas datasets with 500 000 snps and 5000 samples , using one thread and eight threads on a single workstation ( 2.8 ghz intel core imac with 4 gb ram , 4 cpu cores each with 2 threads ) and 256 threads on a computer cluster ( 32 nodes each with 4 cpu cores each with 2 threads ) , respectively .
biforce toolbox took 118.18 , 30.8 and 0.46 h , respectively , for the disease trait , and 293.24 ( using 8 threads on the workstation ) and 6.81 h ( using 256 threads on the cluster ) for the quantitative trait .
in contrast , fastepistasis , a parallel extension of plink ( 21 ) took 29 , 4 or 0.5 days to analyse a quantitative trait in a gwas data set of the same size using 8 , 64 or 512 mpi - bound processors , respectively ( 11 ) ; gboost , a graphical processing unit version of boost ( 12 ) took 1.34 h to analyse a disease trait in a smaller gwas data set ( 351 542 snps and 5003 samples ) on a computer with a nvidia geforce gtx 285 display card ( i.e. 240 cpu cores ) ( 13 ) .
biforce toolbox is a powerful and accessible tool to support high - throughput analysis of epistasis in gwas of disease and quantitative traits on general computer platforms .
it is hoped that with biforce toolbox analysis the study of epistasis in gwas will become a routine exercise and hence improve our understanding of the role of epistasis in the architecture of complex traits .
the biotechnology and biological sciences research council ( bbsrc ) [ bb / h024484/1 to w.h.w . ] ; the medical research council core fund ( to c.a.s . , c.s.h . and w.h.w . ) . funding for open access charge : bbsrc [ bb / h024484/1 ] . | genome - wide association studies ( gwas ) have discovered many loci associated with common disease and quantitative traits . however , most gwas have not studied the gene
gene interactions ( epistasis ) that could be important in complex trait genetics .
a major challenge in analysing epistasis in gwas is the enormous computational demands of analysing billions of snp combinations .
several methods have been developed recently to address this , some using computers equipped with particular graphical processing units , most restricted to binary disease traits and all poorly suited to general usage on the most widely used operating systems .
we have developed the biforce toolbox to address the demand for high - throughput analysis of pairwise epistasis in gwas of quantitative and disease traits across all commonly used computer systems .
biforce toolbox is a stand - alone java program that integrates bitwise computing with multithreaded parallelization and thus allows rapid full pairwise genome scans via a graphical user interface or the command line .
furthermore , biforce toolbox incorporates additional tests of interactions involving snps with significant marginal effects , potentially increasing the power of detection of epistasis .
biforce toolbox is easy to use and has been applied in multiple studies of epistasis in large gwas data sets , identifying interesting interaction signals and pathways . |
hypophysitis is an inflammatory condition of the pituitary gland that is often mistaken for other pituitary mass lesions .
granulomatous hypophysitis is a rare inflammatory disorder , accounting for < 1% of cases involving panhypopituitarism or visual field defects with headaches .
granulomatous hypophysitis commonly presents with enlargement of the pituitary gland that mimics an adenoma , making it difficult to confirm the disease until the time of surgery . in korea , only one case has been previously reported .
we herein report a 31-year - old woman who had normal brain magnetic resonance imaging ( mri ) findings 4 months prior to admission and was diagnosed with granulomatous hypophysitis at the time of surgery .
a 31-year - old woman was admitted to our hospital with severe headache , nausea , and vomiting .
four months before admission to our hospital , she had visited the department of neurology due to a bilateral temporal headache . neurologic examination and
1 ) , so she was treated pharmacologically with a nonsteroidal anti - inflammatory drug .
due to nausea and vomiting for 2 weeks , she was brought to the emergency room .
her blood pressure was 110/70 mmhg , pulse rate was 58 beat / min , respiratory rate was 16 breath / min , and body temperature was 36.6. a neurologic examination revealed bitemporal hemianopsia .
initial complete blood analysis revealed a white blood cell count of 4,860/mm , a hemoglobin level of 12.4 g / dl , and a platelet count of 241,000/mm .
the biochemical test results were as follows : protein , 7.2 g / dl ; albumin , 4.2 g / dl ; aspartate aminotransferase , 16
iu / l ; alanine aminotransferase , 6 iu / l ; blood urea nitrogen , 5.9 mg / dl ; and creatinine , 0.5 mg / dl .
the electrolyte test results were as follows : sodium , 113 meq / l ; potassium , 4.3 meq / l ; and chloride , 82 meq / l .
the thyroid function test results were as follows : serum thyroid - stimulating hormone ( tsh ) level , 0.56 iu / ml ( reference range , 0.27 to 4.2 ) ; free t4 , 0.91 ng / dl ( 0.93 to 1.7 ) ; and t3 , 0.89 ng / ml ( 0.8 to 2.0 ) .
we performed a combined pituitary stimulation function test and rapid adrenocorticotropic hormone ( acth ) stimulation test for further assessment .
we injected regular insulin ( 0.1 /kg ) , thyrotropin - releasing hormone ( 200 g ) , and luteinizing hormone ( lh)-releasing hormone ( 100 g ) ; 2 hours later , the patient 's blood glucose level fell to 60 mg / dl and she complained of hypoglycemic symptoms .
the combined pituitary function stimulation test showed no increase in serum growth hormone , acth , or tsh level .
serum levels of lh and follicle - stimulating hormone were normal over time , and mild hyperprolactinemia was present with normal increments over time ( table 2 ) .
t1- and t2-weighted mri showed an 18 10-mm round mass with isosignal intensity in the sella .
2 ) . prednisolone and levothyroxine were prescribed and the mass was removed using a transsphenoidal approach .
the acid - fast bacilli stain , tuberculosis polymerase chain reaction , angiotensinogen - converting enzyme , and venereal disease tests yielded no abnormal finding .
the patient was finally diagnosed with idiopathic granulomatous hypophysitis , and estradiol and progesterone were added to the prednisolone and levothyroxine for maintenance therapy .
inflammatory pituitary lesions are extremely rare and can be classified into three clinicopathologic entities : lymphocytic , xanthomatous , and granulomatous hypophysitis .
granulomatous hypophysitis , a rare chronic inflammation of the pituitary gland that creates granulomas , has been reported in < 1% of sellar lesions based on surgical findings with the transsphenoidal approach .
lymphocytic hypophysitis is distinguished from granulomatous hypophysitis by the absence of nodular aggregations of epithelioid histiocytes and multinucleated giant cells , but these two diseases show ultrastructural similarities .
the two entities may have the same pathogenetic background or even represent different stages of the same disease , and some authors have suggested that they represent an autoimmune spectrum from a purely lymphocytic form , constituting the predominant early lesion , to a granulomatous form appearing later .
granulomatous hypophysitis , however , differs from lymphocytic hypophysitis in certain epidemiological features , which are mostly associated with pregnancy and autoimmune diseases .
imaging studies can help to distinguish granulomatous hypophysitis from pituitary adenoma . in patients with granulomatous hypophysitis
, mri usually shows symmetric enlargement of the pituitary gland , pre - contrast homogeneity of the pituitary mass , an intact sellar floor , and rapid enhancement of the pituitary stalk after gadolinium administration .
however , these findings are not specific and are quite indistinguishable from other neoplastic or inflammatory hypophyseal processes on imaging .
patients may also present with progressive chiasmal compression , hypopituitarism , amenorrhea - galactorrhea , hyperprolactinemia , fatigue , diabetes insipidus , a sudden onset of aseptic meningitis , and/or blurred vision .
granulomatous hypophysitis is difficult to differentiate from a simple adenoma on the basis of clinical manifestations or imaging findings .
secondary causes of granulomatous hypophysitis include infections ( tuberculosis , syphilis , and fungal ) , systemic inflammation ( sarcoidosis , wegener 's granulomatosis , crohn 's disease , and histiocytosis x ) , and foreign body reactions ( ruptured rathke 's cysts ) .
hypopituitarism resolves after surgery in some cases , but many patients remain on full hormone replacement therapy , as in our case .
lymphocytic hypophysitis was first presumed in the present case because of the rapidly growing sellar lesion observed on mri and the patient 's history of delivery 6 months prior to admission . however , the postoperative biopsy revealed granulomatous hypophysitis , and other examinations ruled out secondary causes of hypophysitis . in summary ,
idiopathic granulomatous hypophysitis is a very rare pituitary condition that is difficult to diagnose preoperatively .
the treatment of this disease is surgery , which helps in diagnosis and treatment , and hormone replacement . | granulomatous hypophysitis is a rare pituitary condition that commonly presents with enlargement of the pituitary gland .
a 31-year - old woman was admitted to the hospital with a severe headache and bitemporal hemianopsia .
magnetic resonance imaging ( mri ) showed an 18 10-mm sellar mass with suprasellar extension and compression of the optic chiasm .
interestingly , brain mri had shown no abnormal finding 4 months previously .
on hormonal examination , hypopituitarism with mild hyperprolactinemia was noted .
the biopsy revealed granulomatous changes with multinucleated giant cells .
we herein report this rare case and discuss the relevant literature . |
pancreatic cancer is estimated as the ninth most frequent cancer and fifth most common cause of cancer - related death in korea ( 1 ) .
pancreatic cancer is rarely diagnosed before 45 yr of age , but its occurrence rises sharply thereafter . the incidence of pancreatic cancer is higher in men than women , and in africans , compared with the caucasian population ( 3 ) .
prognosis for patients with this disease is extremely poor , with a 7.6% 5-yr survival rate in korea ( 1 ) , mainly due to unresectable disease in 80%-90% of patients at the time of diagnosis ( 4 ) .
pancreatic cancer patients seldom exhibit disease - specific symptoms until late in the course of disease progression , and the impact of standard therapy is thus limited .
while the etiology of pancreatic cancer remains to be established , several known genetic and environmental factors are associated with its development .
so far , risk factors accounting for up to 30% of the disease have been established ( 5 ) . among the few risk factors identified to date ,
current smokers are at approximately double the risk as non - smokers , with a trend towards increasing risk according to the frequency or duration of smoking exposure ( 5 ) .
inconsistencies in the patterns of cigarette smoking and incidence between different countries , as well as the low relative risk , suggest that the disease is only partly attributable to smoking ( up to 20% ) , and therefore , other risk factors are likely to be important . diabetes mellitus ( 7 ) and chronic pancreatitis ( 8) are additional predisposing factors of the disease .
however , diabetes as a result of pancreatic cancer development is not infrequent , and chronic pancreatitis explains only less than 3% of pancreatic cancer cases .
an association with obesity has been reported ( 9 ) , but the effects of dietary factors and physical activity are currently unclear .
genetic susceptibility plays a role , with some cases being familial or related to hereditary melanoma , peutz - jeghers syndrome , hereditary breast or ovarian cancer , familial pancreatitis , or hereditary nonpolyposis colon cancer ( 10 ) .
hereditary factors , such as germline mutations , account for only about 10% of the total burden of pancreatic cancer ( 11 ) .
earlier studies have reported a higher association of pancreatic cancer with the non - o blood groups , compared to o ( 12 - 16 ) . a report based on a large
, prospective study involving almost one million subjects with years of follow - up showed a link between abo blood type and pancreatic cancer ( 13 ) . in agreement with
several previously published smaller - scale studies , these investigators showed that non - o blood types accounted for 17% of all new pancreatic cancers .
other recent studies have additionally detected a link between hepatitis b virus ( hbv ) or hepatitis c virus ( hcv ) infection and pancreatic cancer , indicating a stronger relationship with hbv than hcv ( 17 , 18 ) .
however , the findings to date are inconsistent , and the exact mechanism linking pancreatic cancer and blood groups is currently unclear . therefore , further investigation of these risk factors is warranted to determine their significance in pancreatic cancer .
the principal aim of this study was to evaluate the possible association between abo blood type and pancreatic cancer in korea .
in total , 753 patients with pancreatic cancer and 3,012 healthy controls , matched 4 to 1 with pancreatic cancer patients for age and sex , were retrospectively recruited at the national cancer center , korea , between 2001 and 2011 .
our subjects included patients with newly diagnosed pancreatic adenocarcinoma who were evaluated and treated at the national cancer center , korea .
causes of death were ascertained by linking the study patients with the national death certificate database of korea .
exclusion criteria were presence of other types of pancreatic neoplasm , such as neuroendocrine tumor or intraductal mucinous neoplasm of pancreas . among the 2,243 eligible pancreatic cancer patients in the hospital database , 753 ( 34% ) had a historical or current abo blood group available , of which 625 ( 83% ) cases were pathologically confirmed adenocarcinoma .
data on sex , age at diagnosis of cancer , follow - up time after diagnosis , and tumor staging were collected from electronic medical records .
eligible control subjects were selected from individuals in the cancer screening cohort , who were subjected to routine health examinations at the center for cancer prevention and detection of national cancer center , korea , between 2001 and 2011 . in total , 3,012 healthy subjects were included .
control patients were randomly selected from these subjects and matched 4:1 with pancreatic cancer patients by age , sex , and date of admission or visit .
case and control frequencies were matched by age ( 5 yr ) and sex . as part of the health maintenance examination , controls underwent laboratory tests , including tests for hbsag , anti - hbs antibody , anti - hcv antibody , abo blood type , chest radiography , upper endoscopy , occult blood in stool , and abdominal sonography and/or contrast - enhanced computed tomography ( ct ) .
we retrospectively reviewed data , including age , sex , use of tobacco , and diabetes , but were unable to obtain reliable information on pre - diagnosis body mass index , personal or family history of cancer , and alcohol use .
the chemiluminescent microparticle immunoassay ( abbott laboratories , chicago , il , usa ) was used as a screening test for the presence of hbsag in blood .
the presence of antibodies to hbsag ( anti - hbs ) and hcv ( anti - hcv ) was detected using a chemiluminescent immunoassay ( siemens healthcare diagnostics , chicago , il , usa ) .
chronic hbv and hcv infection were defined as the presence of hbsag and anti - hcv antibodies , respectively .
the demographic characteristics and proportions of potential risk factors ( including smoking and diabetes ) were compared among patients and controls .
the t - test was used to compare means of continuous variables between patients and controls , and the chi - square test applied to compare proportions .
multivariate logistic regression analyses , including age , gender , smoking history and diabetes , were performed to estimate the adjusted odds ratios ( aor ) . for each factor , we calculated the aor and 95% confidence interval ( ci ) using maximum likelihood estimation . overall survival ( os ) was defined as the time from initiation of treatment to the date of death .
os were assessed by the kaplan - meier method , and 95% ci for the median time to an event were calculated .
the study was reviewed and approved by the institutional review board of the national cancer center hospital ( irb no .
the study was reviewed and approved by the institutional review board of the national cancer center hospital ( irb no .
the mean age ( standard deviation ) of pancreatic cancer patients was 62.3 9.7 yr and 59.7 7.5 yr for controls .
table 1 presents the abo blood group distribution of 753 pancreatic cancer patients , compared with abo groups of the 3,012 healthy controls .
the frequency distribution patterns of abo blood groups of controls in the current study were similar to those of korean blood donors reported in the korea health and welfare white paper ( 1995 ) . compared with blood type o , the odds ratios ( 95% ci ) in univariate analysis were 1.25 ( 1.04 - 1.53 ; p = 0.02 ) for non - o ( a , b , or ab ) groups after adjusting for age and gender ( table 2 ) .
aor for pancreatic cancer in subjects with non - o blood types was 1.29 ( 95% ci , 1.05 - 1.58 ; p = 0.01 ) .
the odds ratios in univariate analysis were determined as 1.36 ( 1.10 - 1.68 ; p = 0.03 ) for blood type a , 1.21 ( 0.91 - 1.60 ; p = 0.76 ) for type ab , and 1.15 ( 0.92 - 1.44 ; p = 0.79 ) for type b , compared with blood type o , after adjusting for age and gender .
the aors for pancreatic cancer in subjects with blood types a , ab , and b were 1.36 ( 1.09 - 1.71 ; p = 0.08 ) , 1.29 ( 0.96 - 1.74 ; p = 0.47 ) , and 1.20 ( 0.94 - 1.52 ; p = 0.94 ) , respectively .
seropositivity for hbsag was not significantly related to pancreatic cancer , either in univariate ( odds ratio 1.03 ; 95% ci , 0.69 - 1.53 ; p = 0.91 ) or multivariate analysis ( aor , 1.02 ; 95% ci , 0.67 - 1.56 ; p = 0.93 ) ( table 2 ) .
the aor for pancreatic cancer in subjects with seropositivity for anti - hcv was 2.30 ( 95% ci , 1.30 - 4.08 ; p < 0.01 ) .
other risk factors for pancreatic cancer included presence of diabetes ( aor , 2.70 ; 95% ci , 2.20 - 3.31 ; p < 0.01 ) and smoking ( aor , 1.47 ; 95% ci , 1.16 - 1.86 ; p < 0.01 ) .
tnm stages of pancreatic cancer in the present study grouped by blood type are shown in table 3 .
no significant differences in the tnm stages of tumors among patients with various blood groups were evident ( p = 0.413 ) . the median survival times in subjects with blood groups a , b , ab and o were 9.1 , 9.6 , 7.4 , and 7.8 months , respectively , which were not significant , as indicated by the log - rank test ( p = 0.106 ) .
in addition , we observed no marked differences in survival times between the non - o ( a , b and ab ) and o blood groups ( p = 0.428 ) .
studies performed several decades ago initially suggested an association between blood type a and increased risk of pancreatic cancer , compared to blood groups o or b ( 19 - 21 ) .
however , increased pancreatic cancer risk in blood group b patients among 224 cases was observed , compared with a randomly selected group of patients admitted with nonmalignant diseases and blood donors in a more recent study ( 22 ) .
results from two large , independent prospective cohorts of caucasians from the united states suggested that compared with blood group o subjects , those with non - o blood types ( a , ab or b ) were more likely to develop pancreatic cancer ( 13 ) .
similarly , in two large case - control studies on chinese patients , blood group o was associated with a lower incidence of pancreatic cancer , compared with blood groups a and ab ( 16 , 23 ) . in the current investigation involving a large case - control korean study , patients with blood group o had a lower incidence of pancreatic cancer , compared to those with non - o blood groups , consistent with previous reports . to our knowledge , this is the first study showing a correlation between blood group o and pancreatic cancer development in the korean population .
abo blood group antigens are widely distributed throughout the body in addition to their regular occurrence on the red blood cell surface .
the abo phenotype may be associated with risk of gastric cancer , gastric and duodenal ulcer , chronic atrophic gastritis , as well as pancreatic cancer ( 24 ) .
human pancreatic cancer has been shown to express either a or b antigens corresponding to the individual blood group ( 25 ) or lose blood group antigen expression in 80% of the cases ( 26 ) .
deletion of a , b , h or lewis antigens and incompatible expression of a or b antigens has been reported as a cancer - associated event in the pancreas ( 27 ) .
incompatible expression of blood group - related antigens is observed in pancreatic cancer cells , compared with patient blood group type , indicating that lewis antigen expression in pancreatic cancer is independent of the blood group phenotype and may be useful as a tumor marker ( 28 ) .
a recent genome - wide association study revealed an association of a particular abo locus on chromosome 9q34 with susceptibility to pancreatic cancer ( 29 ) .
this snp maps to the first intron of the abo blood type gene . however , limited information is currently available on the role of abo blood group antigens in pancreatic cancer risk in korea .
recently , a retrospective analysis based on the surgical database at washington university in st .
louis showed that abo blood type does not affect overall survival among patients with resected pancreatic adenocarcinomas ( 30 ) .
however , among chinese patients subjected to potentially curative operation , the median survival time of individuals with blood group o was significantly longer than that of those with non - o blood groups ( 16 ) .
the earlier study was limited to a population with resectable disease , accounting for only 10%-20% of patients with pancreatic cancer .
our data showed no association between abo blood type and overall survival in patients with locally advanced and metastatic disease , as well as resected pancreatic cancer .
this result suggested that abo blood type is not associated with the risk for progression in pancreatic cancer .
the hbv antigen has been detected in the pancreatic acinar cells and pancreatic juice in patients with viral replication in the liver ( 31 , 32 ) .
one hypothesis is that hbv can infect and replicate in human pancreatic cells and function in the oncogenesis of pancreatic carcinoma .
however , it is not clear from a biologic viewpoint why resolved or occult hbv infection is associated with elevated pancreatic cancer risk .
previous epidemiological analyses have reported conflicting results on the relationship between chronic hbv infection and development of pancreatic cancer ( 17 , 23 , 33 - 35 ) .
korea is one of the known endemic areas for chronic hepatitis b infection , and seropositivity of hbsag was estimated as 7% to 9% in the 1980s , with about 60% to 70% of adults showing evidence of present or past hbv infection ( 36 ) .
epidemiological findings based on the 1998 korea national health and nutrition survey disclosed hbsag seropositivity of 5.1% in males and 4.1% in females aged 10 yr or over .
this figure was similar among patients with pancreatic cancer and control subjects , resulting in aor for hbsag positivity of 1.02 ( 95% ci , 0.67 - 1.56 ; p=0.93 ) .
further studies are required to confirm this finding , and the potential association between hepatitis b viral infection and pancreatic cancer , although interesting , should be interpreted with caution .
a retrospective cohort study reported that risk of pancreatic cancer is slightly elevated in patients with hcv , which is attenuated after adjusting for alcohol use , pancreatitis , and other variables ( 18 ) . in the present case - control study ,
the aor for pancreatic cancer in subjects with seropositivity for anti - hcv was 2.30 ( 95% ci , 1.30 - 4.08 ; p=0.004 ) .
however , the biological rationale explaining the association between hcv infection and pancreatic cancer is yet to be established .
serum levels of pancreatic enzymes have been shown to increase with liver disease progression in patients diagnosed with viral hepatitis ( 37 , 38 ) .
however , we were limited in terms of our ability to confirm hcv infection based on the presence of serum hcv rna .
serological assays designed to detect antibody to hcv are associated with a high degree of false positivity in regions with low prevalence and low - risk populations .
we were additionally restricted in our ability to adjust for alcohol use , a potential risk factor for pancreatic cancer .
interestingly , the risk of pancreatic cancer in patients with anti - hcv seropositivity was not attenuated after adjusting for smoking and diabetes , which are known risk factors for the disease .
additional epidemiological studies focusing on the association of hcv with pancreatic cancer are therefore necessary .
our results collectively suggest that non - o blood types and seropositivity for anti - hcv , but not hbv infection , may increase the risk of developing pancreatic cancer in korea , where hbv is endemic .
further research is necessary to define the mechanisms by which abo blood type or closely linked genetic variants influence pancreatic cancer risk in korea . | several studies have reported that abo blood group , hepatitis b virus ( hbv ) and hepatitis c virus ( hcv ) infection contribute to the development of pancreatic cancer .
the aim of this study was to evaluate the association between these factors and pancreatic cancer in the korean population .
we retrospectively recruited 753 patients with pancreatic cancer and 3,012 healthy controls , matched 4 to 1 with cancer patients for age and sex , between 2001 and 2011 , at the national cancer center , korea .
a multivariate logistic regression analysis was employed to estimate adjusted odds ratios ( aors ) .
the aor for pancreatic cancer in subjects with non - o blood types ( a , ab , and b ) , compared to blood type o , was 1.29 ( 95% ci , 1.05 - 1.58 ; p = 0.01 ) .
seropositivity for hepatitis b virus surface antigen was not significantly related to pancreatic cancer , either in univariate ( odds ratio 1.03 ; 95% ci , 0.69 - 1.53 ; p = 0.91 ) or multivariate analysis ( aor , 1.02 ; 95% ci , 0.67 - 1.56 ; p = 0.93 ) .
the aor for pancreatic cancer in subjects displaying seropositivity for anti - hcv was 2.30 ( 95% ci , 1.30 - 4.08 ; p < 0.01 ) .
our results suggest that the non - o blood types and anti - hcv seropositivity , but not hbv infection , may increase the risk of developing pancreatic cancer in korea , where hbv is endemic . |
senior citizens aged 75 years and older represent the largest and most frequent users of health care facilities such as hospitals and long - term skilled nursing and residential homes
. since immune systems are often affected by aging and underlying diseases , incidences of hospital - acquired pneumonia ( hap ) induced by methicillin - resistant staphylococcus aureus ( mrsa ) are more frequent in the elderly .
1 improving treatment outcomes for elderly patients with mrsa infections will therefore increase survival and quality of life , and reduce health care expenditure burdens.2 mrsa hap is primarily treated via the intravenous administration of vancomycin ( vcm ) , and the dosage is determined by pharmacokinetic / pharmacodynamic ( pk / pd ) parameters .
however , a previous study involving adult patients with mrsa pneumonia or sepsis failed to show an association between the pk / pd parameters for vcm and treatment outcome.3 martin et al4 recommend a target area under the concentration curve ( auc)/minimum inhibitory concentration ( mic ) for vcm of > 400 g h / ml , while we previously reported that auc / mic values of 250450 g h / ml were acceptable for elderly patients.5 the reason for this discrepancy is currently unclear .
vcm treatment outcomes depend on the characteristics of the patient , and it is well known that hypoalbuminemia is a risk factor for poor treatment outcomes , as reported in a previous study.6 hayashi et al7 reported that hypoalbuminemia was a risk factor with the long - term administration of vcm .
however , the reason that hypoalbuminemia has an influence on the treatment outcome of vcm remains unknown .
since the potency of vcm is dependent on the free unbound form , albumin levels may have an influence on vcm treatment outcomes .
in addition , body fluid volume and renal function are frequently decreased in elderly patients , and such patients show large individual differences in pk / pd parameters .
nevertheless , these parameters in elderly patients with hypoalbuminemia have not been clarified . to elucidate the association between severe hypoalbuminemia and vcm treatment outcomes
the study was conducted at the national center for geriatrics and gerontology hospital , obu , japan .
this 320-bed hospital oversees general ( including emergency ) services , except pediatrics , and admits approximately 5,000 patients per year ( more than 50% of whom are aged over 75 years ) .
mrsa is endemic in this hospital , and the ratio of mrsa isolates per total s. aureus isolates is approximately 70% . over a 7-year period ( from january 2006 through december 2012 ) , all hospitalized patients aged 75 years or older with mrsa pneumonia that had been microbiologically confirmed by sputum or blood cultures , and who had been treated with vcm therapy , were identified using the clinical pharmacokinetics department computer database .
we conducted a retrospective observational study with 28-day mortality as the primary outcome for 94 elderly patients with mrsa pneumonia who had been treated with vcm during the 7-year study period .
the baseline characteristics of the study patients were age , gender , body weight , serum creatinine level , charlson comorbidity index , albumin level , combination antibiotic therapy , diagnosis of pneumonia , and infection severity .
in addition , we assessed the relationship between the effect of vcm pk indices , including serum peak and trough concentrations , auc / mic values , volume of distribution , half - life , clearance of vcm , single and daily dose of vcm , and dose interval of vcm .
the clinical characteristics of the study patients were retrieved from the hospital medical records . for patients with multiple episodes ,
this study was approved by the ethics committee of the national center for geriatrics and gerontology hospital .
the definition of hap was based on american thoracic society guidelines for the management of adults with hospital - acquired , ventilator - associated , and health care - associated pneumonia.2 for the purposes of this study , hap was defined as pneumonia that occurred 48 hours or more after hospitalzation for an acute lung infection characterized by a cough , fever , purulent sputum , and an abnormal chest x - ray that was not deemed to be incubating at the time of admission . among the hap cases , those with mrsa isolated from blood cultures or sputum that showed no signs of improvement after treatment with broad - spectrum antibiotics , such as carbapenem , for more than 3 days
severe hypoalbuminemia was defined as a serum albumin level of < 2.5 g / dl . the severity rating of pneumonia
was defined according to the japanese respiratory society guidelines for the management of hap8 and was used to allocate the patients into severe , moderate , and mild groups .
the severe group was defined as patients with three or more of the following risk factors or conditions : malignancy or immunocompromized status , impaired consciousness , requiring a fraction of inspired oxygen ( fio2 ) > 35% to maintain a saturated oxygen ( sao2 ) level > 90% , men aged 70 years or older , or women aged 75 years or older , and oliguria or dehydration .
the moderate group was defined as patients with any two of the above risk factors , and in addition , at least one of the following secondary risk factors : c - reactive protein ( crp ) 20.0 mg / l or extent of infiltration on a chest x - ray ( cxr ) covering at least two - thirds of one lung .
the mild group was defined as all other patients who did not fit the severe or moderate criteria .
the patients were divided into a severe hypoalbuminemia and a non - severe hypoalbuminemia group before vcm administration .
nephrotoxicity to vcm was defined as an increase in serum creatinine of 0.5 g / dl or a 50% increase over pretreatment levels.2 liver dysfunction was defined according to international consensus meeting criteria.9 the initial treatment schedule for vcm was simulated to achieve a trough concentration 1015 g / ml , with therapeutic drug monitoring ( tdm ) software using patient characteristics , including age , body weight and serum creatinine ( vcm - tdm microsoft excel version 3.0 , shionogi and co , ltd , osaka , japan ) .
the serum concentrations of vcm were determined from samples collected during the fifth day from the start of administration .
the blood samples were obtained twice : before vcm administration ( trough ) and 1 hour after vcm administration ( peak ) .
the predicted 24-hour auc values for vcm were calculated using the tdm software based on peak and trough concentrations .
continuous variables were compared using the student s t - test for normally distributed variables and the welch test for non - normally distributed variables .
categorical variables were compared with the chi - square ( ) test . in these tests ,
a two - sided p - value of < 0.05 was considered to be significant .
the study was conducted at the national center for geriatrics and gerontology hospital , obu , japan .
this 320-bed hospital oversees general ( including emergency ) services , except pediatrics , and admits approximately 5,000 patients per year ( more than 50% of whom are aged over 75 years ) .
mrsa is endemic in this hospital , and the ratio of mrsa isolates per total s. aureus isolates is approximately 70% . over a 7-year period ( from january 2006 through december 2012 ) , all hospitalized patients aged 75 years or older with mrsa pneumonia that had been microbiologically confirmed by sputum or blood cultures , and who had been treated with vcm therapy , were identified using the clinical pharmacokinetics department computer database .
we conducted a retrospective observational study with 28-day mortality as the primary outcome for 94 elderly patients with mrsa pneumonia who had been treated with vcm during the 7-year study period .
the baseline characteristics of the study patients were age , gender , body weight , serum creatinine level , charlson comorbidity index , albumin level , combination antibiotic therapy , diagnosis of pneumonia , and infection severity .
in addition , we assessed the relationship between the effect of vcm pk indices , including serum peak and trough concentrations , auc / mic values , volume of distribution , half - life , clearance of vcm , single and daily dose of vcm , and dose interval of vcm .
the clinical characteristics of the study patients were retrieved from the hospital medical records . for patients with multiple episodes ,
this study was approved by the ethics committee of the national center for geriatrics and gerontology hospital .
the definition of hap was based on american thoracic society guidelines for the management of adults with hospital - acquired , ventilator - associated , and health care - associated pneumonia.2 for the purposes of this study , hap was defined as pneumonia that occurred 48 hours or more after hospitalzation for an acute lung infection characterized by a cough , fever , purulent sputum , and an abnormal chest x - ray that was not deemed to be incubating at the time of admission . among the hap cases , those with mrsa isolated from blood cultures or sputum that showed no signs of improvement after treatment with broad - spectrum antibiotics , such as carbapenem , for more than 3 days were defined as mrsa hap .
severe hypoalbuminemia was defined as a serum albumin level of < 2.5 g / dl .
the severity rating of pneumonia was defined according to the japanese respiratory society guidelines for the management of hap8 and was used to allocate the patients into severe , moderate , and mild groups .
the severe group was defined as patients with three or more of the following risk factors or conditions : malignancy or immunocompromized status , impaired consciousness , requiring a fraction of inspired oxygen ( fio2 ) > 35% to maintain a saturated oxygen ( sao2 ) level > 90% , men aged 70 years or older , or women aged 75 years or older , and oliguria or dehydration .
the moderate group was defined as patients with any two of the above risk factors , and in addition , at least one of the following secondary risk factors : c - reactive protein ( crp ) 20.0 mg / l or extent of infiltration on a chest x - ray ( cxr ) covering at least two - thirds of one lung .
the mild group was defined as all other patients who did not fit the severe or moderate criteria .
the patients were divided into a severe hypoalbuminemia and a non - severe hypoalbuminemia group before vcm administration .
nephrotoxicity to vcm was defined as an increase in serum creatinine of 0.5 g / dl or a 50% increase over pretreatment levels.2 liver dysfunction was defined according to international consensus meeting criteria.9
the initial treatment schedule for vcm was simulated to achieve a trough concentration 1015 g / ml , with therapeutic drug monitoring ( tdm ) software using patient characteristics , including age , body weight and serum creatinine ( vcm - tdm microsoft excel version 3.0 , shionogi and co , ltd , osaka , japan ) .
the serum concentrations of vcm were determined from samples collected during the fifth day from the start of administration .
the blood samples were obtained twice : before vcm administration ( trough ) and 1 hour after vcm administration ( peak ) . the predicted 24-hour auc values for vcm were calculated using the tdm software based on peak and trough concentrations .
continuous variables were compared using the student s t - test for normally distributed variables and the welch test for non - normally distributed variables .
categorical variables were compared with the chi - square ( ) test . in these tests ,
a two - sided p - value of < 0.05 was considered to be significant .
the 94 patients were divided into severe hypoalbuminemia ( n = 23 ) and non - severe hypoalbuminemia ( n = 71 ) groups .
the pk / pd parameters for vcm in the severe hypoalbuminemia and non - severe hypoalbuminemia groups are also summarized in table 2 .
notably , no significant difference was found between the two groups for the mean trough and peak concentrations , clearance of vcm , and whether a single and daily dose of vcm was administered ( table 2 ) .
the percentage of auc values of > 450 g h / ml in the severe hypoalbuminemia group was significantly higher than in the non - severe hypoalbuminemia group ( 9 of 23 patients [ 39% ] vs 6 of 71 patients [ 9% ] ; p < 0.001 ) .
the half - life of vcm in the severe hypoalbuminemia group was significantly longer than in the non - severe hypoalbuminemia group ( 33.2 5.4 hours [ 15135 ] vs 24.9 1.6 hours [ 1068 ] ; p = 0.049 ) .
auc / mic values of 250450 and > 450 g h / ml were significantly associated with 28-day mortality as a primary outcome in the severe hypoalbuminemia patients ( p < 0.001 , figure 1 ) ; statistical power was 0.968 and 0.778 , respectively .
auc / mic values of < 250 g h / ml were not associated ( p = 0.143 , figure 1 ) .
we also examined the adverse effects of vcm treatment among the elderly patients and found that 12 of 94 ( 13% ) developed nephrotoxicity after vcm administration .
we detected a significant difference in increasing the percentage of nephrotoxicity in the severe hypoalbuminemia group ( 6 of 23 patients [ 26% ] ) compared with the non - severe hypoalbuminemia group ( 6 of 71 patients [ 8% ] ; p < 0.001 ) ( table 3 ) . in patients with auc
/ mic values of > 450 g h / ml , the percentage of nephrotoxicity after vcm administration was significantly higher in the severe hypoalbuminemia group than in the non - severe hypoalbuminemia group ( 67% vs 33% ;
p < 0.001 ) ( figure 2 ) , whereas the percentage of nephrotoxicity in the non - severe hypoalbuminemia group was significantly higher than in the severe hypoalbuminemia group for auc / mic values of < 250 g h / ml .
this is the first study to have shown that severe hypoalbuminemia influences the half - life of vcm and treatment outcome .
furthermore , patients with severe hypoalbuminemia with auc / mic values of > 450 g h / ml showed evidence of nephrotoxicity .
the volume of distribution and clearance of other highly protein - bound antibacterials , such as teicoplanin , aztreonam , fusidic acid , or daptomycin , were increased in critically ill patients with hypoalbuminaemia.10 since the protein - binding rate of vcm is lower than that of teicoplanin,11 few studies have investigated whether severe hypoalbuminemia influences the pk / pd parameters of vcm in elderly patients . in the present study ,
the half - life of vcm was longer in elderly patients with severe hypoalbuminemia than in the patients with non - severe hypoalbuminemia .
furthermore , the percentage of auc / mic values of > 450 g h / ml was increased in the patients with severe hypoalbuminemia ( table 2 ) .
although vcm has a relatively low protein - binding rate , these results indicate that severe hypoalbuminemia might affect the concentration of the free form , and prolong the half - life of vcm in elderly patients .
vcm therapeutic guidelines recommend a target auc / mic for vcm of > 400 g h / ml;3 however , our previous report suggested that auc / mic values of 250450 g h / ml were suitable for the treatment of mrsa pneumonia with vcm in elderly patients.4 the reason for this discrepancy is currently unclear . in the present study ,
the half - life of vcm was extended in the patients with severe hypoalbuminemia , and the auc / mic values of vcm were also higher . moreover , with an increase in auc / mic values , the percentage of nephrotoxicity was also increased in patients with severe hypoalbuminemia ( figure 2 ) .
interestingly , auc / mic values of < 250 g h / ml did not influence 28-day mortality in patients with severe hypoalbuminemia , whereas the mortality rate was high in patients with severe hypoalbuminemia with auc / mic values of 250 g h / ml ( figure 1 ) .
these results suggest that elderly patients with severe hypoalbuminemia had a high risk of developing nephrotoxicity and 28-day mortality in the case of target auc / mic values of > 400 g h / ml .
elderly patients with infectious diseases are associated with a high incidence of severe hypoalbuminemia , moreover , hypoalbuminemia can be acute in severely infected patients , and can be entirely independent of baseline nutritional state . since malnutrition leads to poor outcomes for therapy with vcm ,
although we did not recommend a target auc / mic in patients with severe hypoalbuminemia , we consider that elderly patients with low body weight and severe hypoalbuminemia should have their doses individually adjusted by the pharmacist according to those parameters .
first , the targeted vcm trough concentration in this study is about 10 g / ml , which is lower than the guidelines recommended by the infectious diseases society of america / american society of health - system pharmacists : 1520 g / ml.12 moreover , the average weight of patients in the present study was very low .
it was necessary to limit the dosage , because study patients had disuse syndrome or low muscle tone .
we consider that elderly patients with low body weight and severe hypoalbuminemia should have their dose individually adjusted by the pharmacist according to those parameters .
second , a relatively small number of patients with mrsa pneumonia were enrolled in this study .
new , large prospective studies are needed to investigate whether nutritional treatment might be useful for vcm therapy in elderly patients .
this is the first study to indicate that severe hypoalbuminemia influences the half - life of vcm and vcm - related treatment outcomes in patients aged 75 years or older with mrsa pneumonia . to establish more effective and safer treatments , | backgroundvancomycin ( vcm ) treatment outcomes depend on the characteristics of the patient , and it is well known that hypoalbuminemia is a risk factor for poor treatment outcomes , as reported in a previous study .
however , the reason that severe hypoalbuminemia has an influence on the treatment outcome of vcm remains unknown.objectiveto elucidate the association between severe hypoalbuminemia and vcm treatment outcomes , we examined pharmacokinetic / pharmacodynamic ( pk / pd ) parameters in elderly patients with severe hypoalbuminemia.methodswe conducted a retrospective observational study of 94 patients with methicillin - resistant staphylococcus aureus ( mrsa ) hospital - acquired pneumonia who had been treated with vcm between january 2006 and december 2012 .
the 94 patients were divided into severe hypoalbuminemia and non - severe hypoalbuminemia groups .
the pk / pd parameters and treatment outcomes of vcm were compared between the two groups.resultsthe half - life of vcm in the severe hypoalbuminemia group was significantly longer than in the non - severe hypoalbuminemia group ( 33.2 + 5.4 vs 24.9 + 1.6 ; p = 0.049 ) .
area under the concentration curve ( auc)/minimum inhibitory concentration ( mic ) values of 250450 and > 450 g h / ml were significantly associated with 28-day mortality in the severe hypoalbuminemia group ( p < 0.001 ) , whereas auc / mic values of < 250 g h / ml were not associated .
we also detected a significant difference in the increased percentage of nephrotoxicity in the severe hypoalbuminemia group ( 6 of 23 patients [ 26% ] ) compared with the non - severe hypoalbuminemia group ( 6 of 71 patients [ 8% ] ; p < 0.001).conclusionthese findings indicate that severe hypoalbuminemia influences the half - life of vcm and treatment outcomes in elderly patients ( 75 years of age ) . to establish a more effective and safer treatment protocol
, the issue of malnutrition in elderly patients needs to be addressed and improved . |
inverted papilloma is a benign epithelial growth extending into the underlying stroma of the nasal cavity and paranasal sinus .
the tumor is well known for its invasiveness , tendency to recur and association with malignancy . in 1854 , ward first documented the occurrence of inverted papilloma in the sinonasal cavity . however , in 1935 , reingertz histologically described the nature of the tumor and noted its classic inverted nature in underlying connective tissue stroma . in 1971 , hymans reviewed several cases of this tumor and subdivided sinonasal papilloma into inverted , fungiform and cylindrical cell types .
it is a rare benign tumor with incidence rate of 0.6 cases/100,000 people / year .
it usually arises from the lateral nasal wall , in the middle meatus , often extending to the ethmoid and maxillary sinuses . in advance cases ,
extension into all of the ipsilateral peripheral nervous system may occur whereas intracranial growth and dural penetration are rare .
the pathogenesis of this lesion remains unclear although allergy , chronic sinusitis and viral infections have been suggested as possible causes .
a 26-year - old male patient resident of nagpur reported at a private e.n.t clinic with a chief complaint of a mass in the right nasal cavity since 3 years .
the patient also gave history of right nasal blockage and nasal discharge since 3 years .
the patient gave similar history of right sided nasal mass 5 years back , for which he was operated upon in a private clinic and the mass was removed .
after few months , patient started experiencing intermittent nasal obstruction and nasal discharge in the absence of upper respiratory tract infection .
however , as the time passed by these symptoms aggravated and he started experiencing some nasal mass inside right nasal cavity .
on anterior rhinoscopy , a solitary , pinkish , pedunculated , irregular , firm mass was located in right nasal cavity . on probing ,
mass was attached to lateral wall of right nasal cavity and mass did not bleed on touch .
computed tomography ( ct ) scan was advised and it revealed that the tumor was arising from middle meatus of right nasal cavity and there was widening of osteomeatal complex of right side .
based on the clinical features and radiographic findings , a provisional diagnosis of papilloma was given .
the mass was surgically excised by lateral rhinotomy with medial maxillectomy [ figure 1 ] and the excised tissue was sent to the department of oral pathology and microbiology for histopathological examination .
gross examination revealed a single bit of soft tissue specimen of size approximately 3 cm 2 cm , pinkish - white in color , irregular in shape , firm in consistency with rough surface texture . on histological examination , the hematoxylin and eosin stained section showed polypoid tissue covered with pseudostratified columnar ciliated epithelium with admixed mucocytes ( goblet cells ) and intraepithelial mucous cysts at places , which showed inversion into the underlying connective tissue stroma to form large clefts , ribbon and islands .
photograph showing lateral rhinotomy incision scanner view of the section shows an endophytic or inverted growth pattern consisting of markedly thickened squamous epithelial proliferation growing downward into the underlying connective tissue stroma to form large clefts , ribbons and islands ( h&e stain , x40 ) low power view shows the inverted papillomas with an endophytic or inverted growth pattern consisting of markedly thickened pseudostratified ciliated columnar epithelium growing downward into the underlying stroma ( h&e stain , x100 ) high power shows the epithelium to be composed of pseudostratified columnar cells admixed with mucocytes ( goblet cells ) and intraepithelial mucin microcysts ( h&e stain , x400 )
inverted papilloma ( synonym : ringertz tumor , trasitional cell papilloma , fungiform papilloma , cylindrical cell papilloma , schneiderian cell papilloma , epithelial papilloma , papillary sinusitis , soft papilloma and sinonasal - type papillomas ) can be defined as a group of benign neoplasm arising from the sinonasal ( schneiderian ) mucosa and is composed of squamous or columnar epithelial proliferation with associated mucous cells .
the ectodermally derived lining of the sinonasal tract , termed as the schneiderian membrane , may give rise to three morphologically distinct benign papillomas ( schneiderian papillomas ) .
the literature indicates that among sinonasal - type papillomas , the septal papilloma is the most common ; however , practical experience indicates that the inverted type is the most common subtype and the oncocytic type is the least common . in general
, sinonasal - type papillomas occur over a wide age range but are rare in children ; however , septal papillomas tend to occur in a younger age group .
inverted papillomas occur along the lateral nasal wall ( middle turbinate or ethmoid recesses ) , with secondary extension into the paranasal sinuses
typically , the schneiderian papillomas are unilateral ; bilateral papillomas may also occur . in the presence of bilaterality , clinical evaluation to exclude the possibility of extension from unilateral disease should be undertaken .
symptoms vary according to the site of occurrence and include airway obstruction , epistaxis and a symptomatic mass or pain .
the viral etiology has been suggested in few studies and inverted papillomas are reported to be positive for human papillomavirus ( hpv ) on in situ hybridization and/or polymerase chain reaction .
the hpv 6 , hpv 11 , hpv 16 , hpv 18 and epstein-barr virus have been isolated .
whether there is a cause and effect between the presence of hpv and the development of inverted papillomas remains to be determined .
radiological studies are commonly used to evaluate inverted papilloma with ct scan and magnetic resonance imaging ( mri ) scan being the most common .
bony changes including bowing of the bones located near the mass are common ct findings .
tumors involving the maxillary sinus may lead to widening of infundibulum on ct scan , making the uncinate process difficult to discern .
bone - remodeling may be a better term to describe the changes that occur secondary to the constant pressure and mass effect on surrounding bony structures from inverted papilloma , commonly seen at the medial wall of the maxillary sinus and lamina papyracea .
it is postulated that the bony skull base may have limited response to pressure caused by inverted papilloma , leading to more erosive changes rather than remodeling .
mri scan with t1-weighted images with contrast and t2-weighted images can be used to differentiate between tumor mass and postoperative secretions . sometimes , in cases without bony destruction , neither ct nor mri is helpful in the qualitative diagnosis , but they at least provide the suspicion of a neoplasm .
mri is the first imaging modality to perform in the follow - up after removal of inverted papilloma .
however , in this case , mri was not performed since patient was poor . on gross appearance
it is pink to gray in color , with frond - like projections extending from the bulk of the lesion , vary from firm to friable in consistency .
histologically , inverted papillomas have an endophytic or inverted growth pattern consisting of markedly thickened squamous epithelial proliferation growing downward into the underlying connective tissue stroma to form large clefts , ribbons and islands .
the epithelium varies in cellularity and is composed of squamous , transitional and columnar cells ( all 3 may be present in a given lesion ) with admixed mucocytes ( goblet cells ) and intraepithelial mucin microcysts .
a mixed chronic inflammatory cell infiltrate is characteristically seen within all layers of the surface epithelium .
the cells are generally bland in appearance with uniform nuclei and no piling up ; however , pleomorphism and cytoplasmic atypia may be present
. the epithelial component may demonstrate extensive clear cell features indicative of abundant glycogen content .
mitotic figures may be seen in the basal and parabasal layers , but atypical mitotic figures are not seen .
the stromal components vary from myxoid to fibrous , with admixed chronic inflammatory cells and variable vascularity .
intraepithelial mucocytes show intra cytoplasmic mucin positive material , which is mucicarmine positive and diastase resistant , pas positive .
sinonasal inflammatory polyps are clinically similar but histopathologically epithelial alterations are seen in inverted papillomas and not in the inflammatory polyps .
sometimes nonkeratinizing respiratory carcinoma mimics inverted papillomas and then they can be differentiated by the presence of dysplastic features in carcinoma . in verrucous carcinoma , characteristically , cleft - like spaces lined by a thick layer of parakeratin extending from the surface deeply into the lesion which is the hallmark of verrucous carcinoma , is seen and is absent in inverted papilloma .
squamous cell carcinoma may present in the setting of inverted papilloma in three different circumstances .
first , patients may present with small foci of squamous cell carcinoma within inverted papilloma .
the patient may also present with malignancy as a separate synchronous lesion and not within inverted papilloma and finally , the patient may present with metachronous carcinoma in areas of prior resection of benign inverted papilloma .
this association with malignancy , along with a propensity for invasion and recurrence , drives the treatment paradigm for inverted papilloma .
treatment includes complete surgical excision , including the adjacent uninvolved mucosa , as the later is necessary as growth and extension along the mucosa results from the induction of squamous metaplasia in the adjacent sinonasal mucosa .
initially , in 18 century , inverted papillomas were excised via a transnasal closed approach with head light illumination .
in fact , these early procedures mimicked polypectomies . although the intent of these procedures were curative , recurrence rates of 4080% were unacceptably high .
recurrence actually represents residual disease in most cases , so that basic problem facing the clinician is to determine adequate treatment .
open approaches such as the lateral rhinotomy and midfacial degloving procedures allow increased tumor visualization and more complete resection including maxillectomy , which minimizes the recurrence rates .
it is mandatory to resect not only the tumor but also to remove the mucoperiosteum in areas from which the tumor originates using the drill .
intraoperatively histologic control by frozen section is strongly recommended but , unfortunately , it is not performed due to lack of knowledge or negligence on the part of surgeons .
though the inverted papilloma comprises only 0.54% of all primary nasal tumor , one can suspect inverted papilloma if mass in nasal cavity seems to be arising from lateral nasal wall , with involvement of at least 1 paranasal sinus , presenting in male patient of the fifth and sixth decade of life .
common symptoms include unilateral nasal obstruction , epistaxis and sinusitis with nasal discharge . although the intent of surgical procedures were curative , recurrence rates of 4080% were unacceptably high .
recurrence actually represents residual disease in most cases , so that basic problem facing the clinician is to determine adequate treatment .
open approaches such as the lateral rhinotomy and midfacial degloving procedures allowed for increased tumor visualization and more complete resection including maxillectomy , which minimizes the recurrence rates .
| inverted papilloma is a benign epithelial growth in the underlying stroma of the nasal cavity and paranasal sinuses .
the pathogenesis of this lesion remains unclear although allergy , chronic sinusitis and viral infections have been suggested as possible causes .
the tumor is well known for its invasiveness , tendency to recur and association with malignancy .
recurrence rates of inverted papilloma are unacceptably high , which actually represents residual disease in most cases . in this study
, we have presented a case report and reviewed the histological features of sinonasal inverted papilloma . |
ameloblastoma , a benign epithelial odontogenic tumor of the jaws accounting for 1% of all oral tumors and 911% of odontogenic tumors with an incidence of 3/10 million .
the hallmark of this tumor is its locally aggressive behavior owing to its alarming growth rate which may sometimes reach enormous proportions .
in fact , a medline pubmed search for giant ameloblastomas revealed about 31 reported cases , most of which were either of the follicular , plexiform or combined follicular and plexiform variants and only belonged to the granular cell type .
granular cell ameloblastoma ( gca ) is an unusual variant accounting for 3.5% of all ameloblastomas where granular changes generally occur in the central portion of the tumor follicles .
it is a highly aggressive tumor considered to be associated with a high recurrence rate .
however , we report a case of giant gca that showed extensive involvement of mandible , maxilla , temporal bone and dura , but bore a good prognosis .
the demographics of this pathology , raison dtre behind occurrence of granular cells and the differential diagnosis of common oral granular cell lesions is discussed .
a 60-year - old indian male relative of a patient visiting the dental outpatient department was noticed with a massive swelling on the right side of his face .
history revealed a slowly growing painless swelling of the right mandible of 30 years duration that gradually extended to involve the upper jaw , right side of face and temporal region .
the swelling was well - circumscribed , roughly ovoid , lobulated , nontender and firm and measured 18 cm 12 cm 11 cm , extending 3 cm above supraorbital margin superiorly and behind pinna of the ear laterally on the right side .
intra oral examination revealed a huge nontender fleshy mass of the right mandible of differing consistency displacing the tongue , obliterating buccal sulci and extending from lower right canine to retromolar area .
posterior - anterior view of the skull revealed a huge soft tissue mass shadow on the right side of face involving posterior part of the maxilla and with mandibular destruction [ figure 1 ] .
coronal section of computed tomography ( ct ) showed lesion extended from tempero - parietal region till the hyoid bone with displacement of greater wing of sphenoid , temporal bone and maxillary antrum .
axial section of ct scan at a higher level showed erosion of the temporal bone with dural extension .
three - dimensional reconstructed views showed the entire tumor extent that had resorbed the mandible , zygoma and temporal bone on the right side [ figure 2 ] .
baseline laboratory investigations revealed low hematocrit , raised platelet count and erythrocyte sedimentation rate , but normal protein levels .
fine - needle aspiration cytology from the swelling yielded a hemorrhagic , cystic fluid consisting of inflammatory cells in a fibrinous background .
histopathological examination of the tissue specimen obtained by incisional biopsy from the swelling revealed typical ameloblastomatous follicles with varying amounts of granular cell changes , cystic degeneration , and squamous metaplasia in the follicles [ figure 3 ] .
it was thus diagnosed as a gca . after improving patient 's hematocrit , hemimandibulectomy with disarticulation
the tumor mass was resected along with the parotid gland , facial nerve , zygoma and zygomatic arch of the ipsilateral side .
intra operatively , a 2 cm squamous part of the temporal bone was resorbed with a dural tear , but there was no cerebrospinal fluid leakage .
reconstruction was done with pectoralis major myocutaneous flap . the excised specimen measured 17 cm 11 cm 9 cm and weighed 500 g. the interior showed multiple locules , few with whitish nodular projections .
however , our attention was drawn to the unique epithelial changes seen in this case .
there were bland looking epithelial cells with flattened epithelium and whorling resembling squamous odontogenic tumor and conventional ameloblastomatous islands budding from the oral epithelium with focal to extensive proliferations of gca islands form oral epithelium .
granular cell changes were noted extending right across the entire oral epithelium , from the basal and spinous layers up to the superficial layers [ figure 4 ] . to ascertain the nature of granular cells ,
intense positivity for cd68 was shown by granular cells in the follicles , but not by the peripheral ameloblastic cells .
the granular cells showed occasional faint to no reactivity toward ki-67 [ figure 5 ] .
( a and b ) extra- and intra - oral involvement of tumor mass ; ( c ) posterior - anterior view skull showing destruction of posterior part of maxilla and right mandible ( a ) coronal computed tomography ( ct ) scan with contrast showing tumor infiltration into maxilla and temporal region ; ( b ) axial ct scan showing dural extension of tumor ( arrow ) , ( c and d ) three - dimensional reconstructed frontal and occipital views showing entire tumor extent with destruction of right side mandible follicles of granular cell ameloblastoma in the connective tissue stroma ( h and e stain , 10 ) ( a ) postsurgical excised specimen ; ( b ) grossly cut tumor showing whitish nodular projection ( arrow ) within cystic locule , ( c ) granular cells ( arrow ) in overlying oral epithelium ( h and e stain , 10 ) ( a ) intense cytoplasmic positivity to cd68 shown by granular cells ( arrow ) ( 10 ) ; ( b ) moderate reactivity to bcl-2 shown by peripheral ameloblastic cells ( arrow ) in follicles , but absent in central cells ( 4 ) ; ( c ) weak reactivity to ki-67 by ameloblastic cells and occasional granular cells within the follicles ( 4 )
hughes et al . proposed that the term giant ameloblastoma be reserved for lesions that are truly large , cause gross asymmetry , and regional dysfunction . neglected ameloblastomas that become enormous
are rare in developed societies , but can occur in patients who delay treatment due to lack of oral dysfunction or fear of surgery , to which the present case conforms .
ameloblastomas are broadly classified into multicystic , unicystic , desmoplastic and peripheral types and histologically , the multicystic type is further divided into follicular , plexiform , acanthomatous , basal cell and granular cell variants .
gca is a rare subtype of ameloblastoma that shows granular transformation of its cytoplasm and is considered to be a more aggressive variant that occurs at a later age . of the 30 reported cases of giant ameloblastomas , only one gca is recognized , showing its rarity .
present case may be the largest reported gca , to the best of our knowledge . in a series of 20 cases of gca
, the average age was found to be 40.7 years with no gender predilection and they occurred in the posterior mandible .
our case occurred at a much later age , which may have contributed to a particularly large size .
gca is also associated with an increased recurrence rate of 33.3% and higher incidence of malignancy and metastasis .
however , the present case was unique in that it showed neither recurrence nor metastasis .
granular cells are characteristically encountered in other oral tumors also such as granular cell tumor , congenital epulides , granular cell ameloblastic fibroma , central granular cell odontogenic tumor , granular cell variant of odontogenic fibroma , in some mesenchymal tumors , neural tumors , salivary gland tumors , histiocytic tumors , metastatic tumors , and in melanoma .
however , the characteristic ameloblastomatous epithelium in gca helps in ruling out other granular cell lesions . in gca , the granular cells that replace the stellate reticulum like cells in the center of the ameloblastomatous follicles are large oval to polyhedral cells with small pyknotic nuclei , bulky cytoplasm with coarse eosinophillic granules
however , the multifocal continuity of gca islands with oral epithelium and granular changes in oral epithelium observed in the present case was unexpected and may have been as a result of simple fusion between the tumor and epithelium as ameloblastomatous cells have a propensity to fuse with mucosal epithelial cells , mimicking the eruption process .
this nature of occurrence has not been described before , and its impact on the biologic behavior of gca is unknown .
granularity in a normal tissue is an innocuous change and may be encountered even during normal amelogenesis .
ultrastructural , histochemical , and immunohistochemical studies on the nature of granules in gca show that they are lysosomes , but their pathological significance is debatable .
it has also been proposed that granular changes in ameloblastoma represent a dysfunctional status of neoplastic cells , which may be part of an aging process or degenerative process in long - standing lesions .
few authors have stated that the average age of onset of gca is 8 years older than that for conventional ameloblastoma and the tumors have a long duration of symptoms of about 15.3 years compared to 2.35.8 years in the latter .
strong positivity of granular cells to basement membrane proteins like laminin-1 , 5 and fibronectin also suggest an age related transformation .
however , the presence of granular cells in recurrent tumors similar to original tumors and unchanged number of granular cells in follow - up cases negate this hypothesis .
immunohistochemically , granular cells in gca are positive for cd68 , lysozyme and 1 antichymotrypsin , but negative for vimentin , desmin , s-100 , neuron specific enolase and cd15 .
recent immunohistochemical and ultrastructural study by kumamoto and ooya suggested that cytoplasmic granularity in gca is due to apoptotic cell death in neoplastic cells and its following phagocytosis by neighboring neoplastic cells . in our case ,
strong cd68 positivity in granular cells proves their lysosomal nature and absence of anti - apoptotic marker bcl-2 staining in them is indicative of an underlying apoptotic phenomenon .
furthermore , weak to no staining by ki-67 marks a low proliferative index of the granular cells .
the appropriate treatment for gca is marginal resection with 1 cm extension past the radiographic margins or in case of very large tumors , partial jaw resection .
when considering treatment of such extreme lesions , we have to address questions pertaining to its dimensions , weight , involvement of maxilla or mandible , extension to vital structures and the extent of compromise in oral functions . in this case , although tumor size was immense and involved multiple bones including cranium it caused least morbidity and no compromise in oral functions .
it is hypothesized that protein leakage occurs in large cystic ameloblastomas , thus causing secondary hypoproteinemia , but this finding was lacking in the present case .
those lesions showing recurrence and/or metastasis have been associated with long duration of tumor or inadequate treatment at the beginning . | ameloblastomas are rare histologically benign , locally aggressive tumors arising from the oral ectoderm that occasionally reach a gigantic size .
giant ameloblastomas are a rarity these days with the advent of panoramic radiography in routine dental practice .
furthermore , the granular cell variant is an uncommon histological subtype of ameloblastoma where the central stellate reticulum like cells in tumor follicles is replaced by granular cells .
although granular cell ameloblastoma ( gca ) is considered to be a destructive tumor with a high recurrence rate , the significance of granular cells in predicting its biologic behavior is debatable .
however , we present a rare case of giant gca of remarkable histomorphology showing extensive craniofacial involvement and dural extension that rendered a good prognosis following treatment . |
scs as glial cells of the peripheral nervous system are key regulators of the regeneration process of the injured nervous tissue .
they provide structural support and guidance for peripheral nerve regeneration by releasing neurotrophic factors ( 1 ) .
however , there are obstacles on way of clinical application of isolated scs because one or more functional nerves are sacrificed by this aggressive procedure and additional morbidity will also take place .
in addition , cultured scs have also a limited mitotic activity in vitro , so the cell expansion become a time consuming process ( 2 ) .
these cells are able to self - renew with a high growth rate and to differentiate along several mesenchymal cell lineages , including adipocytes , osteoblasts , myocytes , chondrocytes , endothelial cells and cardiomyocytes ( 3 , 4 ) .
ascs have also capability to be induced into neurospheres and neuronal - like cells in vitro ( 5 ) .
previous studies have reported that ascs can be induced into sc - like cells ( 6 , 7 ) .
other studies have also shown that the differentiated ascs can myelinate neurons in vitro and provide functional benefits for peripheral nerve repair ( 2 , 6 ) .
sc - like cells are bi- or tri - polar in shape and immunopositive for nestin and sc markers p75 , gfap and s-100 , like genuine scs ( 8 , 9 ) .
it is noteworthy that many factors have impact on differentiation process of mesenchymal stem cells ( mscs ) .
these include soluble growth factors and cytokines ( 10 , 11 ) , mechanical stimuli ( 12 ) , substrate properties ( 13 ) , and culture conditions ( 14 ) .
initial cell seeding density as one of the culture conditions , has been indicated to have tremendous effect on cell proliferation , differentiation , and extracellular matrix ( ecm ) synthesis ( 1521 ) .
rate of cytokine production is also shown to be dependent on cell density ( 22 ) .
furthermore , it was declared that cell density had impact on biosynthesis of ecm such as collagen ( 23 , 24 ) , yielded higher alkaline phosphatase and produced more mineralization during cell differentiation ( 17 ) . of note ,
the growth patterns of mscs cultures have been reported to be dependent on the initial plating densities .
interestingly , mscs can grow as very dense colonies at low cell density , whereas mscs spread evenly across the culture dish at high cell density ( 17 , 22 ) .
it was reported that human mscs at low density had a high potential for osteogenesis , whereas cells at high density had a propensity to become differentiated into adipocytes ( 25 ) .
some researchers have proved that cell density influences mscs expansion ( 26 , 27 ) .
aforementioned studies imply that cell density as an impressive factor can promote cell proliferation and differentiation .
so far , no literature has revealed the effect of initial cell density on ascs differentiation into sc - like cells .
accordingly , the goal of the current study was to investigate the effect of initial cell seeding density on sc - like cells differentiation of ascs .
ten wistar rats ( male , 8 weeks , weight 150~200 g ) were collectively obtained from laboratory animal research center of ahvaz jundishapur university of medical sciences ( ajums ) for all experimental groups .
they were maintained under standard conditions of controlled temperatures ( 23c ) and a light / dark cycle ( 12/12 hour ) in the animal house of anatomical sciences department , ajums .
it should be mentioned that all experiments were performed in accordance with the protocols approved by the institutional animal care and use committee and with the guidelines for care and use of experimental animals required by ajums .
after rats were sacrificed , their gonadal fat pads were excised , and rat ascs were isolated using a published method ( 12 ) .
briefly , the adipose tissue was carefully dissected and digested using 0.1% collagenase type i ( gibco , usa ) for 50 minutes .
the cell suspension was centrifuged two times to separate the floating adipocytes from the stromal vascular fraction ( svf ) . then
, svf cells were cultivated in dulbecco s modified eagle s medium ( dmem ; gibco , usa ) supplemented with 10% fetal bovine serum ( fbs ; gibco , usa ) 1% penicillin / strepromicine ( sigma , usa ) and 2 mm l - glutamine ( gibco , usa ) .
after 24 hours , the non - adherent cells were discarded by replacing the medium with that of fresh .
of course , the mesenchymal nature of isolated ascs was approved by examining their surface antigens ( cd44 , cd73 , and cd90 ( as positive markers ) and cd45 ( as a negative marker ) ) and testing their differentiation potential into osteogenic and adipogenic cell lineages .
rat ascs at 4 passage were harvested by trypsinization , then the cells were fixed in neutralized 2% paraformaldehyde ( pfa ) ( sigma , usa ) solution for 30 minutes .
the fixed cells were rinsed twice with pbs ( ca / mg - free phosphate - buffered saline , ph 7.2 ) ( gibco , usa ) and incubated with the following antibodies : cd90 , cd73 , cd44 ( positive markers ) and cd45 ( negative marker ) ( all from ebiosciences , usa ) for 30 minutes .
flow cytometry analysis was performed on the fluorescence - activated cell sorting ( facs ) vantage se ( bd biosciences , usa ) and the data were analyzed using flowjo cytometry analysis software ( version 7.6.4 ) .
we used rat ascs at 4 passage to determine the multipotential differentiation capacity of the cells .
cells were grown to at least 80% confluency before being cultured in the induction medium . to commence
the osteogenic differentiation , rat ascs were cultured in dmem supplemented with 10% fbs , 0.1 m dexamethasone , 50 m ascorbate-2-phosphate , 10 mm beta - glycerophosphate ( all purchased from sigma , usa ) for 21 days .
then , cells were fixed with 4% pfa for 30 minutes , rinsed by hank s balanced salt solution ( hbss ) ( gibco , usa ) containing 1% bsa ( gibco , usa ) , and incubated with 0.1% alizarin red s ( sigma , usa ) solution to stain for calcium deposition . for adipogenic differentiation ,
subconfluent passage 4 cultures of ascs were incubated in adipogenic induction medium ( high glucose - dmem containing 10% fbs , 1 m dexamethasone , 0.5 mm methyl - isobutylxanthine , 10 g / ml insulin , and 100 m indomethacin ( all purchased from sigma , usa ) ) for 2 weeks .
cells were fixed with 4% pfa for 60 minutes , rinsed by pbs , and then incubated in 0.3% oil red o ( sigma , usa ) solution in 60% isopropanol for 20 minutes to visualize neutral lipid droplets inside the cells .
after cell counting , cells were cultivated at three different initial cell densities ( 210 , 410 and 810 cells / cm ) on separate poly d - lysine ( pdl ) coated 25 cm flasks overnight and on the next day , were incubated in scs differentiation medium for 14 days as previously described ( 28 ) .
briefly , subconfluent ascs at passage 4 were cultured in a medium supplemented with 1 mm -mercaptoethanol ( sigma , usa ) for 24 hours .
then , the cells were rinsed and fresh medium containing 10% fbs and 35 ng / ml all - trans - retinoic acid ( sigma , usa ) was replaced .
after 72 hours , the cells were rinsed and the medium was replaced with differentiation medium comprising of dmem / ham s f-12 , 10% fbs , 5 ng / ml platelet - derived growth factor ( pdgf ; peprotech , uk ) , 10 ng / ml basic fibroblast growth factor ( b - fgf ; peprotech , uk ) , 14 m forskolin ( fsk ; sigma , usa ) and 200 ng / ml recombinant human heregulin - b1 ( hrg - b1 ; r&d systems , usa ) .
the cells were incubated for 10 days to achieve full differentiation while the medium was changed every 3 days .
finally , immunofluorescence , facs analysis and real time rt - pcr were employed for detection of proteins markers and the number of sc - like cells , respectively .
for immunophenotype characterization of differentiated ascs , the scs markers , including s-100 and gfap , were examined ( 6 , 9 ) .
undifferentiated ( negative control ) and differentiated ascs at three different initial cell densities were fixed by 4% pfa for 20 minute and permeabilized with 0.05% triton x-100 for 10 minute .
nonspecific binding sites were blocked using 3% bovine serum albumin ( bsa ) for 2 hours .
then , anti - gfap ( 1:300 , abcam , uk ) and anti - s-100 protein ( 1:300 , abcam , uk ) were added , and the cells were incubated overnight at 4c .
fitc - conjugated anti - mouse secondary antibody ( sigma , usa ) was incubated with the cells at room temperature for 1 hour .
cell nuclei were labeled with dapi . finally , the cells were observed with a fluorescence microscope ( bx51 , olympus ) and photographed .
glial differentiation was quantitatively analyzed with facs for s100 and gfap . for facs , cells were detached and stained sequentially with primary antibodies ( mouse anti - gfap and anti - s-100 protein ) and secondary antibody ( fitc goat anti - mouse ) .
cells were fixed with 2% formaldehyde until analysis with facs . for detection of intracellular proteins
, cells were permeabilized by ice cold methanol for each sample at 20c for 10 minutes and then centrifuged and rinsed two times in pbs 1% bsa before staining with primary mouse anti - s100 and anti - gfap and fitc - conjugated secondary antibodies .
expression of p75 as a marker of scs was detected by real - time pcr .
p75 primers sequences were designed to span exon / intron junctions using primer express software ( version 2.5 ) as follows : forward 5-cct cat tcc tgt cta ttg ctc cat c-3 and reveres 5-ttc ctc acc tcc tca cgc ttg g-3. total rna was extracted from cells using rneasy mini kit ( qiagen , md , usa ) , subjected with dnase ( qiagen , md , usa ) and quantified .
cdna was synthesized using quantitect reverse transcription kit ( qiagen , md , usa ) according to manufacturer s instruction .
pcr reactions were performed in 96 well plates with an abi prism 7300 sequence detection system ( applied biosystems , ca , usa ) using sybr green to monitor amplification .
real - time pcr data were analyzed with the manufacturer s software ( invitrogen ) .
results were expressed as p75mrna relative expression to -actin as internal reference . to analyze data ,
one way anova was employed to compare the rate of ascs differentiation into sc - like cells at initial cell densities of 2000 , 4000 and 8000 cell / cm .
ten wistar rats ( male , 8 weeks , weight 150~200 g ) were collectively obtained from laboratory animal research center of ahvaz jundishapur university of medical sciences ( ajums ) for all experimental groups .
they were maintained under standard conditions of controlled temperatures ( 23c ) and a light / dark cycle ( 12/12 hour ) in the animal house of anatomical sciences department , ajums .
it should be mentioned that all experiments were performed in accordance with the protocols approved by the institutional animal care and use committee and with the guidelines for care and use of experimental animals required by ajums .
after rats were sacrificed , their gonadal fat pads were excised , and rat ascs were isolated using a published method ( 12 ) .
briefly , the adipose tissue was carefully dissected and digested using 0.1% collagenase type i ( gibco , usa ) for 50 minutes .
the cell suspension was centrifuged two times to separate the floating adipocytes from the stromal vascular fraction ( svf ) . then
, svf cells were cultivated in dulbecco s modified eagle s medium ( dmem ; gibco , usa ) supplemented with 10% fetal bovine serum ( fbs ; gibco , usa ) 1% penicillin / strepromicine ( sigma , usa ) and 2 mm l - glutamine ( gibco , usa ) .
after 24 hours , the non - adherent cells were discarded by replacing the medium with that of fresh .
of course , the mesenchymal nature of isolated ascs was approved by examining their surface antigens ( cd44 , cd73 , and cd90 ( as positive markers ) and cd45 ( as a negative marker ) ) and testing their differentiation potential into osteogenic and adipogenic cell lineages .
rat ascs at 4 passage were harvested by trypsinization , then the cells were fixed in neutralized 2% paraformaldehyde ( pfa ) ( sigma , usa ) solution for 30 minutes .
the fixed cells were rinsed twice with pbs ( ca / mg - free phosphate - buffered saline , ph 7.2 ) ( gibco , usa ) and incubated with the following antibodies : cd90 , cd73 , cd44 ( positive markers ) and cd45 ( negative marker ) ( all from ebiosciences , usa ) for 30 minutes .
flow cytometry analysis was performed on the fluorescence - activated cell sorting ( facs ) vantage se ( bd biosciences , usa ) and the data were analyzed using flowjo cytometry analysis software ( version 7.6.4 ) .
we used rat ascs at 4 passage to determine the multipotential differentiation capacity of the cells .
cells were grown to at least 80% confluency before being cultured in the induction medium . to commence the osteogenic differentiation , rat ascs were cultured in dmem supplemented with 10% fbs , 0.1 m dexamethasone , 50 m ascorbate-2-phosphate , 10 mm beta - glycerophosphate ( all purchased from sigma , usa ) for 21 days .
then , cells were fixed with 4% pfa for 30 minutes , rinsed by hank s balanced salt solution ( hbss ) ( gibco , usa ) containing 1% bsa ( gibco , usa ) , and incubated with 0.1% alizarin red s ( sigma , usa ) solution to stain for calcium deposition . for adipogenic differentiation ,
subconfluent passage 4 cultures of ascs were incubated in adipogenic induction medium ( high glucose - dmem containing 10% fbs , 1 m dexamethasone , 0.5 mm methyl - isobutylxanthine , 10 g / ml insulin , and 100 m indomethacin ( all purchased from sigma , usa ) ) for 2 weeks .
cells were fixed with 4% pfa for 60 minutes , rinsed by pbs , and then incubated in 0.3% oil red o ( sigma , usa ) solution in 60% isopropanol for 20 minutes to visualize neutral lipid droplets inside the cells .
after cell counting , cells were cultivated at three different initial cell densities ( 210 , 410 and 810 cells / cm ) on separate poly d - lysine ( pdl ) coated 25 cm flasks overnight and on the next day , were incubated in scs differentiation medium for 14 days as previously described ( 28 ) .
briefly , subconfluent ascs at passage 4 were cultured in a medium supplemented with 1 mm -mercaptoethanol ( sigma , usa ) for 24 hours .
then , the cells were rinsed and fresh medium containing 10% fbs and 35 ng / ml all - trans - retinoic acid ( sigma , usa ) was replaced .
after 72 hours , the cells were rinsed and the medium was replaced with differentiation medium comprising of dmem / ham s f-12 , 10% fbs , 5 ng / ml platelet - derived growth factor ( pdgf ; peprotech , uk ) , 10 ng / ml basic fibroblast growth factor ( b - fgf ; peprotech , uk ) , 14 m forskolin ( fsk ; sigma , usa ) and 200 ng / ml recombinant human heregulin - b1 ( hrg - b1 ; r&d systems , usa ) .
the cells were incubated for 10 days to achieve full differentiation while the medium was changed every 3 days .
finally , immunofluorescence , facs analysis and real time rt - pcr were employed for detection of proteins markers and the number of sc - like cells , respectively .
for immunophenotype characterization of differentiated ascs , the scs markers , including s-100 and gfap , were examined ( 6 , 9 ) .
undifferentiated ( negative control ) and differentiated ascs at three different initial cell densities were fixed by 4% pfa for 20 minute and permeabilized with 0.05% triton x-100 for 10 minute .
nonspecific binding sites were blocked using 3% bovine serum albumin ( bsa ) for 2 hours .
then , anti - gfap ( 1:300 , abcam , uk ) and anti - s-100 protein ( 1:300 , abcam , uk ) were added , and the cells were incubated overnight at 4c .
fitc - conjugated anti - mouse secondary antibody ( sigma , usa ) was incubated with the cells at room temperature for 1 hour .
cell nuclei were labeled with dapi . finally , the cells were observed with a fluorescence microscope ( bx51 , olympus ) and photographed .
glial differentiation was quantitatively analyzed with facs for s100 and gfap . for facs , cells were detached and stained sequentially with primary antibodies ( mouse anti - gfap and anti - s-100 protein ) and secondary antibody ( fitc goat anti - mouse ) .
cells were fixed with 2% formaldehyde until analysis with facs . for detection of intracellular proteins
, cells were permeabilized by ice cold methanol for each sample at 20c for 10 minutes and then centrifuged and rinsed two times in pbs 1% bsa before staining with primary mouse anti - s100 and anti - gfap and fitc - conjugated secondary antibodies .
expression of p75 as a marker of scs was detected by real - time pcr .
p75 primers sequences were designed to span exon / intron junctions using primer express software ( version 2.5 ) as follows : forward 5-cct cat tcc tgt cta ttg ctc cat c-3 and reveres 5-ttc ctc acc tcc tca cgc ttg g-3. total rna was extracted from cells using rneasy mini kit ( qiagen , md , usa ) , subjected with dnase ( qiagen , md , usa ) and quantified .
cdna was synthesized using quantitect reverse transcription kit ( qiagen , md , usa ) according to manufacturer s instruction .
pcr reactions were performed in 96 well plates with an abi prism 7300 sequence detection system ( applied biosystems , ca , usa ) using sybr green to monitor amplification .
real - time pcr data were analyzed with the manufacturer s software ( invitrogen ) .
one way anova was employed to compare the rate of ascs differentiation into sc - like cells at initial cell densities of 2000 , 4000 and 8000 cell / cm .
rat gonadal adipose tissue was enzymatically digested , centrifuged to isolate the svf from mature adipocytes and plated . after the cells adhered to 25-cm flasks , non - adherent cells , such as red blood cells ,
the initially adherent cells grew into spindle or stellate - shaped cells , which then developed into visible colonies 3 days after the initial plating .
the cells began to proliferate rapidly and formed confluent fibroblast - like monolayers on flasks after approximately 1 week in culture .
cells were passaged by trypsination every 3~4 days whereas they adopted a homogeneous morphology . after 4 passages
, they were plated for differentiation potential assays . after treating ascs by certain induction media
adipogenic differentiation was confirmed by the presence of oil red o - positive vacuoles droplets ( fig .
1a ) and osteogenic differentiation by the production of calcium deposits detected with alizarin red s ( fig .
flow cytometry analysis of rat ascs at 4 passage showed that they were immunologically positive for cd44 ( 17.15% ) , cd73 ( 99.69% ) and cd90 ( 98.11% ) , and negative for cd45 ( 0.47% ) ( fig .
ascs at three initial cell densities were treated with a mixture of glial growth factors for a period of 2 weeks . at the end
, their immunophenotypes were detected for the expression of the specific scs proteins , gfap and s100 .
after commencement of the differentiation , cells morphology started to change so that some cells assumed a bipolar , spindle - shaped cells or tripolar shapes , similar to scs . to investigate whether these morphological changes were resulted from the expression of the scs proteins , we examined the expression of scs markers ( gfap and s100 ) by immunofluorescence ( fig .
immunofluorescence demonstrated that a varying number of cells expressed gfap and s100 in all experimental groups of various initial cell densities .
none of these markers was detected in undifferentiated negative control . to calculate the number of differentiated cells in each cell densities groups accurately ,
3a ) . quantitative analysis indicated that s100-positive cells were significantly higher at cell density of 8000 ( 29.922.3% ) as compared to the density of 4000 ( 17.151.6% ) and 2000 ( 5.881.3% ) ( p<0.001 ) .
similarly , most of the gfap - positive cells existed at cell density of 8000 ( 25.853.5% ) as compared to the density of 4000 ( 16.411.3% ) and 2000 ( 1.440.3% ) ( p<0.001 ) ( fig .
3b ) . p75 mrna expression was upregulated at 2000 , 4000 and 8000 cell density after 14 days ( 7.940.29 , 21.151.12 , and 32.580.75 , respectively ) .
although there was a significant difference among groups at 14 days , p75 mrna expression remained significantly higher at 8000 cell density than those of the other groups ( p<0.001 ) .
the present study demonstrated that a low cell density like 210 cells / cm resulted in a low rate of sc differentiation while the seeding density of 810 cells / cm appeared to be more efficient and appropriate , yielding a highest level of sc differentiation and glial markers expression .
it seemed that the cells probably needed each other and relied on each other to become differentiated . in the recent years
the accessible source , easy harvesting and rapid cell expansion account for the use of ascs over the other sources of stem cells ( 12 , 28 ) .
it seemed that the enhanced cell - cell interaction was necessary for ascs differentiation into sc - like cells .
previously , it was reported that a cocktail of glial growth factors ( containing ggf / hrg , fsk , pdgf and bfgf ) could induce ascs into sc - like cells which had a distinctive morphology and specific markers ( gfap , s100 and p75 ) ( 6 , 8) and promoted nerve regeneration in vivo and in vitro ( 29 ) .
in addition , it was declared that scs expanded when they were cultured with fsk and scs mitogens or serum , whereas cell division was lost when the cells were cultured with fsk alone i.e. without any mitogens and serum ( 30 ) .
camp signal can act as a crucial intracellular signal during several stages of scs development .
in cultured scs , camp elevation can simulate scs performances in the presence of axons during myelination in vivo ( 31 ) .
scs proliferation in response to mitogens was observed only when the cells were also treated with agents elevating camp levels ( 33 , 34 ) .
elevation of camp levels failed to motivate schwann cell expansion in the absence of hrg .
neuregulin-1 is now thought to play important roles in schwann cell survival , differentiation and proliferation during development ( 35 , 36 ) .
it is evident that scs are responsive to hrg , and hrg - dependent growth has been determined to be further stimulated by fsk ( 37 , 38 ) .
in fact , scs treated by hrg / fsk in vitro can grow to very high densities ; however , the rate of growth is markedly decreased at these densities indicating that a cell density - dependent mechanism such as the production of an autocrine inhibitor or contact inhibition alters the sc responsiveness to mitogen .
therefore , it is regarded that hrg / fsk - induced growth of scs in vitro is strongly regulated by cell density .
furthermore , several studies have elucidated that the impact of neural differentiation factor on cells is dose - dependent and that hrg as neural differentiation factor can improve proliferation as long as its concentration remains in a saturated range . when its dose diminished in culture medium after being metabolized by the cells , differentiation commences ( 3539 ) .
it seems that hrg at higher cell density ( 8000 cells / cm ) promotes cell differentiation but its concentration evokes proliferation at lower cell density ( 2000 cells / cm ) .
furthermore , cell density has been shown to influence cell - cell interaction and as a critical factor , controls subsequent cell proliferation and gene expression profiles ( 20 ) .
in addition , cell proliferation is precisely tuned by surface area of cell attachment and contact - inhibition between adjacent cells ( 4042 ) . despite of the fact that high cell density enhances cell - cell contact , contact - inhibition by gap junction alleviates cellular proliferation ( 43 , 44 ) . in addition
, previous studies have shown that low initial cell density of mscs result in higher cell yields and faster expansion ( 27 , 45 ) .
cells seeded at low density yield more doublings in each passage , as growth inhibited by density - dependent mechanism at higher density .
it has also been reported that cell density alters cell shape that changes an adipogenic - osteogenic switch in mscs lineage commitment .
previous studies declared that if the cell density was below the optimal one , increasing the cell density promoted cell function such as bone marker expressions ; when the cell density exceeded the optimal one , a further increase in cell density decreased cell function and tissue regeneration ( 20 , 46 ) .
two competing factors appear to be operative here ; one factor is that the cells communicate with each other for cell - cell interactions , intercellular signal molecules and synthesizing more ecm to maintain their viability and function at a high level .
the other factor was that high cellularity lead to contact - inhibition , lack of oxygen and nutrients and inefficient waste removal .
it seems that high cell density ( 8000 cells / cm ) elicited contact inhibition , attenuated cell proliferation and stimulated ascs differentiation towards sc - like cells .
we concluded that the high cell density like 8000 cells / cm would be optimal for sc differentiation from ascs , yielding a high percentage of sc - like cells with the highest glial markers expression , possibly due to increased cell - cell contact in favor of differentiation induction and cell density - dependent effect of glial growth factors like hrg . | background and objectivesschwann - like ( sc - like ) cells induced from adipose - derived stem cells ( ascs ) may be one of the ideal alternative cell sources for obtaining schwann cells ( scs ) .
they can be used for treating peripheral nerve injuries .
co - culture with scs or exposure to glial growth factors are commonly used for differentiation of ascs to sc - like cells .
however , the effect of initial cell density as an inductive factor on the differentiation potential of ascs into the sc - like cells has not been yet investigated.methods and resultsascs were harvested from rat and characterized .
the cells were seeded into the culture flasks at three different initial cell densities i.e. 2103 , 4103 and 8103 cells / cm2 an overnight and differentiated toward sc - like cells using glial growth factors .
after two weeks , the differentiation rate of ascs to sc - like cells at different densities was assessed by immunofluorescence , fluorescence - activated cell sorting analysis and real time rt - pcr .
expression of the typical scs markers , s-100 proteins and glial fibrillary acidic protein ( gfap ) protein , was observed in all cell densities groups although the number of s100-positive and gfap - positive cells , and the expression of p75ntr mrna , another sc marker , were significantly higher at the density of 8103 cells / cm2 when compared with the other cell densities groups ( p<0.001).conclusionsthe results suggest that the higher differentiation rate of ascs to sc - like cells can be obtained at initial cell density of 8103 cells / cm2 , possibly via increased cell - cell interaction and cell density - dependent influence of glial growth factors . |
singleincision surgery has the main scope to minimize the number of skin incisions , while maintaining similar ergonomics as traditional multiport surgery , and to provide the same or better patient outcomes .
the transition from multiport to single - site laparoscopic surgery had been challenging due to the severe loss of ergonomics , unstable platform , high learning curve , lack of first assistance , and counterintuitive instruments . despite the inferior number of incisions that might translate to better aesthetics than traditional multiport surgery does
, the single - site technique might be associated with increased technical difficulties and possibly adverse outcomes such as trocar - site hernias or visceral injuries .
the robotic single - site platform was introduced in december 2011 as a counterpart for multiport robotic surgery with an intention of overcoming the drawbacks of single incision laparoscopy .
current literature with this platform is limited with initial studies in small series with long operative times in patients with lower body mass index .
because of these limitations , the main aim of this study was to evaluate safety , feasibility , and outcomes for single - site robotic cholecystectomies , in a high - volume robotic center that performs multiport robotic cholecystectomies .
during the period between february 2012 to february 2013 , data on 31 patients who underwent robotic single - site cholecystectomy at a tertiary academic center by a single surgeon were prospectively collected and reviewed retrospectively for mean age , sex , body mass index , previous surgeries , total operative time , port insertion time , docking time , console time , estimated blood loss , closure time , conversion to open or multiport approach , postoperative outcomes for wound infection , bile leak , biliary ductal injury , reoperations , readmission , and mortality .
inclusion criteria included age > 18 years and indications for cholecystectomy were symptomatic gallbladder disease .
all the patients were positioned in reverse trendelenburg , supine position with the da vinci robot ( intuitive surgical , inc . ,
a single - site port was inserted through the umbilicus and positioned with the target anatomy pointing toward the gallbladder .
( the port has 4 lumens that provide access for 2 single - site semirigid robotic instruments , the 8.5-mm 3-dimensional high - definition scope , a 5/10 mm accessory port , and fixed insufflation tubing . )
the robotic arms were attached to the cannulas , with special attention given toward maximizing the range of motion for the surgeon 's arms .
the first assistant then retracts the gallbladder cranially while a grasper and monopolar hook were used to dissect the triangle of calot .
visualization is achieved through an 8.5-mm camera and dissection of the gallbladder is the same as with a multiport cholecystectomy .
hem - o - lok clips ( teleflex incorporated , research triangle park , north carolina ) are placed to transect the cystic duct and artery .
finally , the gallbladder is dissected off the gallbladder fossa and removed through the umbilical port , and the fascial defect was closed with interrupted sutures .
of the 31 subjects , 25 were women ( 80.6% ) and 6 were men ( 19.3% ) with a mean age of 33.613.4 years ( range , 1970 ) , mean body mass index of 32.27 kg / m ( range .
22.3146 ) , and a mean weight of 86.319.2 kg ( range , 52125 ) as shown in table 1 . the most common preoperative and postoperative diagnosis was symptomatic cholelithiasis ( table 2 ) .
pre- and postoperative diagnosis of the patients a 300-mm curved cannula was used in 28 patients ( 90.3% ) and a 250-mm curved cannula in 3 patients ( 9.6% ) .
the mean operative time was 81.421 minutes ( range , 45127 ) and the average time for insertion of the port was 124.8 minutes ( range , 431 ) .
the mean robot docking time was 5.25.9 minutes ( range , 235 ) , and the mean surgeon console time was 3616.7 minutes ( range , 1581 ) .
the average time for closure of fascia and skin was 24.66.7 minutes ( range , 1135 ) .
twenty - four procedures ( 77.4% ) were performed on an outpatient basis . in comparison ,
mean operative time for multiport robotic cholecystectomy for the same surgeon at the same academic setting was 89 minutes .
mean timings of different steps involved during the operation in minutes there were no intraoperative complications .
two patients required placement of an additional 5-mm trocar for a suction catheter in the right midabdomen .
one patient had a superficial wound infection that was treated with a course of oral antibiotics and resolved without any further consequence .
in short - term follow - up of all patients for < 90 days , there were no biliary leaks , no right hepatic artery injury , and no bile duct or other inadvertent injuries to the surrounding structures .
while the cosmetic result of single - site laparoscopic surgery is self - evident , the technique suffers from loss of ergonomics .
this is due to the lesser degree of freedom offered by laparoscopic instruments , compounded by working in a parallel setup with lack of first assistance and an unstable visual field .
together , these factors make single - site laparoscopic surgery an option primarily for expert laparoscopic surgeons .
thus , as a result of this higher learning curve , and subsequent challenge to extend its use to more complex cases , the ability to disseminate this technique remains a concern .
this increased technical challenge for the benefit of fewer trocar - site incisions that might translate into better cosmesis can potentially come with increased perioperative risk of the procedure , such as biliary ductal injury or hepatic arterial injury with long - term increased risk of ventral hernia at the port site .
this platform can help to address the limitations of the laparoscopic technique by providing a stable visual field , with movement of the arms switched by computerized inversion and instruments that offer a higher degree of freedom .
furthermore , unlike the multiport cholecystectomy procedure , docking of the robot for single - site cholecystectomy is recommended from the patient 's right shoulder , with the patient positioned supine in a minimal reverse trendelenburg position ( 15 ) . in our experience , we have found that using more inclination in the reverse trendelenburg ( 3045 ) , with the patient table tilted to minimal left lateral decubitus position , is optimal for unimpeded visualization .
additionally , we first place the patient in the maximum reverse trendelenburg position in order for gravity to allow the transverse colon and bowels to move caudally and then into final operative position .
it has a single - site port with 4 lumens that provide access for 2 single - site semirigid robotic instruments , the 8.5-mm 3-dimensional high - definition endoscope , a 5- to 10-mm accessory port , and an insufflation adaptor .
the single - site port currently comes in only 1 size and fits through a 2.5-cm incision placed through the umbilicus .
the goal of the single - site port is to maximize the range of motion at the end effector of the robot with maximum achievable triangulation of the instruments externally , and with minimal collision .
because the port is of a fixed length , in cases where there is a significant subcutaneous layer , the port may get buried under the skin .
we have not found this to be a hindrance in our experience and , moreover , a u
the curved cannula accommodates instruments used for transection , cutting , ligation , dissection electrocautery , grasping , and suction / irrigation .
it comes in 2 sizes , 5250 mm and 5300 mm , and the choice of which to use depends on the end - organ distance of either 12 to 23 cm or 17 to 28 cm , respectively .
as these are curved cannulas , the size of 300 mm or 250 mm does not equate to 30-cm or 25-cm traditional straight trocars , respectively .
depending on the patient 's body habitus , the estimated distance between the port and the end target ( i.e. , costal margin ) is identified externally , and the appropriate size is selected at the surgeon 's discretion .
mark ; when it is located at the top of the single - site port , it equates to the tip of the cannula coming into laparoscopic view .
the v mark on the 5300 mm cannula also indicates the location of the tip of the 5250 mm .
for the most part , however , the longer cannula tends to work better for retraction of the gallbladder fundus , as the working area is more cranial than the costal margin is .
if the cannula is shorter to the working area , there is internal rotation of the right working hand over the left hand in retracting the infundibulum .
if the cannula is longer , it typically rests over the liver and requires the first assistant to retract the gallbladder cranially .
the da vinci si system software automatically detects and reassociates the surgeon 's hands with the instruments , giving the console surgeon the perception that the left hand is controlling instruments on the left side of the visual field while the right hand is controlling instruments on the right side of the field .
of all the available features , this is an undeniable advantage of the system that makes the approach simple , intuitive , and plausible even for a trainee .
we have confirmed this with fellows who have used the system effortlessly . in our experience , we have found that the procedure can be performed with a 0 camera , without the need to switch to a 30 or 45 camera for optimal visualization of the critical structures , as happens with single port laparoscopic cholecystectomy . additionally , a unique advantage of the robotic platform is the uncompromised 3-dimensional high - definition stable visualization , as opposed to laparoscopic single - site surgery where the visualization is made unstable with every movement of the other instruments .
for this operation , the available instruments include graspers , maryland dissectors , scissors , monopolar cautery , suction irrigator , and hem - o - lok clip appliers . while this limited set of u.s .
food and drug administration approved instruments for cholecystectomy is sufficient to perform the procedure , we have found them to be suboptimal in comparison to regular multiport instruments .
the horizontal segment of the monopolar hook and the hem - o - lok are smaller , particularly for dissection of a dilated cystic duct and require complete skeletonization of the duct for the clip to lock into position . as such , we find the movements and maneuvers allowable with these instruments to be somewhat restrictive when compared with multiport robotic instruments , although still far better when compared with laparoscopic instruments .
the single - site robotic platform was used in all 31 cases of gallbladder disease without exclusion .
all cases were performed to completion without converting to open or to complete multiport setup . in 2 cases ,
an additional 5-mm trocar was placed for a suction / irrigation catheter or for additional retraction and exposure .
in the first case , the patient 's gallbladder was acutely inflamed with empyema along with chronic cholecystitis with challenging dissection and a fellow trainee mostly performed the second case . at short - term follow - up
the single - site robotic cholecystectomy is feasible and safe to perform in patients with acute or chronic cholecystitis , with good short - term perioperative outcomes .
this technique can be used to train residents and surgical fellows , although long - term rates of incisional hernia and cost associated with the procedures still need to be analyzed .
continued use of this platform will also determine if it is possible to perform more complex cases with a single incision . | background : laparoscopic single - incision surgery is fraught with significant technical drawbacks but has witnessed increased growth mainly for its presumed aesthetic advantages . recently , a single - site robotic platform has been introduced to alleviate some of the technical challenges with laparoscopic single - site surgery , although literature on this topic is scant .
the aim of this study is to analyze the experience of a single surgeon with single - site robotic cholecystectomies since the u.s .
food and drug administration gave its approval in december 2011 , and to evaluate the robotic platform 's safety and short - term surgical outcomes.methods:from february 1st 2012 to february 28th 2013 , patients who underwent single - site cholecystectomy at an academic institution in the united states were retrospectively reviewed from a prospectively maintained database .
the following variables were analyzed : age , sex , body mass index , previous surgeries , total operative time , port insertion time , docking time , console time , estimated blood loss , closure time , conversion to open or multiport approach , postoperative outcomes for wound infection , bile leak , biliary ductal injury , right hepatic artery injury , reoperations , readmission , and mortality .
indication for cholecystectomy was symptomatic gallbladder disease .
no exclusion criteria were used and no cost analysis was performed.results:during the study period , 31 patients were enrolled .
the mean patient age , body mass index , weight , and operative time was 33.6 years , 32.2 kg / m2 , 86.3 kg , and 81.4 minutes , respectively .
there were no conversions to the open or traditional multiport approach , and no major complications of biliary ductal or hepatic artery injury , bile leak , reoperations , or mortality occurred .
there was 1 case of superficial wound infection.conclusions:single-site robotic cholecystectomy is feasible and safe and requires a minimal learning curve to transition from traditional multiport to single - port robotic cholecystectomy . |
the blood - brain barrier ( bbb ) is formed by specialized endothelial cells lining capillaries in the central nervous system ( cns ) , and it prevents or slows the passage of some drugs and other chemical compounds , radioactive ions , and disease - causing organisms , such as viruses , from the blood into the cns .
bbb breakdown is associated with many cns - related pathologies , including inflammatory diseases such as multiple sclerosis and chronic and acute cerebrovascular pathology [ 2 , 3 ] .
pathological modifications of the bbb have also been well described in degenerative diseases such as alzheimer disease ; in addition , it has been shown that in brain tumors the bbb is structurally and functionally abnormal
. quantitative investigation of bbb permeability is possible using magnetic resonance imaging ( mri ) , and it has been applied to the study of brain tumors [ 6 , 7 ] . in particular , experimental and clinical studies have demonstrated that dynamic contrast - enhanced ( dce ) mri , with a macromolecular contrast agent ( ca ) , can be used to quantify microvascular permeability in tumors and that permeability increases with increasing histological tumor grade [ 7 , 9 ] .
different theoretical models have been proposed for the analysis of dce - mri data in order to find a more accurate approach for the tumor vascular bed and discriminate blood flow and vascular permeability .
these models include the standard and the extended tofts - kety ( etk ) models [ 11 , 12 ] , the adiabatic tissue homogeneity ( aath ) model [ 13 , 14 ] , the two - compartment exchange model ( 2cxm ) [ 1517 ] , the distributed capillary adiabatic tissue homogeneity ( dcath ) model , and the gamma capillary transit time model ( gctt ) .
the absolute quantification of permeability parameters may substantially differ on the basis of the specific model adopted to fit the dce experimental data .
the purpose of this study was to quantify permeability parameters in patients with different histological types of brain tumors by using two different theoretical algorithms .
to this aim , we compared the volume transfer constant between blood plasma and the extravascular extracellular space ( ees ) ( k ) , the volume of ees per unit volume of tissue ( ve ) ( with 0 ve 1 ) , the vascular volume fraction within the tissue ( vp ) , and the washout rate from the ees back into the blood plasma ( kep ) .
these parameters were calculated using the patlak algorithm , which assumes that kep is small , and therefore negligible , and the etk model .
additionally , the results were evaluated in order to understand if the permeability metrics obtained with the two methods correlated with histological grade .
twenty - five adult patients ( eight females and seventeen males ; mean age standard deviation = 54.6 10.0 years ; age range = 3775 years ) with different brain tumors were enrolled in this study .
the histological grade of each lesion was determined by using the world health organization ( who ) classification of brain tumors .
mri examinations were performed on a 1.5-t clinical system ( siemens magnetom avanto , erlangen , germany ) with an 8-channel head coil , and the following imaging sequences were acquired : t1-weighted ( tr / te = 500/10 ms ; acquisition matrix = 320 384 ; nex = 1 ; field of view = 250 300 mm ; slice thickness = 5 mm , flip angle = 80 ) , t2-weighted ( tr / te = 3800/95 ms ; acquisition matrix = 320 384 ; nex = 2 ; field of view = 250 300 mm ; slice thickness = 5 mm , flip angle = 150 ) , and fluid attenuated inversion recovery ( flair ) ( tr / te = 9000/119 ms ; acquisition matrix = 320 384 ; nex = 1 ; field of view = 250 300 mm ; slice thickness = 5 mm , flip angle = 150 ) . before intravenous injection of the ca , fast low - angle shot ( flash ) axial 3d t1-weighted images ( tr / te = 6.7/1.0 ms ; acquisition matrix = 320 384 ; nex = 1 ; field of view = 250 300 mm ;
slice thickness = 5 mm ) were acquired with multiple flip angles ( 5 , 10 , 15 , 20 , and 30 ) .
acquisition of a dce - mri sequence was started immediately after intravenous administration of a gadolinium - based ca ( gadobutrol 0.1 ml / kg , gadovist ) by a power injector ( spectris solaris ep medrad ) at a rate of 5 ml / s .
dynamic axial 3d t1-weighted turboflash images ( tr / te = 6.7/1.0 ms ; acquisition matrix = 320 384 ; nex = 1 ; field of view = 250 300 mm ; slice thickness = 5 mm ; flip angle = 30 ) were acquired for 30 time points .
permeability maps were created as described in the theory and data analysis section , and the mean of k , ve , and vp was obtained for each patient in a region of interest ( roi ) within the tumor . the patlak algorithm and the etk model were used in order to calculate different permeability parameters . the tracer concentration in tissue , ct , and the tracer concentration in arterial blood plasma , cp ,
are related by the differential equation
( 1)dctdt = ktranscpkepct ,
where kep = k / ve is the rate constant .
the solution of ( 1 ) , using the initial conditions cp = ct = 0 at t = 0 , is
( 2)ct(t)=ktranscp()exp[kep(t)]d ,
where t represents the current time step , is the variable of integration , ct(t ) is the time course of the ca concentration in the tissue compartment , and cp(t ) is the time course of the ca concentration in the plasma ( aif ) [ 22 , 23 ] . equation ( 2 ) is the standard tofts - kety model and is acceptable in tumors with no large increase in blood volume ; however , it is not valid in other contexts , for instance when blood volume can increase markedly in neoplasms .
it is possible to extend ( 2 ) to include the concentration of ca in the blood plasma :
( 3)ct(t)=ktranscp()exp[kep(t)]d+vpcp(t ) .
equation ( 3 ) is the etk model , where vp is the vascular volume fraction within the tissue .
equations ( 2 ) and ( 3 ) are the basis of the most of dce - mri experiments currently being reported in the literature .
the general approach is to measure cp and ct time courses and perform a nonlinear least squares fit of these equations to such data . by varying the parameters in those equations , it is possible to obtain estimates on k , ve and vp . for this study , postprocessing analysis for the etk model was performed using in - house software .
this analysis assumes that the rate constant between ees and blood plasma ( kep ) in ( 3 ) can be ignored due to low permeability and short measuring time . in these cases ,
( 3 ) can be reduced to
( 4)ct(t)=vpcp()+ktranscp()d.
using the patlak analysis , it is possible to linearize ( 4 ) to obtain a graph of the ratio ct(t)/cp(t ) versus cp()d/cp(t ) in order to calculate the values of k and vp .
it is useful to keep in mind that this model does not take into account the backflow and , therefore , the results could have some limitations . for this work ,
postprocessing analysis for the patlak algorithm was performed using toppcat ( t - one weighted perfusion imaging parameter calculation toolkit ) [ https://dblab.duhs.duke.edu/ ] , which is a free permeability analysis software available on internet .
the calculation of the permeability parameters by using ( 2 ) , ( 3 ) , or ( 4 ) requires the values of s0 , the equilibrium longitudinal magnetization , and t1 pregadolinium mapping for each voxel .
precontrast t1 mapping of tissue can be obtained with different approaches ; a common method employs multiple 3d gradient echo acquisitions at multiple variable flip angles . for this purpose , pulse sequence and flip angle
must be chosen to maximize the sampling rate within the constraint of an adequate signal - to - noise ratio ( snr ) .
another frequently used method to calculate the t1 baseline values utilizes 2d t1-weighted inversion recovery scans at various inversion times [ 27 , 28 ] .
this method is considered more accurate than the variable flip angle method , but it is usually more time consuming . in our study , we chose to use acquisitions at multiple variable flip angles to obtain s0 and t1 values . in flash mri with a complete spoiling of the transverse magnetization , the steady - state signal is given by
( 5)s=s0(1exp[tr / t1])sin1cos exp[tr / t1 ] ,
where tr is the repetition time and is the flip angle for tr > > t2 * . to construct the t1 and s0 maps , it is necessary to fit the data from flash sequence by a linearization of ( 5 ) .
the t1 and s0 values are then utilized to estimate the voxel r1 time courses , or relaxation rates , from the acquired signal intensity time courses ,
( 6)r1(t ) = 1tr ln[(1(s(t)s(0)s0sin+1m1(mcos ) ) ) (1cos(s(t)s(0)s0sin+(1m1(mcos))))1 ] ,
where m = exp[tr / t1 ] , is the flip angle of the dce - mr sequence , and s(0 ) and s(t ) are the signal intensities at time t = 0 and time t , respectively .
the relaxation rate r1(t ) is related to the tracer concentration ct(t ) by
( 7)r1(t)=r10+r1c(t ) ,
where r10 is the relaxation rate before tracer injection and r1 is the relaxivity of the ca , which varies with the molecule 's contrast type ( in our case r1 = 4.3 mms ) .
the kolmogorov - smirnov test of normality was used to determine whether the distribution of values was normal , and comparisons between these two theoretical models were performed using the student 's t - test .
a p value lower than 0.05 was considered to indicate statistical significance for all comparisons .
the study group consisted of twenty - five patients , whose histological diagnoses were as follows seventeen glioblastoma multiforme ( who grade iv ) , two oligoastrocytoma ( who grade iii ) , two anaplastic astrocytomas ( who grade iii ) , one pleomorphic xanthoastrocytoma ( who grade iii ) , and three astrocytomas ( who grade ii ) .
differences in permeability measurements were found between different tumor grades , with higher histological grades characterized by higher permeability values . in particular , for grade iv tumors , the mean k value calculated by using the patlak algorithm was 0.039 0.016 min , and the mean k value calculated by using the etk model was 0.051 0.015 min ( figure 1 ) . for grade iii tumors , the mean k value was 0.032 0.011 min for the patlak algorithm and 0.043 0.022 min for the etk model .
in grade ii tumors , we obtained a mean k value of 0.010 0.006 min with the patlak algorithm and a mean k value of 0.011 0.007 min with the etk model .
statistical analysis showed significant differences in the mean k values obtained with the patlak algorithm versus those obtained with the etk model for grade iv tumors ( p = 0.049 , t = 2.04 ) and for high grade tumors ( i.e. , who iv and who iii grouped together ) ( p = 0.031 , t = 2.02 ) .
no significant differences were found between mean k values obtained with the two algorithms for grade ii tumors .
in addition , we did not find significant differences in mean vp values between the two methods in both high - grade tumors and low - grade tumors .
additionally , we calculated ve only for the etk model , finding for high - grade tumors ( who iv and who iii grouped together ) a mean ve of 0.15 0.05 , whilst a mean ve of 0.017 0.006 was found for low - grade tumors . the histological grade of tumors for these patients was found to have a significant correlation with k values .
for the patlak algorithm , we found a pearson correlation coefficient r = 0.54 ( p = 0.004 ) , and for the etk model we found a pearson correlation coefficient = 0.58(p = 0.002 ) .
over the past two decades , important progress has been made in the development of a robust method to noninvasively quantify the microvascular permeability of the bbb for clinical use .
bbb can be altered in brain tumors neoangiogenesis because of new vessels , which are structurally and functionally abnormal .
this abnormality impairs effective delivery of therapeutic agents to all regions of tumors , creates an abnormal microenvironment ( e.g. , hypoxia ) that reduces the effectiveness of radiation and chemotherapy , and selects for more malignant cells .
bbb disruption caused by tumors is heterogeneous , thus , the permeability can vary widely in different areas of the same tumor .
creation of parametric maps with a k value for each voxel can be of practical importance and useful , for instance , to guide the biopsy target .
different theoretical models can be used to fit dce - mri experimental data , and the resulting permeability parameters can be influenced by the model used . in this study , 25 patients with different brain tumors underwent dce mri .
the patlak algorithm , which assumes that the washout rate from the ees back into the blood plasma is small and therefore negligible , and the etk model were applied to estimate kinetic parameters .
our results showed that k mean values in rois within high - grade tumors were significantly different between the two methodologies , with higher k mean values calculated with the etk model . in many studies ,
k is estimated by applying the tk model or the etk model . however , the main disadvantage of the tk model is that it overestimates k in highly vascularized regions , since the contribution of intravascular ca to the signal enhancement is mistaken as tracer that enters the ees and thus appears to reflect permeability .
used the tk and etk models to estimate permeability on 18 high - grade gliomas , and they found that k values calculated by using the tk model were considerably higher than the k values obtained using the etk technique . on the other hand , port et al .
estimated the difference between noncompartmental , tk and etk models on 20 patients with recurrent glioblastoma , finding significant differences in the results obtained with the different models . in our study , we found statistically different k values for high - grade tumors , but no significant difference for low - grade tumors .
this result may be related to the low number of patients with grade ii tumors in this study , but biological differences between high - grade tumors and low - grade tumors can also be associated with these findings .
both techniques reveal a significant correlation between the histological grade of tumors and their k values .
these data are in line with other results that can be found in the literature [ 35 , 36 ] .
in conclusion , we demonstrated that dce acquisition is feasible in patients with brain tumors and that the choice of the postprocessing tool can influence the permeability metrics .
in particular , the use of the patlak algorithm versus the etk model can lead to statistically significant differences in the k values . in our small sample | the purpose of this study was to assess the feasibility of measuring different permeability parameters with t1-weighted dynamic contrast - enhanced ( dce ) magnetic resonance imaging ( mri ) in order to investigate the blood brain - barrier permeability associated with different brain tumors .
the patlak algorithm and the extended tofts - kety model were used to this aim .
twenty - five adult patients with tumors of different histological grades were enrolled in this study .
mri examinations were performed at 1.5 t. multiflip angle , fast low - angle shot , and axial 3d t1-weighted images were acquired to calculate t1 maps , followed by a dce acquisition .
a region of interest was placed within the tumor of each patient to calculate the mean value of different permeability parameters .
differences in permeability measurements were found between different tumor grades , with higher histological grades characterized by higher permeability values . a significant difference in transfer constant ( ktrans ) values
was found between the two methods on high - grade tumors ; however , both techniques revealed a significant correlation between the histological grade of tumors and their ktrans values .
our results suggest that dce acquisition is feasible in patients with brain tumors and that ktrans maps can be easily obtained by these two algorithms , even if the theoretical model adopted could affect the final results . |
skeletal muscle produces efficient contractile force because they contain thin and thick filaments of well - defined lengths that are organized into regular , symmetric arrays that interdigitate .
filament length is an important aspect of muscle function because a muscle generates force in proportion to thin and thick filament overlap .
whereas thick filament length is considered a constant 1.6 m , thin filament lengths are fine - tuned at ~1.01.3 m , depending on species and muscle type ( littlefield and fowler , 2008 ) to overlap with thick filaments and to meet the muscle s physiological demands ( granzier et al . , 1991 ;
thick filament overlap and impact a muscle s force generating capacity at a given sarcomere length : thus , thin filament length is a key aspect of muscle function .
since length is not an intrinsic property of actin filaments ( actin monomers assemble in vitro to highly variable polymer lengths ( pollard and borisy , 2003 ) , thin filament length is likely to be specified in vivo by an actin - binding protein ; for this , nebulin has been considered for a long time a prime candidate ( wang and wright , 1988 ; labeit et al . , 1991 ; labeit and kolmerer , 1995 ) , but critical evidence has been lacking up until recently .
nebulin is a giant protein ( mw 700800 kda ) expressed in skeletal muscle , and makes up 23% of the myofibrillar protein mass .
immuno - electron microscopy revealed that a single nebulin molecule spans the thin filament with its c - terminus anchored at the z - disk and its n - terminal region directed toward the thin filament pointed - end ( for a schematic representation , see figure 1 ; wang and wright , 1988 ) .
the first evidence for nebulin s proposed role in specifying thin filament length came from the analysis of nebulin s cdna sequence .
this revealed that the bulk of the molecule is comprised of modules with the centrally located modules , m9 to m162 , each thought to represent individual actin - binding motifs , and organized into seven - module super - repeats that match the repeat of the actin filament ( figure 1 ) .
this precise arrangement is thought to allow each nebulin module to interact with a single monomer of the actin filament ( labeit et al . , 1991 ; labeit and kolmerer , 1995 ) , and each nebulin super - repeat to associate with a single tropomyosin ( tm)/troponin ( tn ) complex ( jin and wang , 1991 ; mcelhinny et al . , 2003 ;
extreme n - terminal modules m1m3 ( figure 1 ) contain a high - affinity binding site for the thin filament pointed - end capping protein tropomodulin ( mcelhinny et al . , 2001 ) .
tropomodulin , in addition to binding nebulin s n - terminus , binds actin and tropomyosin with high - affinity and prevents actin filaments from elongating or shortening at the pointed - end ( dos remedios et al . , 2003 ) .
furthermore , earlier studies revealed that the electrophoretic mobility of nebulin from different muscle types correlates with thin filament length ( kruger et al .
top : three schematics of the skeletal muscle sarcomere depicting the three proposed models of nebulin in the z - disk : the overlap model in which nebulin penetrates the z - disk creating a ~75 nm wide nebulin overlap zone in the center of the z - disk ; the no - overlap model in which nebulin penetrates the z - disk by only about 25 nm , and nebulin filaments from neighboring sarcomeres do not overlap ; the cross - linking model in which nebulin fully penetrates the z - disk and cross - links thin filaments from adjacent sarcomeres . also note that a single nebulin molecule spans most of the length of the thin filament , but that according to recent studies the thin filament pointed - end is nebulin - free .
the length of the nebulin - free thin filament extension is variable ( as indicated in the drawing by the dotted nebulin filament ) and depends on species and muscle type .
nebulin has a highly modular structure , with in the central region ( m9m162 ) seven modular repeats arranged into 22 super - repeats .
although the findings discussed above were consistent with the hypothesis that nebulin is involved in specifying thin filament length , direct evidence was lacking .
more conclusive evidence for a role for nebulin in specifying thin filament length required studies of muscle that lack nebulin . to test the role of nebulin in skeletal muscle in vivo ,
nebulin ko mouse models were generated ( bang et al . , 2006 ; witt et al .
the first work on these models revealed that in nebulin - deficient skeletal muscle the thin filaments are on average shorter , thus supporting a role for nebulin in the in vivo regulation of thin filament length ( bang et al .
witt et al . ( 2006 ) performed an immuno - electron microscopy study and reported that thin filament lengths in wildtype tibialis cranialis muscle are a constant 1.2 m , but in nebulin - deficient muscle are on average ~0.8 m , and range from ~0.4 to 1.2 m .
that such reduction in thin filament length greatly affects force production was illustrated by ottenheijm et al .
( 2009 ) by plotting force as a function of sarcomere length for both wildtype and nebulin - deficient muscle . in these experiments ,
skinned muscle fibers were activated by exogenous calcium at various sarcomere lengths and the force response was measured ( note that in skinned fiber preparations factors outside of the myofilaments , e.g. , calcium handling by the sarcoplasmic reticulum , do not contribute to force production ) .
the force - sarcomere length relation of wildtype muscle is characterized by a force plateau reflecting optimal thick thin filament overlap , followed by a descending limb at higher sarcomere lengths reflecting the decreased filament overlap . that the descending limb in wildtype muscle starts at a sarcomere length of ~2.6 m and ends at ~4.0 m suggests a thin filament length of ~1.2 m , which is in line with the previously mentioned electron microscopy data . in nebulin - deficient muscle , the shortened thin filaments reduce thin thick filament overlap at a given sarcomere length , impairing force production and resulting in a leftward shift of the force - sarcomere length relation ( see figure 2b ) .
furthermore , when thin filaments are non - uniform in length no optimal thick thin filament overlap exists , and therefore the force - sarcomere length relation of nebulin - deficient muscle lacks the characteristic plateau .
consistent with these findings on demembranated muscle , studies on intact nebulin - deficient muscle from another nebulin knockout model ( gokhin et al .
, 2009 ) , in which muscles were activated at various lengths by electrical field stimulation , also revealed a leftward shift of the force - muscle length relation of nebulin - deficient muscle .
thus , the force - length relation of nebulin - deficient muscle is altered in a manner that is consistent with the presence of shorter thin filament lengths .
( a ) force ca characteristics of murine skinned nebulin - deficient muscle fibers from m. tibialis cranialis ( neb - ko , left panel ) , and of skinned muscle fibers from m. quadriceps of patients with nebulin - based nemaline myopathy ( neb - nm , right panel ) . note that the force generated in response to incubation with incremental increase of [ ca ] is significantly decreased in both neb - ko and neb - nm tissue , resulting in a rightward shift of the force
ca relationship ( figures adapted from chandra et al . , 2009 ; ottenheijm et al . ,
( b ) left : the force - sarcomere length relation of murine wt ( neb - wt ) fibers has a characteristic force plateau followed by a descending limb .
the force - sarcomere length relation of neb - ko fibers is shifted leftward compared to control fibers , and the force plateau is absent .
right : the force - sarcomere length relation of muscle fibers from nm - neb patients is shifted to the left and is very similar to that found in neb - ko fibers ( figures adapted from ottenheijm et al . , 2008 ) .
( 2006 ) on their nebulin ko model , using confocal microscopy on 1-day - old mice , indicated that in the absence of nebulin thin filament lengths are reduced from ~1.15 to 1.3 m ( depending on muscle type ) in wildtype muscle to a consistent ~1.0 m in all muscles types .
these findings led to the proposal ( littlefield and fowler , 2008 ) that a nebulin - independent mechanism specifies uniform thin filament lengths of ~1.0 m in all muscle types , whereas nebulin is responsible for specifying longer thin filament lengths in a muscle - specific manner .
( 2009 ) who used immuno - fluorescence microscopy on rabbit muscle and concluded that nebulin specifies the minimum thin filament length ( ~1.0 m ) with a nebulin - independent mechanism regulating the final length according to the requirements of a particular muscle .
( 2012 ) , showing that human thin filaments have nebulin - free pointed - end extensions that comprise up to 30% of total thin filament length , and by pappas et al .
( 2010 ) who showed that when endogenous nebulin is replaced with a mini - nebulin in skeletal myocytes , thin filaments extend beyond the end of mini - nebulin .
detecting differences in thin filament length , especially length gradients , with high precision is challenging and might be hard to accomplish with confocal microscopy , as was done in the two aforementioned studies .
in contrast , witt et al . ( 2006 ) used electron microscopy and decorated thin filaments with gold beads ( attached to actin monomers with the actin - binding peptide phalloidin ) .
this made it possible to determine thin filament length gradients and showed that thin filaments varied in length and were on average shorter than in wildtype muscle .
( 2006 ) that there is a nebulin - independent mechanism that sets a constant thin filament length of 1.0 m , but is consistent with castillo et al . that there is a nebulin - dependent mechanism that sets a minimum thin filament length . to resolve these discrepancies additional studies on a range of mouse muscle types
are needed that measure by electron microscopy thin filament length and the location of nebulin s n - terminus .
it is clear , however , from the above referenced studies that nebulin does play a critical role in regulating thin filament length : in its absence the average thin filament length is shorter and force is reduced .
to understand the layout of the c - terminal region of nebulin in the z - disk , immunoelectron microscopy ( iem ) has been used on human soleus muscle and nebulin s c - terminus has been labeled with the nebulin - specific anti - sh3 antibody and the more n - terminal m177181 domains with anti - neb177181 ( millevoi et al . , 1998 ) .
results showed that the nebulin sh3 domain is located about 25 nm inside the z - disk , and the repeats neb176 to neb181 near to the edge of the z - disk .
these results are consistent with two distinct models of the layout of nebulin in the z - disk ( for details , see millevoi et al . , 1998 ;
, nebulin penetrates the z - disk by only about 25 nm , and nebulin filaments from neighboring sarcomeres do not overlap ( no - overlap model ) . in the second model , nebulin penetrates the z - disk by about 100 nm and this creates a ~75 nm wide nebulin overlap zone in the center of the z - disk ( overlap model ) .
it has been argued that the no - overlap model is more likely to be correct ( millevoi et al . , 1998 ) , but definite experimental evidence for either model is lacking . in a more recently proposed model ,
nebulin fully penetrates the z - disk and cross - links thin filaments from adjacent sarcomeres ( pappas et al . , 2008 ) .
a drawback of this model is that it predicts that the sh3 epitope is further from the center of the z - disk than the m177181 epitope , which is opposite of what has been measured .
it also remains to be explained why nebulin leaves one actin filament to which it is anchored and moves to a neighboring filament , and how the thin filament spanning region of nebulin accommodates the myofilament lattice spacing changes that occur during muscle contraction .
clearly further work is needed to map the layout of nebulin in the z - disk .
z - disks of different muscles can vary greatly in width , from less than 100 nm in fast skeletal muscle to more than 150 nm in slow skeletal muscle ( tonino et al . , 2010 ) .
the importance of regulation of z - disk width is illustrated by muscle from patients with nemaline myopathy , which displays greatly widened z - disks , including the characteristic nemaline rods ( wallgren - pettersson et al .
previously titin has been suggested to play a role in z - disk assembly ( gautel et al . , 1996 ) .
the z - disk region of titin contains a family of differentially expressed repeats , the titin z - repeats ( gautel et al . , 1996 ) .
these z - repeats are a family of -actinin - binding motifs , which are differentially expressed in a tissue- and developmental - stage - specific fashion ( gautel et al . , 1996 ) .
as previously pointed out , it is unlikely that the differential expression of the titin z - repeats alone can determine the z - disk width , because too few isoforms exist to account for the wide range of different z - disk widths ( millevoi et al . , 1998 ) .
cdna sequencing of rabbit nebulin has demonstrated that the z - disk region of nebulin is differentially expressed ( millevoi et al . , 1998 ) .
several different isoforms have been identified that result from the skipping of various combinations of seven z - disk domains ( millevoi et al . , 1998 ) , leading to the suggestion that nebulin is one of several proteins that are important for z - disk width regulation ( millevoi et al . , 1998 ) .
consistent with this notion , the ultrastructural characterization of the z - disk in neb - ko mice revealed that average z - disk width is increased by 4080 nm depending on muscle type ( bang et al .
in addition , inclusion - like bodies of mis - assembled z - disks are detected that resemble the rod bodies that are a hallmark of nemaline myopathy ( bang et al .
further support was obtained by a recent study on nebulin in a range of muscle types during postnatal development of the mouse in which transcript studies were performed with a mouse nebulin exon microarray ( buck et al . , 2010 ) .
during postnatal development of the soleus muscle major changes in splicing were detected in the z - disk region of nebulin .
three differentially spliced z - disk exons were upregulated during postnatal development of soleus muscle and this correlated with a significant increase in z - disk width .
the increase in z - disk width of the soleus muscle might reflect the increasing stress exerted on this muscle due to the rapid increase in body weight during postnatal development .
together , these findings support a model in which titin and nebulin together specify z - disk width , with titin constructing the central region of the z - disk , including the number and positions of -actinin cross - links and nebulin determining the ending of the z - disk structure and its transition to the i - band , i.e. , nebulin functions as a z - disk terminator .
the mechanism by which nebulin terminates the z - disk might involve interaction between nebulin and z - disk - localized proteins , such as capz .
capz is a barbed - end actin capping protein that binds near the c - terminus of nebulin ( pappas et al . , 2008 ) .
in muscle fibers devoid of nebulin ( witt et al . , 2006 ; pappas et al . , 2008 ) , capz does not localize properly , allowing the barbed ends of thin filaments to continue to grow beyond the z - disk resulting in widened z - disks .
when this feature of nebulin is not present , z - disks widen , ultimately culminating in the formation of nemaline rods .
in addition to regulating z - disk width and structure , nebulin is suggested to play a role in laterally linking myofibrils at the z - diks .
longitudinally , sarcomeres are connected by z - disk lattices that anchor thin filaments and transmit force along the myofibril . in the transverse direction ,
linkage of myofibrils at the z - disks allows for lateral force transmission and limits the degree to which adjacent myofibrils translocate relative to each other during active contraction or passive stretch , thereby preventing damage to intermyofibrillar membrane systems , such as t - tubules and the sarcoplasmic reticulum .
an important protein involved in linking adjacent z - disks is the intermediate filament protein desmin , which forms a network of filaments that surrounds myofibrils at the level of the z - disk ( wang and ramirez - mitchell , 1983 ; capetanaki et al . , 2007 ) .
the subunit proteins of desmin filaments are elongated coiled - coils with extensive intermolecular ionic and hydrophobic interactions between individual subunits , giving rise to filaments with high tensile strength as well as plasticity ( costa et al . , 2004 ) . that desmin tethers adjacent z - disks is supported by work on a desmin ko mouse in which z - disk misalignment was shown to occur in stretched muscle ( shah et al . , 2002 ) .
in vitro work , using a yeast two - hybrid approach , suggested that desmin binds to the c - terminal region of nebulin ( bang et al . , 2002 ) , which is anchored in the z - disk , and recently it was shown that nebulin modules m160170 are involved in this interaction ( conover et al . , 2009 ; conover and gregorio , 2011 ) .
these findings lead bang et al . to speculate that this desmin - nebulin interaction links myofibrillar z - disks to the intermediate filament system , thereby forming a lateral linkage system which maintains adjacent z - disks in register .
this role for nebulin in intermyofibrillar connectivity was tested recently by studies using a nebulin ko mouse model ( tonino et al . , 2010 ) .
in these studies it was found that upon stretch , myofibrils devoid of nebulin translocate to a much higher degree than wt muscle , resulting in much larger z - disk displacement .
although desmin is present in muscle devoid of nebulin , it is reduced in the intermyofibrillar spaces that surround the z - disks , suggesting that nebulin is required for proper localization of desmin at the z - disk .
consistent with this , both knockdown of nebulin with sirna and overexpression of m160m170 did not interfere with the formation of normal striation patterns but did prevent desmin localization at the mature z - disk .
thus , nebulin is required to laterally link myofibrils at the z - disk by desmin filaments ; in the absence of nebulin myofibrillar connectivity is significantly reduced leading to z - disk displacement .
exciting recent work revealed that nebulin s c - terminal sh3 domain is involved in the induction of muscle hypertrophy .
( 2010 ) found that insulin - like growth factor 1 induces skeletal muscle maturation and hypertrophy by forming a complex of nebulin and n - wasp at the z - disks of myofibrils by interfering with glycogen synthase kinase-3. their results convincingly show that n - wasp interacts with nebulin s sh3 domain , and that n - wasp is rapidly recruited to the z - disk after treatment with insulin - like growth factor 1 , which stimulates myofibrillogenesis and suppresses sarcomere breakdown via the pi3k akt pathway .
the notion that nebulin is important in maintaining muscle mass is consistent with the previous observation that nebulin - deficient mice display severe muscle fiber atrophy ( bang et al .
, 2006 ; witt et al . , 2006 ) although thin filament instability caused by nebulin - deficiency is likely to be involved as well in the mechanisms underlying muscle atrophy .
recent work shows that nebulin s role is not merely structural , but that nebulin also regulates contraction .
muscle contraction is driven by the cyclic interaction between the myosin - based crossbridges and actin and the level of force a muscle generates is proportional to the force generated per crossbridge and the number of crossbridges in the force generating state .
it is generally accepted that this interaction between actin and myosin is regulated through a steric hindrance mechanism in which tropomyosin and troponin control the conversion between interaction permissive and non - permissive states ( gordon et al . , 2000 ) .
, 2009 ; chandra et al . , 2009 ) recently identified that nebulin contributes to the regulation of crossbridge cycling kinetics , with one of the two ( chandra et al . , 2009 ) also identifying a role for nebulin in the calcium sensitivity of force generation . during the crossbridge cycle , unbound non - force generating crossbridges move to an actin - bound force generating state followed by atp - driven crossbridge release back to the non - force generating state ( huxley and simmons , 1971 ; lymn and taylor , 1971 ) .
( brenner , 1988 ) proposed an analytical framework in which this transition between force and non - force generating crossbridge states can be described by two apparent rate constants ; one for crossbridge attachment ( fapp ) and one for crossbridge detachment ( gapp ) .
these two rate constants determine the fraction of force generating crossbridges during activation , and a change in one or both will affect this fraction and thus force production .
gapp is directly proportional to the atp consumption rate normalized to tension generation ( i.e. , tension cost ) , and can therefore be estimated from the simultaneous determination of atp consumption rate and tension in activated muscle fibers .
such studies on nebulin - deficient muscle ( chandra et al . , 2009 ) revealed significantly higher tension cost in nebulin - deficient muscle , thus indicating a faster gapp and crossbridge detachment rate when nebulin is absent .
likewise , studies on another nebulin ko mouse model ( bang et al . , 2009 ) reported higher velocity of unloaded shortening in nebulin - deficient muscle , also suggesting that gapp is higher when nebulin is absent .
it is also important to highlight that these findings are consistent with results of in vitro motility assays in which nebulin fragments were found to reduce the sliding velocity of f - actin over myosin ( root and wang , 1994 ) . in brenner s framework ( brenner , 1988 )
, the rate constant of force redevelopment ( ktr ) is proportional to fapp + gapp , and the fraction of force generating crossbridges to fapp/(fapp + gapp ) .
the rate constant of force redevelopment can be estimated by imposing a rapid release - restretch protocol on an activated fiber , mechanically disengaging all bound crossbridges so that force drops to zero and then measuring force redevelopment .
such experiments revealed that force redevelopment is slower in nebulin - deficient muscle ( bang et al .
thus , the decrease in ktr of nebulin - deficient muscle , together with the notion that gapp is increased , indicates that fapp must be reduced and that the reduction must be larger than the increase in gapp .
combined , this leads to the conclusion that the fraction of force generating crossbridges [ fapp/(fapp + gapp ) ] is reduced in nebulin - deficient muscle .
furthermore , stiffness measurements indicated that the force per crossbridge was not affected by the absence of nebulin ( bang et al .
, recent studies suggest that nebulin increases the rate of crossbridge attachment and reduces the rate of crossbridge detachment , and that as a result the number of force generating crossbridges is increased .
although the mechanism by which nebulin affects crossbridge cycling needs further investigation , previous work ( root and wang , 1994 ) has shown that nebulin associates with the actin n - terminus in subdomain 1 , where also the myosin crossbridge binds .
thus , the presence of nebulin at or near the s1 binding site might enhance the binding of crossbridges and slow their detachment .
chandra et al . ( 2009 ) estimated that the effect of nebulin on crossbridge kinetics enhances a muscle s force generating capacity by ~50% , and increases the economy of contraction by ~35% .
( 2009 ) , and they can largely account for the more pronounced leftward shift of the measured force - sarcomere length relation of nebulin - deficient muscle when compared to the predicted relation based on only thin filament length measurements . clearly , nebulin is a major factor in determining the level of force and the energetic cost of force production in skeletal muscle . in line with this role of nebulin in the regulation of crossbridge cycling kinetics , recent studies on muscle fibers from patients with nemaline myopathy with
severely reduced nebulin protein levels revealed that in addition to altered thin filament length , changes in crossbridge cycling kinetics contribute to the muscle weakness observed in these patients ( ottenheijm et al . , 2010 ) .
chandra et al . ( 2009 ) also measured active force at a range of calcium levels and the obtained force - pca relations were markedly shifted to the right in nebulin - deficient muscle fibers ( see figure 2a ) , with a 0.16 unit reduction in pca50 ( pca that gives the half - maximal force level ) . to rule out a possible difference in the troponin complex ( isoform composition and posttranslational modification ) between wt and ko fibers ,
these studies were carried out on wildtype and ko fibers that had been reconstituted by the same recombinant troponin complex .
an analysis of expression levels of tropomyosin , myosin heavy chain , myosin light chain did not reveal a significant difference in these proteins between the fibers , suggesting that the absence of nebulin is the most likely explanation for the lower calcium sensitivity of the ko fibers .
it is possible that the discrepancy is due to the fact that those studies did not carry out a troponin exchange and that differences in the troponin complex between wt and ko fibers could have negated nebulin s effect on calcium sensitivity .
the two studies that did not detect a difference in calcium sensitivity were carried out at long sarcomere lengths [ ~2.5 m ( witt et al . , 2006 ) and ~2.6 m ( bang et al . , 2009 ) ] whereas the study that did show a difference ( chandra et al . , 2009 )
was performed at ~2.0 m . the implication is that nebulin plays a role in the length dependence of activation with a much larger pca50 in the nebulin ko fibers than in wt fibers .
it is a well known phenomenon that as sarcomere length increases muscle becomes more calcium sensitive .
this length dependence of activation is most prominent in cardiac muscle ( and is thought to underlie the frank starling law of the heart ) but is much less pronounced in skeletal muscle ( konhilas et al . ,
the presence of nebulin provides an explanation for why skeletal muscle has less length dependence : the presence of nebulin increases calcium sensitivity at short length .
the structure and protein binding properties of nebulin support a role in thin filament activation .
nebulin contains ~200 domains of ~35 amino acids that are characterized by the actin - binding sequence sdxxyk ; these domains make up seven domain super - repeats characterized by the tm / tn binding motif , wlkgigw ( mcelhinny et al . , 2003 ) .
biochemical studies have shown that a single nebulin module interacts with a single actin monomer and that each nebulin super - repeat interacts with a tm / tn complex of the thin filament ( ogut et al . ,
2003 ) , binding characteristics that support that nebulin follows the helical path of f - actin .
it is interesting that similar to tm , nebulin appears to have different binding sites on f - actin with one site in close proximity to both the strong binding site for myosin and the blocked state of tropomyosin ( lukoyanova et al . , 2002 ) .
this leads to the intriguing possibility that nebulin acts in concert with tm and that it promotes the transition of contractile regulatory units ( tm / tn ) from the non - permissive to the permissive states , thereby increasing myofilament calcium sensitivity .
it is striking that nebulin - deficient skeletal muscle shares the low calcium sensitivity and low maximal active force with cardiac muscle , where stoichiometric levels of nebulin are absent ( bang et al .
cardiac muscle contains the nebulin - homolog nebulette ( moncman and wang , 1995 ) .
however , nebulette is much smaller than nebulin ( ~100 vs ~800 kda for nebulin ) and its location is restricted to the z - disk and the near z - disk i - band region ( millevoi et al . ,
although it can not be ruled out that nebulette induces long - range conformational effects that propagate along the thin filament to affect its function , such effects would have to be first demonstrated in a direct manner , and in absence of such findings we consider it not likely that nebulette is intimately involved in thin filament activation .
cardiac muscle has multiple mechanisms for enhancing thin filament activation , such as , enhanced length - dependent activation , and the presence of multiple cardiac - specific phosphorylation sites in various thin and thick filament based protein sites ( solaro and de tombe , 2008 ) , which allow cardiac muscle to grade its force response to different loading conditions .
since skeletal muscle lacks the aforementioned features unique to cardiac muscle , nebulin might be essential for tuning thin filament activation for optimal skeletal muscle function .
a striking upregulation of sarcolipin ( sln ) , an inhibitor of serca , occurs in neb - ko mice ( bang et al . , 2006 ; gokhin et al . , 2009 ;
this upregulation might be viewed as an adaptation in neb - ko mice as an attempt to increase cytosolic calcium levels and counteract reduced myofilament calcium sensitivity .
the mechanism by which nebulin - deficiency upregulates sln requires further future studies , as does the functional role of sln upregulation , for example by crossing sln - ko mice and nebulin ko mice .
the above discussed studies clearly indicate that nebulin is not merely involved in regulation sarcomere structure , but also acts as a regulator of muscle contraction .
dysfunctions of the skeletal muscle thin filament have emerged as important causes of skeletal myopathies of which nemaline myopathy is the most common disease with a previously estimated prevalence of 0.002% ( sanoudou and beggs , 2001 ) . however , because of its heterogeneous nature and often milder phenotypes , the actual frequency of nemaline myopathy in older patients may be considerably higher .
genetically , nm is heterogeneous and so far seven genes have been identified as nm - causing , namely alpha - tropomyosin-3 and beta - tropomyosin ( tpm3 and tpm2 ) , neb , actin alpha 1 ( acta1 ) , troponin t type 1 ( tnnt1 ) , cofilin-2 , and kbtbd13 ( laing et al . , 1992 , 1995 ; nowak et al . , 1999 ;
, 2007 ; sambuughin et al . , 2010 ) . an unbiased linkage analysis of 45 nm families derived from 10 different countries implicated nebulin mutations as disease causing in 41 of these families . the typical form of nm presents with early onset in infancy , has a non - progressive or slowly progressive course and is caused by mutations in the nebulin gene .
these mutations are often missense mutations , but in addition a 2502-bp deletion causing a 33-residue in frame deletion of exon 55 was identified as a prominent recessive disease causing allele initially in the ashkenazi jewish population , and more recently also in non - jewish patients ( anderson et al . , 2004 ; lehtokari et al . , 2006 , 2009 ) .
recent studies on muscle fibers from patients with nemaline myopathy due to the deletion of nebulin exon 55 showed that these patients have severely reduced nebulin protein levels and that they show remarkable phenotypic similarities to fibers from nebulin ko mice , i.e. , shorter and non - uniform thin filament lengths ( figure 2b ) and significantly impaired force generating capacity ( ottenheijm et al . , 2009 ) .
thus , loss of thin filament length regulation appears to be an important contributor to muscle weakness in patients with nemaline myopathy .
in addition to altered thin filament length , changes in crossbridge cycling kinetics and reduced calcium sensitivity of force production contribute to the muscle weakness observed in these patients ( see figure 2a ; ottenheijm et al . ,
2010 ) , in line with the role of nebulin in these processes ( chandra et al . , 2009 ; ottenheijm and granzier , 2010 ) .
thus , nebulin s role in thin filament length regulation and contraction deduced from work on neb - ko mice provides a mechanism for the first time to explain severe muscle weakness in patients with nemaline myopathy .
interestingly , recent work form our lab demonstrates that patients with nemaline myopathy due to mutations in the tropomyosin gene ( tpm3 ) display a contractile phenotype that is distinct from that of patients with neb - based myopathy ( ottenheijm et al . , 2011 ) . whereas both show severe myofilament - based muscle weakness , the contractile dysfunction in tpm3-based myopathy
can be largely explained by changes in crossbridge cycling kinetics , whereas dysregulation of thin filament length and altered crossbridge cycling kinetics play a prominent role in neb - based myopathy ( ottenheijm et al . , 2009 ) .
furthermore , the loss of force generating capacity in tpm3-based myopathy appears to be partly compensated by enhanced calcium sensitivity of force generation , whereas decreased calcium sensitivity of force generation further depresses the capacity for force production in neb - based myopathy ( ottenheijm et al . , 2010 ) .
whereas these findings support the existence of distinct genotype - phenotype correlations in nm , it is yet unclear whether they extrapolate to other patients with mutations in neb or tpm3 , and how they compare to the phenotype of nm patients with mutations in acta1 , tpm2 , tnnt1 , cfl2 , and kbtbd13 .
understanding the genotype - phenotype correlations is important , as it allows the development of genotype - targeted treatment strategies .
for example , analogous to current clinical studies that address whether contractility during heart failure can be augmented by actomyosin activating small molecules ( malik et al . , 2011 )
the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest . | the sliding filament model of the sarcomere was developed more than half a century ago .
this model , consisting only of thin and thick filaments , has been successful in explaining many , but not all , features of skeletal muscle .
work during the 1980s revealed the existence of two additional filaments : the giant filamentous proteins titin and nebulin .
whereas the role of titin rapidly progressed , nebulin s role in muscle structure and function remained long nebulous .
an important feature of muscle structure and function that has remained relatively obscure concerns the mechanisms that are involved in regulating thin filament length .
filament length is an important aspect of muscle function as force production is proportional to the amount of overlap between thick and thin filaments .
recent advances , due in part to the generation of nebulin ko models , reveal that nebulin plays an important role in the regulation of thin filament length , most likely by stabilizing f - actin assemblies . another structural feature of skeletal muscle that has been incompletely understood concerns the mechanisms involved in maintaining z - disk structure and the regular lateral alignment of adjacent sarcomeres during contraction .
recent studies indicate that nebulin is part of a protein complex that mechanically links adjacent myofibrils .
in addition to these structural roles in support of myofibrillar force generation , nebulin has been also shown to regulate directly muscle contraction at the level of individual crossbridges : cycling kinetics and the calcium sensitivity of force producing crossbridges is enhanced in the presence of nebulin .
thus , these recent data all point to nebulin being important for muscle force optimization .
consequently , muscle weakness as the lead symptom develops in the case of patients with nemaline myopathy that have mutations in the nebulin gene . here ,
we discuss these important novel insights into the role of nebulin in skeletal muscle function . |
we studied patients admitted to the prince of wales hospital , hong kong special administrative region , who had laboratory - confirmed acute hepatitis e during 2002 2007 .
acute hev infection was diagnosed on the basis of clinical manifestations of acute hepatitis , elevation of hepatic parenchymal enzyme levels , and presence of anti - hev immunoglobulin ( ig ) m as determined by an hev igm kit ( biotec laboratories ltd . , suffolk , uk ) .
clinical data were retrieved from patients records in the computerized clinical management system of the hospital .
hev rna was then amplified from patients stored serum by using a nested reverse transcription
pcr ( rt - pcr ) specific for open reading frame ( orf ) 2 with external primers consorf2-s1 ( 5-gacagaattratttcgtcggctgg-3 ) and consorf2-a1 ( 5-cttgttc rtgytggttrtcataatc-3 ) and internal primers consorf2-s2 ( 5-gtygtctcrgccaatggcgagc-3 ) and consorf2-a2 ( 5-gttcrtgytggttrtcataatcctg-3 ) ( 5 ) .
positive samples were subjected to another nested rt - pcr targeting orf1 with external primers he5 - 1 ( 5-tcgatgccatggaggccc-3 ) and hevorf1 - 1as ( 5-ggccattgcctccgcaacatc-3 ) and internal primers he5 - 2 ( 5- gccytkgcgaatgctgtgg-3 ) and hevorf1 - 2as ( 5- accatcaaagcagtaagtccg-3 ) ( 6 ) .
the orf2 ( 145-bp ) and orf1 ( 364-bp ) pcr products were sequenced , and sequence fragments were aligned by clustalx 2.0 ( 7 ) .
phylogenetic trees were constructed by using the neighbor - joining method ( 8) , and rooted phylogenetic trees were generated by using paup * version 4.0b ( 9 ) .
bootstrapping values obtained from 1,000 resamplings of the data were performed to assess the robustness of trees ( 10 ) .
nucleotide sequence similarity among isolates was analyzed by using the bioedit software ( 12 ) .
the sequence data were deposited in genbank under accession nos . fj438395fj438427 and fj438428fj438460 , respectively .
nineteen were females ( none pregnant ) 1482 years of age ( median age 57 years , interquartile range [ iqr ] 4374 years ) ; 38 were men 2676 years of age ( median age 52 years , iqr 3867 years ) .
no significant difference in age was observed ( p = 0.323 by mann - whitney u test ) .
eleven patients were hepatitis b carriers , 1 had cirrhosis , and 1 was a hepatitis c carrier .
forty - eight ( 84% ) patients had no history of travel during the prior 6 months .
nine ( 16% ) had traveled outside hong kong ( 6 to people s republic of china , 1 to macau , 1 to south korea , 1 to the united states ) 14 weeks before illness onset .
the highest liver function levels recorded for the women were serum alanine aminotransferase ( alt ) 2616,500
iu / l ( median 1,280 iu / l , iqr 4348,322 iu / l , reference < 58
serum alkaline phosphatase ( alp ) levels ranged from 111 to 469 iu / l ( median 214 iu / l , iqr 178268
iu / l ) , and total serum bilirubin levels ranged from 10 to 565 mol / l ( median 85 mol / l , iqr 30146 mol / l , reference , < 15 mol / l ) . the highest liver function levels recorded for the men were serum alt 2534,525
iu / l ( median 1,714 iu / l , iqr 1,0432,608 iu / l , reference < 58
iu / l ( median 188 iu / l , iqr 132261 iu / l , reference 35100 iu / l ) , and total serum bilirubin 10544 mol / l ( median 112 mol / l , iqr 72227 mol / l , reference < 15 mol / l ) .
thirteen patients had other medical conditions including end - stage renal failure , diabetes , ischemic heart disease , colon cancer , system lupus erythematous , thyroitoxicosis , hepatitis b liver cirrhosis , and previous liver transplantation .
patients with a medical condition had significantly higher levels of serum alp ( median 267 iu / l , iqr 861,703 iu / l ) than did those without medical conditions ( median 192 iu / l , iqr 131164 iu / l ; p = 0.03 by mann - whitney u test ) .
phylogenetic analyses of the orf2 fragments from 46 patients and orf1 fragments from 33 patients showed complete agreement ( figure ) , with most ( 45 [ 98% ] ) belonging to genotype 4 .
the remaining isolate was genotype 3 ( hk14 ) obtained from a woman who had no history of travel .
most of the hong kong isolates clustered closely with a swine isolate reported from guangxi province , china ( accession no .
furthermore , the orf2 phylogenetic tree showed our isolates were closely related to those reported recently from beijing , china ( accession nos .
phylogenetic tree
showing the relationship of hepatitis e virus ( hev ) isolates from hong kong .
trees were constructed by the neighbor - joining method based on the partial nucleotide sequence of the open reading frame ( orf ) 2 ( a ) and orf1 ( b ) regions of hev samples .
bootstrap values for the various branches are shown as percentages of trees obtained from 1,000 resamplings of the data .
sequences used for phylogenetic analysis were isolates from genotype 1 : burma ( genbank accession nos .
m73218 ; d10330 ) , pakistan ( m80581 ) , india ( x98292 ; x99441 ) , and china ( d11092 ; d11093 ) ; genotype 2 : mexico ( m74506 ) ; genotype 3 : usa ( af060668 ; af060669 ) , japan ( ap003430 ) ; and genotype 4 : china ( ab108537 ; ab197674 ; af082083 ; af082084 ; af082087 ; af082095 ; af082096 ; aj272108 ; eu107466 - 74 ) , japan ( ab074915 ; ab074917 ; ab080547 ; ab091395 ; ab097812 ; ab099347 ; ab161717 - 19 ; ab220971 - 79 ; ab253420 ; ab369688 ; ab369690 ) .
branches of swine hev genotype 3 sequences ( ab238522 ; af455784 [ experimentally infected swine ] ) and swine hev genotype 4 sequences ( ay723745 ; dq450072 ; ef570133 ; eu366959 ; eu676172 ) are included in the analysis . accession numbers in boldface are swine isolates .
all isolates from the current study have a prefix hk followed by a number in italics .
we further analyzed the sequence variation of 32 hev genotype 4 isolates for which orf1 and orf2 sequences were generated in the current study .
the nucleotide sequence similarity was 79.9% for the orf1 region ( nt 170448 ) , and 86.4% for orf2 ( nt 64096504 ) . for comparison ,
the sequence similarity for hev isolates collected elsewhere that had been deposited in genbank was 73.8% for orf1 ( nt 170448 ) and 74.0% for orf2 ( nt 64096504 ) .
regions within nt 171221 , 280310 , and 6,4616,495 were most conserved and represented the best targets for primer or probe design .
our study showed that most hev cases in hong kong were sporadic and autochthonous . although a substantial proportion ( 21% ) of patients were hepatitis b or c carriers , all diseases had a self - limiting course .
this finding could be related to the circulation of relatively mild genotypes 3 and 4 in this locality .
the epidemiology of hev in hong kong resembled industrialized countries with a predilection for older adults , rather than older children and young adults as occurs in developing countries .
patients with hev infections were older than those with hepatitis a , which peaked in persons 2029 years of age , as reported by chau et al .
the distribution of hev genotypes is related to geographic location and the mode of spread ( 2 ) .
genotype 3 is widely distributed and has been isolated from humans in north and south america , europe , japan , and the pacific region and in domestic pigs in many countries except in africa .
genotype 4 has been isolated from humans in china , japan , taiwan , and vietnam and from domestic pigs , boar , and deer in many countries ( 2,5,6 ) .
genotype 4 , the predominant genotype in hong kong , is less virulent ; it is responsible for occasional cases of clinical hepatitis in industrialized countries .
a recent study from guangzhou in southern china showed that most ( 39/41 ) hev isolates found there were similar to burmese - like isolates ( genotype 1 ) ( 15 ) .
this finding could imply that the poor hygienic conditions required for sustaining the circulation of genotypes 1 and 2 do not exist in hong kong .
the sporadic nature , older age of affected patients , and predominance of genotype 4 correspond with the epidemiology in industrialized countries where zoonosis is the major source of infection .
public health control should focus on zoonotic , especially swine , foodborne transmission as a source of human hev infection in hong kong . | most acute cases of infection with hepatitis e virus ( hev ) in hong kong were autochthonous , sporadic , and occurred in older adults .
all except 1 isolate belonged to genotype 4 ; most were phylogenetically related to swine isolates .
the epidemiology is similar to that in industrialized countries , where zoonosis is the major source of hev infection in humans . |
myo - inositol ( myo - ins ) is the major component of a family of nine hexahydroxycyclohexane inositol ( ins ) stereoisomers broadly distributed in eukaryotic tissues and cells , where they are involved in basic biological functions .
ins is incorporated into cell membrane phosphoinositides or phosphatidyl - glycerides as phosphatidyl - inositol ( pi ) , which is phosphorylated by a set of specific phosphoinositide-3-kinases ( pi3k ) to phosphatidyl - inositol phosphates .
phosphatidyl - inositol trisphosphate ( pip3 ) can be further metabolized by phospholipase c ( plc ) to inositol trisphosphate ( ins1,4,5p3 , insp3 ) , which acts as second messenger for various hormones such as fsh , tsh , and insulin [ 2 , 3 ] .
inositol is the precursor of other membrane components and glycosyl - phosphatidyl - inositol ( gpi ) protein - anchor molecules ( reviewed by ) , signaling molecules ( reviewed by [ 5 , 6 ] ) , chromatin - remodeling , and transcription - modulating molecules .
increasing evidence shows that myo - ins has a physiological role in mammalian gametogenesis and embryonic development and a positive clinical impact on human medically assisted reproduction . together with its isomer d - chiro - inositol ( d - chiro - ins ) ,
myo - ins acts as second messenger of insulin and has been widely studied for its involvement in metabolic syndrome ( reviewed by ) as well as for the treatment of polycystic ovary syndrome ( pcos ) ( reviewed by [ 11 , 12 ] ) , one of the most common female endocrine disorders strictly associated with insulin resistance .
recent reports strongly suggest that both ins isomers can be positively associated in the management of pcos patients enrolled in assisted reproduction procedures , with significant encouraging clinical outcomes . while d - chiro - ins does not appear to have a clear role in the ovary or in the testis , myo - ins affects gametogenetic and embryogenetic processes at several levels , having a protective , positive role in reproduction and development ( reviewed by ) .
myo - ins concentration in the reproductive tracts of male mammals is higher than in blood serum being produced by fsh - responsive sertoli cells [ 18 , 19 ] . studies performed on pathological sperm samples have shown that myo - ins is crucial in spermatogenesis by positively affecting sperm cell motility and mitochondrial membrane potential , an apoptotic marker directly associated with cell viability . since these parameters are predictors of good embryos quality , the use of myo - ins in the andrology laboratory is now employed to improve the recovery of sperm cells after swim - up in in vitro fertilization ( ivf ) cycles . as for female gametogenesis , myo - ins
is actively concentrated in mammalian follicular and tubal fluid , being higher than in blood serum .
respective values of myo - ins concentration in human follicular fluid and serum are 30-to-40 m and 10-to-20 m . in addition , a positive correlation exists between follicular myo - ins content and oocyte quality as well as pregnancy outcome . at the ovarian level ,
however , although it is known that myo - ins metabolism is severely deregulated in follicle cells of pcos patients , effects of this molecule on somatic ovarian cells still need to be evaluated .
we have hypothesized that in theca and granulosa cells myo - ins may sustain steroidogenic activity during the ovarian cycle by modulating the dynamics of cytoskeletal structures .
myo - ins is transported into mammalian cells , including growing and fully - grown oocytes and early preimplantation embryo blastomeres , by a sodium cotransporter and a sodium independent transporter . in oocytes ,
activity of myo - insderived insp3 on the modulation of intracellular ca concentration in response to the action of the hormones lh and fsh [ 29 , 30 ] is well known .
it acts via cell - specific receptors ( insp3-r1 ) and appears to play a key role in meiotic maturation .
cultured preovulatory oocytes from outbred mice in minimal essential medium supplemented with myo - ins and observed increased rates of maturation , ivf , and development to the two - cell stage with respect to oocytes cultured in medium alone , suggesting that higher availability of myo - inositol increases both meiotic and activation efficiencies of the oocyte . after transfer to foster mothers , the implantation rate and postimplantation viability of these embryos were also increased in the myo - ins treated groups . during mammalian preimplantation development , activity of myo - ins transporters
ensures a robust uptake , which progressively increases from the one - cell to the blastocyst stage [ 34 , 35 ] , suggesting a parallel increase in cellular requirement of the molecule .
it has been shown that , in preimplantation embryo blastomeres , myo - ins is rapidly incorporated into phosphoinositides , leading to raised intracellular insp3 levels .
this led several groups , including ours , to investigate whether myo - ins could also have a positive action during preimplantation development in both the laboratory mouse and farming species .
myo - ins supplementation of culture media was found to improve rabbit and bovine blastocyst formation and expansion [ 35 , 36 ] .
culture of rabbit embryos at the morula stage in medium containing myo - ins at the optimal concentration of 75 m resulted in blastocysts expansion with a fourfold increase in diameter when compared to that provided by standard culture conditions .
similar observations were obtained on bovine zygotes matured and fertilized in vitro , after culture in synthetic oviduct fluid medium in the presence or absence of 2.77 mm myo - ins . as a result ,
blastocyst rate was higher among embryos developed in the presence of myo - ins . to evaluate postimplantation effects of this treatment ,
ten blastocysts grown in the presence of myo - ins were transferred to foster cows and developed to term , producing five healthy animals . with a clear interest in human assisted reproduction technology ( art ) , positive data on myo - ins in mammalian preimplantation development prompted us to hypothesize that inclusion of myo - ins in embryo culture media would result in increased numbers of high quality embryos produced by ivf / intracytoplasmic sperm injection ( icsi ) .
we tested this hypothesis in a previous work , using the mouse embryo model [ 39 , 40 ] , by investigating the effects of myo - ins supplementation of sequential human embryo culture media , starting 30 minutes after fertilization ( p.f . ) and for the whole length of preimplantation development ( day 4 p.f . ) .
in that study , we cultured embryos obtained by icsi from gametes of inbred c57bl/6n mice , in which icsi results in suboptimal rates of oocyte survival and blastocyst development , when compared to hybrid mice .
after fertilization in unmodified fertilization medium , embryos were cultured in cleavage medium in the presence or absence of 10 mm myo - ins ( myo - ins+ and myo - ins , resp . ) and monitored daily for developmental progression . at all - time intervals monitored , ( p.f . )
, myo - ins+ embryos displayed a faster cleavage rate , with a higher percentage of embryos at the most advanced stage . in these embryos , early differentiative events such as compaction and blastulation occurred at the proper developmental stage , excluding apparent toxic effects of myo - ins .
although results scored on day 4 have already been published , those recorded at intermediate times have not been presented elsewhere and are reported in figures 1 and 2 , together with embryo morphology observed on day 4 . embryos cultured in the presence of myo - ins were developmentally advanced with respect to control embryos , being mostly represented by expanded blastocysts with a higher number of blastomeres , as shown by hoechst 33343 nuclear staining .
we concluded that myo - ins supplementation represents an improvement of culture conditions reducing the developmental gap typically observed between embryos obtained and cultured in vitro and those developed in vivo , further supporting its possible use for human embryo preimplantation culture .
one of the issues left uncovered by these experiments concerns the nature of biochemical pathways induced by exposure to myo - ins in preimplantation of embryo blastomeres .
existing information suggests that possible pathway(s ) elicited by myo - ins in mouse embryo blastomeres and responsible for a reduced length of cell cycles and a more efficient proliferation activity are initiated via rapid incorporation into phosphoinositides , namely , pip3 , enzymatically fostered by activity of pi3k . in turn
, pip3 is converted by plc into diacylglycerol and insp3 , which , by mobilizing ca stores , has already been recognized to speed up several cellular processes , including proliferation [ 42 , 43 ] .
pi3k is also responsible for the activation of the pkb / akt pathway , which is known to promote proliferation of mouse embryo blastomeres .
as myo - ins has been shown to increase akt phosphorylation and hence its activity in mouse skeletal muscle cells , we hypothesized that a critical step in enhancing embryo preimplantation development may be represented by akt activation induced by myo - ins supplementation .
we here provide the first evidence , obtained by immunofluorescence analysis , that myo - ins supplementation of culture media increases serine phosphorylation of pkb / akt in mouse preimplantation embryos .
a second open issue in current research concerns the safety of preimplantation embryo exposure to myo - ins for postimplantation and postnatal development . besides the reported observations on bovine embryos
, it could be argued that preimplantation embryos , including human embryos , were once routinely cultured in undefined media supplemented with commercial bsa preparations or batches of bovine or human sera , which contain variable amounts of myo - inositol , without reporting any apparent negative effect .
in addition , myo - ins appears important for normal development ( reviewed by ) .
its concentration in foetal human serum is severalfold higher than in adults , lowering only around birth , and its administration during pregnancy has positive effects on pathological conditions in both humans [ 49 , 50 ] and rodent species [ 5154 ] .
all data hitherto reported suggest that preimplantation embryo exposure to myo - ins would have no detrimental effects on further development . to obtain more direct information on this issue , we transferred blastocysts produced in vitro in the presence of myo - ins into recipient foster mothers and allowed their development to term . by this approach
, we obtained healthy offspring that appeared normal in the sex ratio and , at least until weaning , somatometrically .
these experiments provide additional data on myo - ins effects on mammalian preimplantation embryos and strongly suggest that it can be considered safe for embryo development to term .
animals ( charles river italia , calco , va , italy ) were housed in a temperature - controlled facility ( 22 1c ) on a 12/12 h light / dark cycle , inside standard cages with unlimited access to food and water .
forty - to-60-day - old c57bl/6n mice were used as donors of oocytes and male studs .
forty - to-60-day - old cd/1 mice were used as foster mothers and vasectomized male studs .
all experimental procedures were conducted in accordance with the official european guidelines for the care and use of laboratory animals ( 86/609/eec ) .
experimental protocols and related procedures were approved by the italian ministry of public health . in order to model human preimplantation embryo culture ,
all procedures were performed in commercial quinn 's advantage media and supplements ( biocare europe , napoli , italia ) , which do not contain myo - ins , as described .
female mice were hormonally induced by intraperitoneal injections of 5 iu pregnant mare serum gonadotropin ( nhpp , torrence ca , usa ) and , 4648 h later , of 5 iu human chorionic gonadotropin ( corulon , intervet italia , aprilia , lt , italy ) . after mating with male mice on ,
females carrying a vaginal plug were sacrificed by cervical dislocation and cumulus - oocyte complexes were collected from the oviducts , freed from cumulus cells by treatment with 0.1% hyaluronidase in hepes buffered quinn 's protein plus advantage htf - medium containing 5% human serum albumin , rinsed in htf - medium , and incubated at 37c in quinn 's protein plus cleavage medium ( c - medium ) under a humidified atmosphere of 5% co2 in air .
one - cell embryos were positively scored by the presence of two pronuclei and then divided into two groups .
one group was further cultured in 14 l / ml myo - inositol ( 10 mm , andrositol lab lo.li .
pharma , italy ) in c - medium and the other one in 14 l / ml phosphate buffered saline in c - medium .
embryo culture was performed in 500 l medium inside falcon 4 well ivf plates without medium replacement , as described .
preimplantation embryos at the morula or blastocyst stage were washed in 0.1 m phosphate buffered saline , ph 7.4 ( pbs ) , containing 0.3% bovine serum albumin ( bsa ) ( pbs - bsa ) , and were then fixed in 4% paraformaldehyde in pbs for 30 min at room temperature ( rt ) . after three washes in pbs - bsa
louis , mo ) for 15 min at rt and washed three times in pbs - bsa .
permeabilized embryos were processed for immunostaining by an overnight incubation at 4c in the presence of the primary antibody , rabbit monoclonal anti - phospho - akt ( ser473 , # 4080 ) , anti - phospho - akt ( thr308 , # 4056 ) or anti - akt ( total , # 9272 ) antibody ( cell signaling technology , beverly , ma , usa ) ( 1 : 75 ) , followed by washing in pbs - bsa and 1 hr final incubation with the secondary , and alexa flour-488 conjugated secondary antibodies ( invitrogen ) , diluted 1 : 200 in pbs - bsa for 1 h at rt . after washing with pbs - bsa
finally , embryos were washed three times for 10 min each in pbs - bsa and mounted in pbs and glycerol ( 1 : 1 v / v ) using coverslips .
fluorescent signals were detected using a zeiss axioplan fluorescence microscope ( carl zeiss , oberkochen , germany ) at 400x magnification .
for semiquantitative analysis of fluorescence , embryos at various developmental stages were immunostained after pooling in the same drops .
fluorescence emission was collected under similar excitation conditions and then quantitatively analyzed by using the imagej software ( imagej 1.47v , wayne rasband , national institutes of health , usa . http://imagej.nih.gov/ij/ ) . in ten replicate experiments ,
embryos developed to the blastocyst stage after 4 days of culture in myo - ins+ c - medium or myo - ins c - medium were transferred to the uteri of pseudopregnant foster mothers mated 2.5 days earlier with vasectomized males and carried to term .
on the day of delivery , newborn animals were weighed , checked for gross abnormalities , and left to be nursed by their moms until weaning .
preweaning morphological analyses included body growth at one week , fur appearance , and eye opening .
mice were finally weighed and sexed at weaning ( three weeks of age ) . where not stated otherwise , chemicals were purchased from sigma - aldrich co. ( st . louis , mo , usa ) .
data were analyzed by using student 's t - test or tests with yates continuity correction .
statistical analyses were performed using r : a language and environment for statistical computing ( r development core team , r foundation for statistical computing , isbn 3 - 900051 - 07 - 0 , 2008 , vienna , austria .
we have determined presence and phosphorylation of akt in late preimplantation stage embryos cultured in the presence or absence of myo - ins by immunofluorescence analysis .
this approach revealed the presence of serine 473- and threonine 308-phosphorylated akt in both morula and blastocyst embryos ( figure 3 ) .
phosphorylated akt was localized prominently in blastomere cytosols but a limited nuclear localization was also observed .
the relative content of phosphorylated akt at the same stages was also measured by quantification of immunofluorescence data ( table 1 ) . the level of serine 473-phosphorylated akt did not appear to be modified in embryos cultured in the presence of myo - ins at the morula stage ( t4 = 1.75 , p = 0.15 ) ( figure 3(a ) ) but it was increased at the blastocyst stage ( t4 = 3.61 , p = 0.02 ) ( figure 3(c ) ) . on the contrary ,
the level of threonine 308-phosphorylated akt did not vary significantly depending on the presence or absence of myo - ins ( morulae , t4 = 0.670 , p = 0.54 , figure 3(b ) ; blastocysts , t4 = 0.640 , p = 0.56 , figure 3(d ) ) .
amounts of total akt did not vary under different conditions ( not shown ) . in ten replicate experiments , all embryos that had developed in vitro to the expanded or nonexpanded blastocyst stage in the presence or absence of myo - ins
were transferred to foster mothers and allowed to develop through birth and until weaning . of 105 myo - ins+ and 76 myo - ins blastocysts transferred , 59 ( 56.2% ) and 33 ( 43.4% ) , respectively ,
although this difference was not significant , when we compared numbers of delivered animals with numbers of fertilized oocytes cultured under the two conditions , 154 myo - ins+ one - cell embryos and 147 myo - ins one - cell embryos , we obtained a significant improvement in the overall efficiency of the treatment ( = 8.91 , p = 0.003 ) .
a similar difference was observed by comparing numbers of fertilized oocytes with numbers of transferred embryos ( = 8.52 , p = 0.003 ) .
body weights at birth were 1.42 0.01 g for myo - ins+ and 1.41 0.02 g for myo - ins ( t90 = 0.645 , p = 0.52 ) . during the first week
, six and four pups were found dead in the myo - ins+ and myo - ins groups , respectively .
somatometric development appeared similar in mice of both groups , with appropriate acquisition of body fur and eye opening .
weights at one week of age were also similar ( myo - ins+ , 2.06 0.16
g , myo - ins , 2.08 0.19 g ; t80 = 0.286 , p = 0.78 ) .
sex distributions in the two conditions were both casual ( myo - ins+ , 27 males , 26 females , myo - ins , 14 males , 15 females ) .
finally , weights at three weeks of age were similar for sexes and embryo culture conditions ( males : myo - ins+ , 10.20 0.21 g ; myo - ins , 10.29 0.28
g , t39 = 0.790 , p = 0.43 ; females : myo - ins+ , 9.90 0.19 g ; myo - ins , 9.75 0.40 g , t39 = 1.214 , p = 0.23 ) .
akt represents a major downstream effector of growth factors , cytokines , and adhesion receptors capable of promoting cell survival and proliferation [ 56 , 57 ] and is regulated , among others , by pip3 , the second messenger product of pi3k , via recruitment of pdk1 .
pdk1 induces direct phosphorylation at threonine 308 and appears to be also implicated in the negative regulation of phosphorylation at serine 473 , by direct binding to this hydrophobic site .
however , pdk1 can be displaced by an appropriate signal [ 59 , 60 ] , including activation of integrin like kinase ( ilk ) , allowing akt autophosphorylation or phosphorylation by other kinases at serine 473 .
this pathway makes akt phosphorylation of serine 473 inducible by upstream signals ; on the contrary , phosphorylation of threonine 308 appears to have a constitutive nature .
we have previously shown that , during the first two stages of embryo development , akt phosphorylation is not prevented by inhibition of either pi3k or pdk1 and concluded that the phosphorylation state and the intracellular distribution of akt in two - cell embryos are independent of the activity of both kinases .
this suggested that akt is inherited from the oocyte in its phosphorylated / dephosphorylated form at both serine 473 and threonine 308 .
we now show by quantitative immunofluorescence analysis that serine 473 phosphorylation of akt can be increased in late preimplantation embryos by the presence of myo - ins in the culture medium .
it thus appears that , after the initial stages of development , new phosphorylation of akt may occur in mid - to - late preimplantation stages depending on availability of myo - ins . in agreement with previous observations that development of mouse preimplantation embryos requires pi3k activity from the 8/16-cell stage
, it would be reasonable to hypothesize that this finding can be ascribed to the increased production of phosphoinositides and consequent increased production of pip3 by pi3k .
an increase in phosphorylation of akt may be responsible for the faster developmental rate of embryos cultured in the presence of myo - ins .
the lack of inducibility of threonine phosphorylation of akt by myo - ins is also in agreement with previous observations .
it is puzzling that myo - ins exerts opposite effects on both pi3k and akt activities when added to cancer cell cultures , in which it has been found to reduce pi3k levels as well as akt activity by inhibiting its phosphorylation . to this respect ,
it is therefore tempting to speculate that such effect could be considered context - dependent and that some other still unknown factors are likely to participate in modulating the pi3k / akt pathway . in order to obtain more specific information on this pathway
, we are currently analyzing the effects of myo - ins both on development and on akt phosphorylation in preimplantation embryo in the presence of inhibitors of pi3k and pdk1 .
further experiments will address the involvement on proliferative activity of preimplantation embryos blastomeres of pro- and anti - apoptotic factors of the bcl-2 family .
data here produced represent a first assessment of the effect of preimplantation embryo exposure to myo - ins on mouse development to term .
so far , information on this issue in mammals is limited to one finding obtained on bovine embryos cultured in the presence of 2.77 mm myo - ins .
in that study no comparison was made between embryos cultured in the two conditions , but blastocysts that had developed after preimplantation exposure to myo - ins were transferred producing healthy animals .
present results obtained in the mouse show ( a ) the apparent absence of early toxic effects of myo - ins , as suggested by normal prenatal and short - term postnatal development , and ( b ) a significant increase in the overall rate of live births obtained after preimplantation embryo culture in myo - ins and subsequent transfer into foster mothers . if the first observation was expected in light of the body of information here reported on the positive effects displayed by myo - ins on mammalian gametogenesis and development , the second one deserves particular attention . in fact
, it supports the possibility that a regular use of myo - ins as culture supplement provides high efficiency in the production of viable preimplantation embryo in vitro both in the mouse and in farming specie with promising outcome for both scientific and economic purposes .
in addition , it strengthens the hypothesis that the use of myo - ins would have a similar positive role in the culture of in vitro produced human embryos , with obvious medical consequences . to this end , however , additional assessments of myo - ins effects are necessary at least at three different levels : ( a ) on the expression of imprinted genes during development ; ( b ) on the acquisition of sensory / motor / behavioral functions during early development ; and ( c ) on long - term consequences on the whole organism . | myo - inositol ( myo - ins ) has a physiological role in mammalian gametogenesis and embryonic development and a positive clinical impact on human medically assisted reproduction .
we have previously shown that mouse embryo exposure to myo - ins through preimplantation development in vitro increases proliferation activity and blastocyst production , representing an improvement in culture conditions .
we have herein investigated biochemical mechanisms elicited by myo - ins in preimplantation embryos and evaluated myo - ins effects on postimplantation / postnatal development . to this end
naturally fertilized embryos were cultured in vitro to blastocyst in the presence or absence of myo - ins and analyzed for activation of the pkb / akt pathway , known to modulate proliferation / survival cellular processes . in parallel , blastocyst - stage embryos were transferred into pseudopregnant females and allowed to develop to term and until weaning .
results obtained provide evidence that myo - ins induces cellular pathways involving akt and show that ( a ) exposure of preimplantation embryos to myo - ins increases the number of blastocysts available for uterine transfer and of delivered animals and ( b ) the developmental patterns of mice obtained from embryos cultured in the presence or absence of myo - ins , up to three weeks of age , overlap .
these data further identify myo - ins as a possibly important supplement for human preimplantation embryo culture in assisted reproduction technology . |
proprioception provides sensory feedback from the body to the nervous system ; therefore , it
contributes to the maintenance of optimal body alignment1,2,3,4 .
it includes several
submodalities such as joint position sense , kinesthesia , and sense of tension .
joint
position sense is used for not only recognizing the location of joints , but also for
measuring proprioception .
sense of tension is also used to measure proprioception , and
kinesthesia is considered to perceive active and passive movements2 , 5 .
various receptors
such as ruffini receptors and parcinian corpuscles are involved in the relay of information
to the central nervous system ; however , the receptors in the muscle called muscle spindles ,
particularly play a major role in proprioception2 ,
6 .
changes in the muscle length caused
by poor posture for a sustained period of time result in musculoskeletal problems such as
forward head posture ( fhp)7 , 8 .
fhp is defined when that the head is anterior to a vertical line through the center of
gravity .
it is considered a common postural disorder related to abnormalities in
musculoskeletal balance9 , 10 .
many studies have reported that fhp is correlated to
headache , temporomandibular disorders , myofacial pain syndrome , and abnormal scapular
movement .
neck pain resulting from a reduction not only in the length of muscle fibers , but
also in the capacity to generate tension in muscles is also attributed to fhp11,12,13 . since there are many mechanoreceptors in
the cervical muscle , the cervical region is considered to play a crucial role in the
transmission of information .
multifactorial problems in this region are mainly caused by
decreased joint sense7 , 14 , 15 . thus far , many studies have been conducted investigating the correlation between fhp and
pain8 , 9 ,
16 , 17 .
however , investigations concerning whether fhp can affect
proprioception are few . in the current study , we investigated the effect of fhp on
proprioception via the measurement of cervical position - reposition error .
thirty - nine subjects with no history of neuromuscular disorder , fracture , or moderate or
severe scoliosis were recruited for this study .
they were divided into two groups in
accordance with their craniovertebral angles : the fhp group ( n=19 ) and the control group
( n=20 ) ( table 1table 1.general characteristics of the subjectsfhp groupcontrol groupgender ( m / f)7/1212/8age ( years)22.21.922.72.1height ( cm)166.07.4169.77.3weight ( kg)63.812.364.012.6values are expressed as the meansd . ) .
the craniovertebral angle of all subjects was measured , and when the angle
was less than 53 , subjects were included in the fhp group10 , 11 , 18 .
all subjects were informed of the purpose of this study and
provided their written informed consent prior to their participation .
a very common method for the assessment of fhp is taking a picture of the lateral view of
the subject18 , 19 .
the base of the camera was set at the
height of the subjects shoulder .
the tragus of the ear was marked , and a plastic pointer
was taped to the skin overlying the spinous process of the c7 vertebra .
we measured the
craniovertebral angle , which is defined as the angle between a horizontal line passing
through c7 and a line extending from the tragus of the ear to c7 .
the error value of cervical position sense was evaluated using a head repositioning
accuracy ( hra ) test20,21,22,23 .
the hra test was used to measure the difference between the start
( 0 position ) and return positions .
a laser pointer attached to a cycling helmet was firmly
placed on the subjects heads . with their head in a natural resting position , the subjects
were requested to focus on a target that was positioned at eye level .
all subjects were then
instructed to close their eyes , and the target was moved so that the laser pointer s beam
projected onto the target .
the subjects were told to memorize this position because this was
the reference position .
then they performed a cervical full flexion at their preferred speed
and held this position for 5 seconds . following this , the subjects , with their eyes
still
closed , were instructed to return to the reference position at their preferred speed .
the absolute error value was
measured as the distance between the two marked points .
the same procedure was followed to
assess extension , right rotation , and left rotation , which were performed at random .
data satisfying the normal distribution were examined with a parametric test . to assess the
differences in the error values of the joint position sense ( cervical flexion , extension ,
and rotation ) between the fhp and control groups , the independent t - test was performed . in
addition , pearson correlation coefficients were used to assess the degree of correlation
between the craniovertebral angle and the error value of each joint position sense .
there were significant differences between the error values of the joint position sense
( cervical flexion , extension , and rotations ) of the fhp and control groups ( p<0.05 ) . in
addition
, there was an inverse correlation between the craniovertebral angle and the error
values of position sense ( p<0.05 ) .
the statistical values are shown in tables 2 and
3table 2.comparison of the cervical position sense errors of the fhp and control groups
( unit : cm)fhp groupcontrol groupflexion6.231.91 * 4.141.74extension6.232.20 * 4.561.62right rotation7.082.27 * 5.131.51left rotation5.602.03 * 4.231.62values are expressed as the meansd . an asterisk ( * ) indicates a significant
difference ( p<0.05).table 3.the correlations between the craniovertebral angle and the error values of
position senseflexionextensionright rotationleft rotationpearson s correlation ( r)0.597*0.421*0.428*0.389*an asterisk ( * ) indicates a significant correlation ( p<0.05 ) .. values are expressed as the meansd .
an asterisk ( * ) indicates a significant
difference ( p<0.05 ) . an asterisk ( * )
nowadays , the use of visual display terminals ( vdt ) of computers and smart phones in almost
all homes and organizations is very common24 .
excessive use of vdts results in musculoskeletal disorders . among
these disorders , fhp is one of the most common conditions10 , 16 , 25 .
these
changes in the cervical region may lead to musculoskeletal dysfunction such as an
upper
crossed syndrome resulting from maintaining poor head position for a long duration of
time24 , 26 ,
27 .
these pains cause a reduction of joint sense which influences
abnormal proprioception and poor postural balance7 .
the present study examined the position - reposition error of the cervical region in order to
investigate whether fhp affects joint position sense .
higher error rates were shown by the
group with fhp compared to the group without fhp . in all movements ( flexion , extension , and
rotation ) , there were significant differences in repositioning errors between the two
groups .
the present study also
demonstrated that there is a correlation between the degree of fhp and joint position sense ,
additionally suggesting that as fhp becomes more severe , joint position sense becomes
worse .
it is the
ability to recognize the joint location , and influences body alignment and joint
stability2,3,4,5 .
muscle spindles have a primary ending which responds to changes in
the length and speed of muscle stretch as well as a secondary ending that responds only to
changes in the muscle length2 , 28,29,30 . in general
, fhp leads to changes in the lengths of the anterior and posterior neck muscles ,
and prolonged fhp causes sustained loading on the cervical spine8 , 26 . in this regard ,
the results of the present study imply that changes in the lengths of muscles , as a result
of fhp , have a bad influence on the activity of the muscle spindles , and this is the reason
why poor joint position sense is induced by fhp .
further studies of the effect of
therapeutic exercise for improving position sense of subjects with fhp should be
encouraged . in conclusion ,
this result implies that a
change in the muscle length caused by fhp affects decreases joint position sense . | [ purpose ] the purpose of this study was to investigate the effect of forward head posture
( fhp ) on proprioception by determining the cervical position - reposition error .
[ subjects
and methods ] a sample population was divided into two groups in accordance with the
craniovertebral angle : the fhp group and the control group .
we measured the
craniovertebral angle , which is defined as the angle between a horizontal line passing
through c7 and a line extending from the tragus of the ear to c7 .
the error value of the
cervical position sense after cervical flexion , extension , and rotation was evaluated
using the head repositioning accuracy test .
[ results ] there were significant differences
in the error value of the joint position sense ( cervical flexion , extension , and rotation )
between the fhp and control groups .
in addition , there was an inverse correlation between
the craniovertebral angle and error value of the joint position sense .
[ conclusion ] fhp is
associated with reduced proprioception .
this result implies that the change in the muscle
length caused by fhp decreases the joint position sense .
also , proprioception becomes
worse as fhp becomes more severe . |
ghrelin , a 28-amino acid peptide , is produced mainly in the stomach and to a lesser extent in the small intestine , pancreas , and hypothalamus and is the endogenous ligand for the growth hormone secretagogue receptor ( ghs - r ) [ 19 ] .
serine-3 of ghrelin is acylated with an octanoic acid , the process catalyzed by a recently discovered ghrelin o - acyltransferase ( goat ) [ 10 , 11 ] .
the acylation is thought to be required for its biological activity , although desacyl ghrelin has been reported to exert several effects .
administration of pharmacological doses of acylated ghrelin (= ghrelin ) to intact animals increases food intake , induces body weight gain , and causes obesity [ 1318 ] .
the orexigenic and body fat promoting properties of ghrelin and growth hormone ( gh ) secretagogue ( ghs ) are thought to be independent of gh and mediated primarily by the hypothalamic neuropeptide y ( npy ) and agouti - related protein ( agrp ) systems [ 1416 , 1922 ] .
although pharmacologic effects of ghrelin are well documented , physiological role of endogenous ghrelin is poorly understood .
it has recently been reported that knockout of either the ghrelin gene or ghrelin receptor gene exerts no or minor effects on body weight and body composition [ 2325 ] .
however , the lack of phenotypic changes in knockout mice might reflect compensatory mechanisms that are known to operate occasionally . in humans , gastrectomy results in loss of body weight of about 10% within the first six months after surgery , primarily due to reduced body fat .
in addition , gastrectomized patients often complain of loss of appetite , general fatigue , and in some cases impaired bone quality such as osteopenia and osteomalacia [ 9 , 27 ] . at present there are no satisfactory mechanistic explanation and treatment for any of these symptoms .
the effects of gastrectomy on food intake and body composition have been poorly documented in rodents .
loss of ghrelin could be implicated in these symptoms , since as much as 80% of circulating ghrelin is lost following surgical removal of the glandular stomach or the acid producing part of the stomach in rats and humans [ 4 , 28 , 29 ] . in this study
, we performed total gastrectomy in rats and examined whether the recovery from gastrectomy - associated anorexia depends on the circulating ghrelin level or other factors and studied temporal changes in the biosynthesis and orexigenic ability of ghrelin following gastrectomy .
male 4-week - old wistar rats ( slc , japan ) were maintained on a 12-hour light / dark cycle and given conventional food and water for 2 weeks and not deprived of food before gastrectomy .
they were operated at 6 weeks of age with body weight around 130180 g. rats were anesthetized with an intraperitoneal injection of pentobarbital ( 40 mg / kg ) and a median abdominal incision was made .
after the stomach was separated from the greater and lesser omentum , the duodenal bulb was ligated and transected .
after the left gastric artery was ligated , the esophagus was clamped above the esophagogastric junction and the stomach was resected .
an end - to - side anastomosis between esophagus and jejunum at the 4 - 5 cm anal side from treitz ligament was performed with 70 monofilament polyglyconate synthetic absorbable string ( maxon ; johnson & johnson inc .
usa ) using interrupted suture ( billroth ii ( b - ii ) reconstruction ) . on the other hand ,
jejunum was transected at the 2 - 3 cm anal side from the treitz ligament . then
, an end - to - side anastomosis between the esophagus and jejunum was performed with 70 maxon using an interrupted suture .
jejunojejuno anastomosis was also done by end - to - side method ( roux - en - y ( r - y ) reconstruction ) . the abdominal wall and skin closure was made by 50 nylon ( johnson & johnson inc .
after the operation , the rats were allowed only clear water without food for 3 days , dry milk from the 3rd day , and from the 7th day a conventional pellet diet with free access to water .
after the operation , food intake for 24 hours and body weight were measured once a week for 3 months .
the rats were sacrificed at three months after operation . to measure plasma ghrelin concentrations ,
duodenum samples of about 3 cm were taken from 1 cm anal side of duodenal stump .
jejunum samples of about 3 cm were taken from 1 cm anal side of esophagojejunostomy .
one antiserum was raised against the cooh - terminally cys - extended rat ghrelin ( position 1 - 11 ) in new zealand white rabbits ( # g606 ) that was shown to specifically recognize ghrelin with n - octanoylated ser 3 ( acylated ghrelin ; ghrelin ) . by the radioimmunoassay ( ria ) using this antiserum , designated nh2-terminal ria ( n - ria ) , the concentration of acylated ghrelin was obtained .
the other antiserum was raised against the nh2-terminally cys - extended rat ghrelin ( position 13 - 28 ) ( # g107 ) that was shown to recognize both acylated ghrelin and desacyl ghrelin . by the ria using this antiserum , named as cooh - terminal ria ( c - ria )
these two ghrelin - specific rias were used to measure acylated ghrelin and desacyl ghrelin contents in several tissues . the bound and free ligands were separated using a second antibody .
acylated ghrelin is expressed as ghrelin and acylated ghrelin plus desacyl ghrelin is expressed as
plasma concentrations of acylated ghrelin and desacyl ghrelin were measured using elisa kits ( mitsubishi kagaku iatron , tokyo , japan ) .
ghrelin ( peptide institute , osaka , japan ) or saline was administered subcutaneously to control normal rats ( eight - weeks - old ) and those received gastrectomy with b - ii reconstruction . for ghrelin treatment at 2 and 6 weeks after gastrectomy
control and gastrectomized rats were divided into two groups ; one group was treated with ghrelin and the other with saline , and food intake and body weight were measured .
the injection of ghrelin ( 10 nmol / kg body weight ) was carried out once a day for seven days , and food intake and body weight were measured . for ghrelin treatment at 12 weeks after gastrectomy , the following procedures were used .
single injection of ghrelin or saline was followed by measurements of food intake for the subsequent 24 hours . in the next week , two groups were changed : one group that had first received ghrelin was injected with saline , and the other group that had first received saline was injected with ghrelin .
food intake after the first injection and that after the second injection were pooled and averaged .
statistical analysis was performed using student 's paired and unpaired t - tests . values of p < .05 were considered statistically significant .
food intake for 24 hours and body weight were measured in total gastrectomized and control rats during 12 postoperative weeks .
gastrectomy with b - ii ( n = 40 ) and r - y reconstruction methods ( n = 35 ) both markedly decreased food intake . in both b - ii and r - y groups , at the 1st week after operation food eaten for 24 hours decreased to a level around 50% of that in control rats ( figure 1(a ) ) . in b -
in contrast , food intake in r - y group increased gradually during 4 postoperative weeks .
the daily food intake averaged for postoperative 12 weeks was significantly ( p < .05 ) reduced in b - ii and r - y groups ( figure 1(a ) ) .
food intake in r - y group was significantly ( p < .05 ) greater than in b - ii group throughout postoperative 12 weeks .
body weight significantly decreased after gastrectomy in both b - ii and r - y groups ( figure 1(b ) ) .
body weight in r - y group was significantly greater than that in b - ii group from the 2nd through 12th postoperative week ( p < .05 ) .
the correlation between body weight , 24 hours food intake , and plasma ghrelin concentrations at the 12th postoperative week was studied .
food intake and body weight were correlated with each other irrespective of reconstruction methods and were plotted on a single regression line ( figure 2(a ) ) .
in contrast , there was no correlation between food intake and plasma concentrations of total ghrelin ( figure 2(b ) ) .
we next examined whether body weight correlates with plasma concentrations of ghrelin or total ghrelin , by using n - ria and c - ria .
body weight did not correlate with plasma ghrelin and total ghrelin levels ( figures 2(c ) and 2(d ) ) .
furthermore , no significant difference between r - y and b - ii groups was observed in averaged ghrelin levels ( b - ii : 46.3 7.2 fmol / ml ( n = 40 ) , r - y : 35.9 4.6 fmol / ml ( n = 35 ) ) and total ghrelin levels ( b - ii : 393.0 40.0 fmol / ml ( n = 40 ) , r - y : 349.8 23.2 fmol / ml ( n = 35 ) ) , though some rats in b - ii groups showed higher levels of ghrelin and total ghrelin .
these results indicate that plasma ghrelin levels are significantly reduced in r - y and b - ii groups and that greater food intake in r - y than in b - ii is related to the method of reconstruction but not plasma ghrelin levels .
plasma ghrelin concentrations were markedly reduced in both r - y and b - ii groups to the levels of about 30% of those in control rats ( figure 3(a ) ) .
therefore , the reduction of plasma ghrelin levels is due to lack of release of ghrelin from stomach .
we examined whether the reduction of ghrelin due to gastrectomy could influence the production of ghrelin in the intestine and pancreas , the tissues known to produce ghrelin .
ghrelin concentrations in the duodenum increased approximately by 100% in r - y and b - ii groups ( figure 3(b ) ) , while those in the jejunum did not change ( figure 3(c ) ) .
ghrelin concentrations in the pancreas were much smaller than those in the intestine , however , they dramatically increased approximately by 500% in b - ii and by 400% in r - y groups ( figure 3(d ) ) . on the other hand ,
plasma concentrations of total ghrelin were approximately five times higher than those of ghrelin , and they were markedly reduced by gastrectomy in both groups ( figure 3(e ) ) .
contents of total ghrelin in the duodenum and pancreas , but not jejunum , increased in both r - y and b - ii groups ( figures 3(f)3(h ) ) .
the gastrectomy - associated relative changes of total ghrelin in the circulation , duodenum and pancreas were similar to those of ghrelin .
these results suggest that gastrectomy - induced reductions in circulating ghrelin levels promote synthesis of ghrelin in the duodenum and pancreas , which may have compensatory roles including the restoration of circulating ghrelin levels . in consistence with this inference , plasma levels of ghrelin and total ghrelin that were markedly reduced at 2 weeks after gastrectomy with b - ii reconstruction ( ghrelin : 25.0 5.3 fmol / ml ( n = 4 ) , total ghrelin : 295.8 34.6 fmol / ml ( n = 4 ) ) were significantly ( p < .05 ) elevated at the 12th postgastrectomy week ( ghrelin : 46.3 7.1 fmol / ml ( n = 40 ) , total ghrelin : 393.0 40.0 fmol / ml ( n = 40 ) ) .
we examined whether reduced levels of plasma ghrelin after gastrectomy could be altered by fasting , the physiological regulator of ghrelin release .
at the 12th postoperative week , blood samples were taken from ad - lib fed and 48 hour fasting rats .
effects of fasting on plasma levels of ghrelin and desacyl ghrelin were examined by using elisa , since ria did not allow us to determine exact levels of desacyl ghrelin .
.05 ) elevated by fasting approximately by 3 times in both b - ii and r - y groups ( figures 4(a ) and 4(c ) ) . desacyl ghrelin concentrations were also significantly ( p < .05 ) elevated by 3 - 4 times by fasting in b - ii and r - y groups ( figures 4(b ) and 4(d ) ) .
thus , fasting induced similar fold increases in both ghrelin and desacyl ghrelin levels in the circulation in gastrectomized rats .
rats aged 8-weeks were divided into two groups and received subcutaneous injection of either saline or ghrelin once a day for seven continuous days .
injection of ghrelin increased food intake , in which significant ( p < .05 ) difference was obtained at the 2nd , 4th and 5th days ( figure 5(a ) ) .
the cumulative food intake during the treatment period of 7 days was significantly ( p < .05 ) increased by ghrelin ( figure 5(b ) ) . at 2 weeks after total gastrectomy
, rats were divided into two groups and received subcutaneous injection of either saline or ghrelin once a day for 7 continuous days .
the 24 hours food intake averaged for the treatment period of 7 days was not significantly different between ghrelin - and saline - treated groups ( figure 5(c ) ) .
essentially the same results were obtained in rats at 6 weeks after total gastrectomy ; 24 hours food intake averaged for the treatment period of 7 days was not different between ghrelin - and saline - treated groups ( figure 5(d ) ) . at the 12th week after total gastrectomy
one group received daily subcutaneous injection of saline for a week followed by that of ghrelin for the next week .
vice versa , the other group received ghrelin for the first week followed by saline for the next week .
the amount of 24 hours food intake in rats injected with ghrelin was significantly ( p < .05 ) greater than that with saline ( figure 5(e ) ) .
these results indicate that ghrelin injection increases food intake in the later stage of 12 weeks after gastrectomy but not earlier .
ghrelin is secreted primarily from the stomach [ 9 , 30 ] , and stimulates food intake and body weight gain .
gastrectomy commonly decreases food intake , body weight , fat mass and bone mass , and is also accompanied by a marked reduction in circulating ghrelin levels .
however , whether ghrelin levels are causally related to food intake and/or body weight after gastrectomy is not well understood . in the present study
, we produced total gastrectomized rat models and examined postoperative changes in ghrelin levels , food intake and body weight .
plasma ghrelin levels were markedly reduced by gastrectomy irrespective to whether the reconstruction was performed by b - ii or r - y method . however , the recovery from gastrectomy - induced reductions in food intake and body weight was much greater in r - y than in b - ii group .
the results indicate that the recovery from reduced food intake and body weight after gastrectomy is not correlated with circulating ghrelin levels but strongly depends on the reconstruction method .
the mechanism for the better recovery of food intake and body weight with r - y method after total gastrectomy remains unclear .
ghrelin contents in the duodenum , jejunum and pancreas at 12th postoperative week were not different between b - ii and r - y methods . it is speculated that in b - ii method bile juice may directly reflux the esophagojejunostomy and consequently decrease food intake .
alternatively , shorter distance between esophagojejunostomy and jejunojejunostomy in r - y method than in b - ii method may yield smaller reflux of bile juice .
it is suggested that selecting the reconstruction method with less reflux of bile juice may contribute to better recovery from the gastrectomy - induced anorexia . at the 12th week after gastrectomy ,
ghrelin production was markedly enhanced in the duodenum , the organ known to produce the second largest amount of ghrelin .
this result was essentially the same between b - ii and r - y groups , suggesting that the enhanced ghrelin production is not related to the reconstruction method but likely due to the removal of gastric ghrelin .
it is therefore suggested that the enhanced ghrelin production in the duodenum could partly compensate for gastrectomy - associated reductions of the circulating ghrelin and its endocrine functions . in another line of experiments , at 12th postoperative week fasting markedly increased plasma ghrelin levels in gastrectomized rats , indicating that the tissue other than stomach , most likely the duodenum , secretes ghrelin in response to fasting , the physiological regulator of ghrelin secretion [ 9 , 32 ] .
furthermore , the feeding response to peripheral ghrelin administration was once eliminated by gastrectomy but restored at 12th postoperative week .
these data suggest that ghrelin can be released and stimulate food intake under fasted conditions at this later period after gastrectomy .
collectively , the markedly upregulated production of ghrelin in the duodenum could contribute to the recovery of food intake and body weight in the later postoperative period . in this study ,
since the ghrelin content in the pancreas and its increment due to gastrectomy are much smaller than those in the duodenum , they may neither significantly contribute to the circulating ghrelin levels nor operate endocrine functions .
however , previous studies using ghrelin receptor antagonists and ghrelin - deficient mice have demonstrated that ghrelin in the pancreatic islets inhibits insulin release in an autocrine / paracrine manner and consequently regulate blood glucose levels [ 33 , 34 ] .
the late postprandial dumping syndrome accompanying gastrectomy is characterized by hypoglycemia principally due to excessive insulin release .
the mechanism for excessive insulin release remains unclear , lack of the stomach - derived ghrelin could be implicated .
if so , it is possible that upregulated pancreatic ghrelin compensates lack of stomach - derived ghrelin and attenuate insulin release , thereby counteracting the hypoglycemia in the late dumping syndrome .
further study is necessary to elucidate the role and mechanism for the upregulation of ghrelin production in the pancreas after gastrectomy .
it has been well documented that administration of pharmacological doses of ghrelin to intact animals increases food intake , induces weight gain , and causes obesity [ 9 , 1318 ] .
ghrelin injection failed to stimulate food intake in the early stages of 2nd to 6th week after operation , but increased it in the late stage of 12th week .
the results suggest that some machinery that links ghrelin reception to feeding dysfunctions after gastrectomy but can be restored .
it has been reported that the peripheral injection of ghrelin does not increase food intake after cutting vagus nerve [ 15 , 30 , 35 ] .
it is thought that peripheral ghrelin signal is transmitted to the feeding center at least partly via afferent vagal nerves [ 15 , 30 , 35 ] . in our study ,
it is speculated that regeneration of vagus nerves takes place not immediately but time - dependently , allowing reception of injected ghrelin and transmission of its signal to the feeding center .
in addition , chronic hypoghrelinemia in gastrectomized status was reported to induce hypersensitivity to ghrelin as judged by secretion of growth hormone .
if this is also the case for the orexigenic action of ghrelin , the hypersensitivity to ghrelin could make the ghrelin injection more potent and/or efficacious in correcting anorexia after gastrectomy . collectively , in the certain period
when vagus nerves are regenerated and sensitivity to ghrelin is elevated , ghrelin treatment is expected to effectively stimulate feeding . in fact , the fractional increase of food intake in response to ghrelin injection at 12th postoperative week was similar to that in control rats ( figures 5(e ) versus 5(a ) ) .
thus , our results raise a possibility that ghrelin replacement therapy given in later stages could be effective in promoting food intake and correcting some symptoms associated with gastrectomy in humans .
an effective treatment of severe obesity is bariatric surgery , in which gastric bypass is a representative method .
gastric bypass , as well as gastrectomy , markedly reduces appetite and body weight , suggesting that changes in plasma ghrelin levels are involved .
however , studies on changes in plasma ghrelin levels after gastric bypass have yielded inconsistent results ; decreased , unchanged , or increased [ 3840 ] . in our study , better restoration in feeding and body weight after gastrectomy was obtained with r - y than b - ii method , while plasma ghrelin levels were similar .
these observations by us and others collectively suggest that feeding and body weight in the earlier period after gastric surgery , are not associated with plasma ghrelin levels but determined by other factors , possibly reconstruction methods associated with specific histological conditions . in summary ,
recovery from gastrectomy - induced reductions in food intake and body weight is not related to plasma ghrelin levels but depends on the reconstruction method in rats . at postoperational 12th week , ghrelin production is increased in the duodenum and pancreas , and circulating ghrelin level is elevated by fasting , suggesting that ghrelin could be released from extra - stomach tissues and play a compensatory role .
ghrelin administration stimulates feeding at postoperational 12th week but not earlier , suggesting that ghrelin treatment in later periods could be effective in treating patients with gastrectomy - induced anorexia and associated symptoms . | gastrectomy reduces food intake and body weight ( bw ) hampering recovery of physical conditions .
it also reduces plasma levels of stomach - derived orexigenic ghrelin .
this study explored changes in orexigenic ghrelin system in rats receiving total gastrectomy with billroth ii ( b - ii ) or roux - en - y ( r - y ) method . feeding and bw were reduced by gastrectomy and subsequently recovered to a greater extent with r - y than b - ii while plasma ghrelin decreased similarly . at postoperative 12th week , ghrelin contents increased in the duodenum and pancreas , plasma ghrelin levels increased upon fasting , and ghrelin injection promoted feeding but not in earlier periods . in summary , gastrectomized rats partially recover feeding and bw , in a reconstruction - dependent manner . at 12th week
, ghrelin is upregulated in extra - stomach tissues , plasma ghrelin levels are physiologically regulated , and orexigenic effect of exogenous ghrelin is restored .
this time - related recovery of ghrelin system may provide a strategy for promoting feeding , bw , and thereby physical conditions in gastrectomized patients . |
hashimoto s encephalopathy ( he ) is an acute or subacute encephalopathy with elevated anti - thyroid antibodies in patients with hashimoto s thyroiditis . the age of onset varies , but most frequently occurs to people in their 40s .
its clinical features are as follows ; decreased cognitive function , abnormal behaviors , myoclonus gait apraxia language disorders , confusion and psychosis.1,2 the diagnosis should be considered in patients with encephalopathy with elevated thyroid autoantibody level and without any other etiologies .
it is known to show a good response to steroids and other immune therapies.2,3 the cognitive dysfunction of he is often observed and it usually has progressive course . if cognitive dysfunction lasts for a prolonged period of time , it is related with poor response to treatment .
it is very rare that it occurs all of a sudden.4 we report a case of he presenting acutely developed cognitive decline and convulsion .
a 65-year - old man visited the hospital due to a sudden onset of cognitive decline .
two weeks prior to visiting to the hospital , the patient caught a cold for one week .
he could nt remember where the razor was and was unable to find the bathroom .
he confirmed that he indeed brushed his teeth when asked in case certainly he did nt brush his teeth and did nt use toothpaste when brushing his teeth . at work , he was unable to find the office keys nor able to recognize his co - workers . as well , he was unable to recall the road he routinely walked along , where he parked his car and if he owned a car
. he did nt give the hospital staff proper attitude while saying there was something when he saw the electric light turned on .
besides , he showed the nervousness as serious as being restless and after that he presented urinary incontinence .
he collapsed turning his body around to the left as neurological examination were performed and lost consciousness with the convulsion to move his head to the right for about 20 seconds when he visited the hospital .
when he visited to the hospital , his vital signs showed a blood pressure of 135/80 mmhg , respiration rate of 22 times per minute , pulse of 80 times per minute and a body temperature of 36.5c .
his physical check - up showed no hair loss , edema , goiter , and etc .
korean version of mini - mental state examination ( k - mmse ) showed a 2/5 point in time orientation , 4/5 in place orientation , 0/3 in the three word recall and 4/5 point in serial seven substraction test .
there were no abnormal results among complete blood count , serum chemistry and electrolytes , serum tumor markers , paraneoplastic syndrome antibodies , and autoimmune disease and connective tissue disease test .
thyroid function test confirmed the asymptomatic hypothyroidism ; the level of triiodothyronine was 62.98 ng / dl ( reference value : 65150 ) , that of thyroxine 0.88 ng / dl ( reference value : 0.781.54 ) , that of thyroid stimulating hormone 5.85 iu / ml ( reference value : 0.554.78 iu / ml ) .
the level of thyrotropin - releasing hormone was normal . according to the thyroid autoantibody tests , the level of anti - thyroglobulin antibody was 77 iu / ml ( reference value : 060 ) and that of anti - thyroid microsomal antibody was > 1,300 iu / ml ( reference value : 060 ) .
the cerebrospinal fluid ( csf ) showed a slightly increased level of protein ( 58 mg / dl ) and the normal level of white blood cells .
and there were no abnormalities in virus including herpes simplex , bacteria and mycobacterium tuberculosis tests .
seoul neuropsychological screening battery ( snsb ) carried out 4 days after he was hospitalized revealed a decrease in language and visual memory and language fluency , but there were no abnormalities in the concentration and time and place orientation .
2 ) . single photon emission computed tomography ( spect ) showed decreased in perfusion in the bilateral temporal lobes ( fig .
3 ) . ultrasonography demonstrated an increased vascularity and irregular echoing in the bilateral thyroid lobes .
it was diagnosed as hashimoto s encephalopathy based on the clinical features and test results .
after oral steroids ( prednisolone 60 mg / day ) and anticonvulsant ( levetiracetam 1,000 mg / day ) administration , he showed a gradual recovery of cognitive function . therefore , on the 14th day after he was hospitalized , the score of k - mmse became normal to 30/30 .
since then , the patient s cognitive dysfunction gradually improved and showed no suffering from any convulsions .
we presented a patient with hashimoto s encephalopathy who had a sudden cognitive deterioration and convulsion .
the sudden decline of cognitive function is a rare clinical feature and it is unusual for the he to affect the old man .
the common cold he caught before the onset of the disease is considered to affect the symptoms beginning .
its pathophysiological mechanism is not clearly known , but the factors considered to cause it are as follows ; vasculitis by autoimmune mechanism , antineuronal antibody mediated reaction , and the direct toxicity of thyroid stimulating hormone releasing hormone to the central nervous system.2,3 the elevated serum level of anti - thyroid antibody is essential in diagnosing he and it means the autoimmune mechanism of the thyroid gland.2 he may not be related to the functional status of thyroid gland and it can appear in various ways .
it shows asymptomatic hypothyroidism 2335% , hypothyroidism 1720% , hyperthyroidism 7% and normal thyroid 1845%.2 it often shows the normal thyroid function , a little decreased function of thyroid gland and a high level of thyroid gland autoantibody.2,3 we presented a case with asymptomatic hypothyroidism and the high titer of autoantibody .
clinical manifestations of he are various and non - specific , but it is generally classified into two types .
one consists of about 25% presenting focal neurological deficits repeatedly such as stroke and the other consists of about 75% showing diffuse progressive course such as dementia , confusion , and hallucination.1,2,5 two clinical manifestations can occur simultaneously in the course of he.5 here , it occurred in the way that convulsion is added while the sudden decline of cognitive function is the main symptom .
it is considered to happen in the way the focal neurological abnormality is combined to the diffuse clinical pattern of the disease .
the two thirds of patients with he may have focal or generalized seizure and the generalized one is more common.1,2 it rarely happens to have status epilepticus.2,6 the eeg characteristics are various and mostly show non - specific features.1,2 the slow wave is the most common and appears generally or restricted to forehead or temporal area .
focal spike , sharp wave and transient epileptic discharges were rare.1,6 these eegs are related to the severity of the disease and may show improvement according to the response to medical treatment.6 the cognitive decline is often observed in he and it has relatively gradual clinical course .
the neuropsychological studies which were carried out before and after treatment are rare . as the cognitive decline starts , all domains of cognition may be affected . with progression
, there are high probability of decreased executive function and procedural memory.7 with the worsening of cognition , frontal lobe dysfunction become more common.7 compared with mild alzheimer s disease , he shows impairment in episodic memory concentration , visuospatial function , and executive function but showed normal in naming test.8 in this patient , there were some problems in orientation , recall , concentration , and calculation . on the day
and in the snsb carried out 4 days after visiting a hospital , it showed mainly the impairment in memory and partial frontal lobe dysfunction .
brain imaging studies frequently reveal non - specific findings.4,9 the brain mri is sensitive to find the lesion and it is likely that signal changes can appear in subcortex , basal ganglia , cerebellum , and medial temporal lobe.1,2,4,9 spect may show focal , multifocal , or global hypoperfusion.10 the brain imaging may show reversible improvement after treatment for he.9,10 this case handles he with sudden onset of cognitive deterioration and convulsion , which abruptly appeared after the patient caught a common cold without the clinical evidences of hypothyroidism . when the sudden cognitive function decrease or convulsion appears , he should be considered regardless of age and gender . | hashimoto s encephalopathy is an immune - mediated disorder characterized by acute or subacute encephalopathy related to increased anti - thyroid antibodies .
clinical manifestations of hashimoto s encephalopathy may include stroke - like episodes , altered consciousness , psychosis , myoclonus , abnormal movements , seizures , and cognitive dysfunction .
acute cognitive dysfunction with convulsion as initial clinical manifestations of hashimoto s encephalopathy is very rare .
we report a 65-year - old man who developed acute onset of cognitive decline and convulsion due to hashimoto s encephalopathy . |
regaining balance ability is a fundamental component of the clinician s goals for patients
with a variety of disorders1 , 2 .
although controlling distal segments such as the ankle ,
knee , and hip is known to be a major component of balance ability , co - contraction of the
abdominal and paraspinal muscles also contributes to maintaining the center of mass within
the base of support1,2,3,4 .
therefore , facilitating trunk musculature is one of the exercise
assessments for enhancing balance ability3 , 4 .
previous findings have suggested that impairments in postural control during walking are a
major risk factors for falling5 , 6 .
kim et al . also suggested that spine stabilization exercise
protocols could be helpful for improving overall postural stability in a standing
posture6 .
although various trunk
stabilization exercises have been suggested in the clinical literature and studies , however ,
there still remains a question regarding which exercise form is effective for enhancing
balance ability through trunk exercises7 , 8 .
proprioceptive neuromuscular facilitation ( pnf ) interventions classically suggest two
stabilizing exercise techniques , which are referred to as stabilizing reversal ( sr ) and
rhythmic stabilization ( rs)9 .
sr is
characterized by alternating isotonic contractions opposed by resistance to prevent motion ,
which allowing small movements .
rs is characterized by isometric contractions against the
resistance provided by the therapist , with the subject trying to maintain their position as
the therapist changes the direction of resistance9 .
the primary purpose of the present study was to investigate whether the sr and rs
techniques are effective for enhancing balance ability .
the secondary purpose was to
determine which exercise is effective for improving trunk stability .
thirty - four asymptomatic subjects were recruited from a local university . they were 20 to
26 ( 21.9 2.8 ) years old ,
their mean height and weight were 174.9 5.6 and 66.5 5.3 kg
respectively .
participants were excluded if they had any history of musculoskeletal problems
within the last 6 months .
this study was approved by the choonhae university faculty of health sciences human
ethics committee .
the center of pressure ( cop ) data for under the feet were collected by
using a pedoscan ( international gmbh , schlangenbad , germany ) . for the pre - exercise balance
test
, each subject stood on the pedoscan plate under the following conditions : double - leg
stance with the eyes open ( dls - o ) , double - leg stance with the eyes closed ( dls - c ) , one - leg
stance with the eyes open ( sls - o ) , and one - leg stance with the eyes closed ( sls - c ) .
measurement was performed under each balance test condition during an 11 s period , and the
data for the first and last three second seconds of the period were eliminated .
the primary
researcher performed stabilizing reversal and rhythmic stabilization for each subject to
facilitate trunk musculature in a randomized order .
the period was 3 min for each exercise ,
and the period between exercise conditions was one day .
immediately after the sr and rs
exercise , the cop was measured again under the during four balance test conditions using the
pedoscan .
significance was accepted for values of p<0.05 , and pasw statistics
18.0 was used for statistical analyses .
descriptive statics of the balance test and total cop movement are represented in table 1table 1.the average and standard deviation ( sd ) of total anterior - posterior center of
pressure ( a - p cop ) movement values before and after stabilizing reversal ( sr ) , and
post rhythmic stabilization exercisestrialtotal a - p cop movement ( mean sd , mm)dls with eyes opendls with eyes closedsls with eyes opensls with eyes closedbefore exercise ( mm)43.4 17.253.9 22.692.8 49.6158.9 58.7*after sr ( mm)44.5 13.757.0 31.981.4 27.9132.6 48.9*after rs ( mm)38.2 9.645.4 17.876.9 29.8149.7 53.1*dls : double - leg stance , sls : one - leg stance .
* significant difference between
conditions .. there were significant difference in movement of cop when compared according
to both exercise and balance test conditions ( p<0.05 ) .
subsequent post hoc testing
revealed that rs significantly reduced total cop movement compared with the pre - exercise
values ( p<0.05 ) . in factor of the balance test ,
the total cop displacements were
gradually altered in serial conditions of double leg stance with eyes - open , double - leg
stance of eyes - closed , one - leg stance with eyes - open , one - leg stance with eyes - closed
( p>0.05 ) .
evaluation of balance ability in a standing posture is clinically well evidenced , and this
includes evaluation under one - leg and double - leg stance and presence of vision1 , 6 , 8 .
our data suggested that the double - leg
stance with eyes open and double - leg stance with eyes closed , one - leg stance with eyes open
and one - leg stance with eyes closed were well differentiated between conditions according to
difficulty of conditions .
mitchell et al . reported that the presence of vision critically
affects standing balance10 , but the
present results indicated that a narrow base of support has a major effect on the balance
ability of young subjects .
rs significantly reduced anterior - posterior cop displacement , compared with the values
after sr and those before exercise .
some previous studies have reported that rs increased
co - contraction , which is necessary for enhancing upright trunk posture11 . theoretically , balance is defined as the ability to
maintain the center of mass ( com ) within the base of support12 .
furthermore , the com is positioned approximately anterior to the
sacrum in a standing position12 .
therefore , co - contraction of the trunk muscle could reduce com displacement through the rs
technique .
although the results of the present study showed significant decreases in cop displacement
with the rs technique , the present study could not affirm that sr is less useful than rs . in
elderly subjects or patients with insufficient muscle strength , sr , which induces
the
present study suggests that standing balance could be improved with the rhythmic
stabilization technique in asymptomatic subjects .
and balance tests with variations of
vision and support surface could be well differentiated and could be applied for stepwise
assessment . | [ purpose ] the purpose of this study was to evaluate the immediate effect of stabilizing
exercise using the pnf technique on standing balance in one - leg and double - leg stances .
[ subjects and methods ] the present study recruited 34 healthy participants from a local
university .
the participants performed four balance tests ( double - leg stance with and
without vision , one - leg stance with and without vision ) , before and after exercise .
the
exercise consisted of exercises performed using pnf techniques ( stabilizing reversal and
rhythmic stabilization ) , which were applied to facilitate trunk musculature . to examine
balance ability , total displacement of the center of pressure
was measured during balance
tests .
[ results ] the total anterior posterior center of pressure displacement was
significantly reduced after applying rhythmic stabilization compared before exercise
regardless of the balance test conditions .
[ conclusion ] the present results suggest that
trunk stability exercise using rhythmic stabilization could effectively enhance balance
ability under one - leg and double - leg conditions . |
in recent upsurge of interest for an adequate scientific knowledge about human development in the arctic , an academic division of labour , the legacy of cartesian dualism , has left the material body to the natural and medical sciences , with social sciences and humanities for the most part occupied with intersections of individual and collective , autonomy and agency , culture and the social ( 8) . relevant literature addressing climate impacts on human health is dominated by natural and medical sciences .
the studies of climate change impacts on human health , as a consequence of the division of labour , have often been split into a number of areas .
there are those whose concern is to point out the possible direct and indirect consequences of climate and environment change for the health of population in the north , e.g. the impacts of various forms of contamination , thermal stress , ultraviolet radiation in the arctic ; the effects of environmental changes on wildlife and their impacts on the food chain , nutrition and dietary patterns ; climate change effects on local environment and their subsequent impact on the traditional lifestyles and human health in the arctic ( 917 ) .
this has become a very powerful trend , which tries to establish links between climate and environment change and human health in the arctic .
others focus on health vulnerabilities of women and men to the effects of climate change .
the physical condition of both women and men , especially of women , is connected to the health of their children and , consequently , to the health of their communities ( 7 , 18 ) .
some studies have proven links between climate change , changing living - conditions and negative health outcomes among arctic population , especially indigenous peoples , also indicating differences between men and women ( 1925 ) .
climate change - driven transformations of living conditions and the disruption of traditional lifestyles contribute to mental and social stress associated with the loss of community and culture .
indirect effects of climate change include changes in the environment and potential changes in bacterial and viral diseases as well as access to quality water and food sources ( 15 , 17 )
. some studies also show associations between mental and social stress , violence or sexual abuse and current health problems , which are more common in women and children ( 7 , 9 ) .
evidence - based research provides data showing that ( a ) the effects of climate change are not gender neutral and ( b ) direct and indirect impacts of climate change and health risks vary for women and men ( 26 ) .
globally , women and men face different vulnerabilities due to their different gender roles ( 2628 ) .
women form a disproportionately large share of the poor all over the world and especially in developing countries . in southern countries , women are more dependent than men ( e.g. in agriculture ) on primary resources that are threatened by climate change because of their responsibility to secure water , food and energy for cooking and heating ( 27 , 28 ) . as a result ,
women more often come in contact with poor - quality water and are more vulnerable to water - related diseases ; women also bear the main burden of caring for those who are ill ( 26 ) .
women are vulnerable to extreme weather effects in particular ways before , during and after the extreme event : women 's traditional roles ( looking after children and elderly ) and cultural restrictions may hamper their self - rescue efforts ( 28 ) . women 's mortality , especially of elderly women , related to heat waves is higher than the mortality of men ( 27 ) .
being male or female has an impact on individual health since the natural course of a disease may be different for women and men ; individuals may respond differently to illness and society may react differently to sick women and men ( 26 ) .
women may have less access to vital information on mitigation or adaptation strategies because of time constraints due to the caring and other domestic responsibilities .
still preet et al find it as a general tendency that gender perspective is hardly represented in the research and policies on climate change and health ( 5 ) .
anyway , the point here is not to claim that gender perspectives remain of marginal interest within the climate change studies or in the arctic research on climate change and human health .
rather , the point is to state that we still continue to observe a disciplinary division of labour .
when present in studies , gender is often used as one of the variables in making the statistical data rich . very often gender paragraph
of course , gender is but one , albeit important , perspective alongside with other perspectives that are important in climate change studies .
still gender is hardly ever a keyword introducing the articles contents in the scientific journals , such as the international journal of circumpolar health , even when there are paragraphs or sections on gender there . and
the problem here is that medical and natural science papers often operate with the category of
gender implying women and men as sexes , in a purely demographic sense ( see e.g. 2931 ) . within social sciences , and gender studies in particular , the category of the category of gender has connotations of power misbalances between and within men and women and consequent differences in their social roles and positions .
gender approach towards climate change impacts on human health would imply exploring , e.g. how gender power relations affect and are involved into bigger contexts of climate and environment changes impacts on human health ; what dispositions are available to men and women ; which adaptation , resilience strategies are at the disposal of women and men ; how health risks , health rights and health security are perceived by women and men and , in turn , how their awareness affects their situation and agency . in no way
the message here is that qualitative studies that operate with gender / sex as a variable have little value and significance or that the discussions on power should be a part of each paper mentioning women and men .
the criticism here is mainly addressed to the traditionalism of the approaches in knowledge production and the terminology confusion that arises from discipline segregation .
human development is complex ( 2 , 32 ) and in order to grasp this complexity , the diversity of populations and their lifestyles , health risks and adaptation strategies we need to develop more sophisticated tools via multi / transdisciplinary research . yet
the blame can not be entirely laid on climate change and health sciences , as i would further show , social sciences and humanities likewise show little interest in studying intersections of climate change impacts , human health and gender .
within social sciences and humanities it is acknowledged that climate change has an impact on well - being of people in local communities ( 1 , 2 , 32 , 33 ) .
indigenous people , elderly , women and children are considered particularly vulnerable groups in arctic communities ( 4 , 34 ) . by focusing on cultural and social aspects of knowledge production in / on the arctic ,
a number of researchers point to the fact that the arctic has an image of a male world ( 6 , 35 , 36 ) or , what is more , of masculinity and whiteness ( 37 , 38 ) .
first , arctic exploration was predominantly male - driven : expeditions ; search of natural resources ; search for the northwest passages ; fur - trading , whaling , hunting , mining and military activities were all male - centred activities .
second , most literature written on peoples of the arctic was written by men ( mostly white ) and mostly about men , transmitting male values ( 35 ) . the scientific language employed in the arctic research
women 's contribution to local communities survival , the decision - making , hunting / fishing economy and spirituality of relations has been underestimated ( 3941 ) .
in russian pomor traditional culture , in canada and greenland , we find evidence that women played a key role in household , family and local community life ; women were the ones who distributed food , organised everyday routines , hold families together and raised strong leaders for their communities ( 1 , 2 ) .
as some scholars have mentioned , among the indigenous populations , the arctic was home for at least as many women as men .
provide examples when inuit women have dominant economic and governmental positions in their communities , territories and global political forums , and yet social sciences focus on observations by male inuit hunters ( 6 ) .
some scholars suggest that climate change affects women harder in the south , while in the north it is men who experience the effects of climate and environment change more dramatically ( 42 ) .
the disruption of traditional roles for men has been identified in a number of studies as a reason for profound problems in male identity and loss of men 's self - esteem which , in turn , leads to a lot of psycho - social disorders among men , including higher suicide rates and alcoholism ( 1 , 34 , 43 ) .
also , men 's loss of identity and self - worth , societal tension and issues of power and control have been identified in some studies as contributing factors to increased violence against women and children , increased human trafficking and prostitution ( 1 , 2 , 43 ) .
another effect of climate and environment change on well - being of people in arctic communities observed is a pattern of out - migration by young adult females in a number of northern regions , including alaska , greenland , the faroe islands , iceland , norway , newfoundland and russia ( 33 , 34 , 39 , 44 ) .
researchers point to education , marrying outsider men , access to services , including health care services , employment opportunities and search for security as the major reasons affecting women 's decision to move from small rural places and communities ( 2 ) . following consequences of female out - migration
were highlighted in the studies : the lack of reproduction in the communities contributes to their stagnation .
development of homosocial bachelor cultures as a result of men 's need for sustaining certain aspects of masculine identity is another feature ( 45 ) .
ageing of population and increased poverty are also possible consequences ( 33 ) . in many parts of the arctic , economic cutbacks by national governments have often negative impact on small , rural and remote places , reducing the standard of living and the quality of life in these areas through limited employment opportunities , low wages and poor infrastructure and social services .
according to hoogensen , this leads to increased feeling of insecurity among women for their own future and the future of their children in the current place of residence and becomes one of the factors forcing women to migrate ( 45 ) .
thus , what social sciences and humanities mainly contribute with is proving to the fact that the picture of human development in the arctic generally and the studies on climate and environment impacts on human health is to be understood in the light of gender approach .
a gender approach in this respect contributes to knowledge building essential for human development of the arctic ( 2 ) .
what is more inspired by the traditions of post - structuralism and post - colonialism , social sciences and humanities turned their attention to the complexity of phenomena as well as concepts , representations and interpretations used to describe them . to grasp the complexity of human development in the artic , analysis of intersections of gender , ethnicity and of various processes that create differences between people in the arctic brings very important perspectives into both research and policy agenda .
though the research addressing intersections of human health , climate change and gender is indeed scarce , there are some multi / transdisciplinary studies where these intersections are explored .
one can mention , for example , the research carried out by joanna kafarowski on gendered dimensions of environmental health and contaminants in nunavik in canada ( 3 , 46 , 47 ) .
kafarowski studies the differences between women 's and men 's perceptions of contaminants threat to human health and shows that men and women not only have different perceptions of health risks but also develop different adaptation strategies .
she concludes that both gender and ethnicity affect people 's visions and are important for developing efficient environmental health policies and strategies in response to the contaminants in small native communities .
another example is sandra owens research on inuit indigenous women in nain , canada , and the ways indigenous people experience climate change through their daily activities . in her study
, she particularly focuses on how indigenous women experience changes in the dietary patterns as a constantly decreasing access to traditional foods as healthy and nutritious ones ( 48 ) .
women 's access to decision - making processes is seen as crucial when encompassing reproductive health , sexual health and environmental justice ( 46 , 49 ) .
some researchers suggest , even though women develop original solutions to complex environmental issues in the arctic , they , nevertheless , mostly advance the agenda at the grassroot level , within social and environmental activism ( 2 , 6 ) . beyond the grassroots level , women are less visible in environmental politics .
men tend to assume positions of responsibility and power in the public sphere and , therefore , it is likely that environmental decision making itself is deeply gendered .
the contributions of women are valued less than the contributions of men in western , non - indigenous societies and women are less likely to attain decision - making positions ( 39 , 43 , 44 , 49 ) . what is of particular value with these studies is that they do their research at intersections of gender , ethnicity and the variety of processes that create differences in peoples health rights and climate justice in the artcic .
the only limitation is that the studies linking climate and health with gender and ethnicity , to our knowledge , mostly have been carried out in alaska and arctic canada .
it affects people and their environment with potential consequences for communities and individuals psychosocial well - being and health ( 1517 ) . at the same time
, any discussion of power relations and gender roles must also recognize the social and cultural diversity across the circumpolar north and the fact that many different perspectives can be applied when analyzing these roles ( 2:187 ) .
vulnerability of communities and individuals will vary depending on differences in climate alterations , distances , infrastructure , resources , etc .
climate change impacts on human health also vary between and within communities in the arctic , and the examinations of gender relations illustrate the complexity of communities in the differences of perspectives that abound within and between them .
various academic disciplines within arctic research provide data showing that ( a ) climate change effects are not gender neutral and ( b ) direct and indirect effects of climate change and health risks vary for women and men .
gender perspectives on climate change and health is formally recognised as an important field of research .
the analysis , undertaken in this article , illustrates that gender issues remain of marginal interest for environmental , climate change and public health studies in the arctic . yet
the blame can not be completely laid on climate change and health sciences as social sciences and humanities have likewise often ignored gendered dimensions of climate change impacts on human health . or ,
rather , gender studies show little interest to climate change and the arctic population health issues . as a result of the division of labour between disciplines , little dialogue is established cross - disciplinary , which results in narrow use of concepts and the disciplinary - bound pictures of human development in the arctic .
the arctic population health is an important dimension and an indicator of monitoring human development .
there are different starting points for investigation of peoples lives and climate change effects on their health .
awareness of differentiated climate and environment effects on health of women and men has to be incorporated in knowledge production in order to understand the processes taking place in the arctic human development and to plan the future strategies .
attempts to implement a gender perspective will be successful only when different disciplines work together in multi / transdisciplinary research .
transdisciplinarity is a way to challenge academic / disciplinary homogeneity and their boundaries , to take advantage of the diversity of approaches and methods in the production of new integrated knowledge .
it is also a way to escape reproduction of trivial approaches towards complex phenomena and to develop new indicators for monitoring human health and elaborating effective and adequate policies and strategies to the benefit of both women and men in the arctic .
the authors have not received any funding or benefits from industry or elsewhere to conduct this study . | backgroundclimate change and environmental pollution have become pressing concerns for the peoples in the arctic region .
some researchers link climate change , transformations of living conditions and human health .
a number of studies have also provided data on differentiating effects of climate change on women 's and men 's well - being and health.objectiveto show how the issues of climate and environment change , human health and gender are addressed in current research in the arctic .
the main purpose of this article is not to give a full review but to draw attention to the gaps in knowledge and challenges in the arctic research trends on climate change , human health and gender.methodsa broad literature search was undertaken using a variety of sources from natural , medical , social science and humanities .
the focus was on the keywords.resultsdespite the evidence provided by many researchers on differentiating effects of climate change on well - being and health of women and men , gender perspective remains of marginal interest in climate change , environmental and health studies .
at the same time , social sciences and humanities , and gender studies in particular , show little interest towards climate change impacts on human health in the arctic . as a result
, we still observe the division of labour between disciplines , the disciplinary - bound pictures of human development in the arctic and terminology confusion.conclusionefforts to bring in a gender perspective in the arctic research will be successful only when different disciplines would work together .
multidisciplinary research is a way to challenge academic / disciplinary homogeneity and their boundaries , to take advantage of the diversity of approaches and methods in production of new integrated knowledge .
cooperation and dialogue across disciplines will help to develop adequate indicators for monitoring human health and elaborating efficient policies and strategies to the benefit of both women and men in the arctic . |
there is evidence that the supportive relationship in education between student and teacher can be as a basis for student 's health promotion .
nursing students start clinical training from the second semester and this is run concurrently with theoretical courses until the end of the 3 year .
they learn in the clinical environment under the direct guidance and supervision of a male or female nurse teacher who had master degree in nursing for the first 3 years . in the final year
many factors such as the number of students in training groups , long time clinical experiences and hard work may affect teaching and learning situations and their quality .
this may distinguish clinical situation from other educational situations . among the other influential factors are student and teacher characteristics , learning experience of theoretical courses and relationship in clinical setting .
indeed , clinical education is a face - to - face education in which the quality of relationship plays a key role in its promotion .
this relationship is valuable in clinical nursing education and should be facilitated in various ways .
considering the fact that teaching and learning are mutual interactive processes between students and teachers , these processes will be certainly facilitated via supportive approaches .
in fact , the type of relationships can help make a qualified clinical placement scenario .
as much as the teachers have more support in their relationship with students , students are more convienent in the learning settings and they have more involvement in learning activities . also ,
the students who experience supportive relationships from their teachers , exppress high quality of recieved education . despite the importance of student - teacher relationship and its salient effects on self confidence , learning motivation and promotion of professional skills in students , positive learning experiences , decrease in students fear and anxiety , reduction of the possibility of educational failure , promotion of professional identity and reception of more support during care provision by students ,
webb and shakespeare ( 2008 ) cited bennett ( 2002 ) who found that students have potentially damaging experiences such as being ignored and unattended by clinical educators .
the findings of savage 's and favret study ( 2006 ) are indicative of students experiences of their educators insulting them in front of others . in iran
students points of view regarding effective factors in establishing communication between students and faculty members was accomplished .
now , student - teacher relationship with regard to the paradigm shift in nursing education and its emphasis on centrality of relationship has been changed from what it has been in the past .
the exploration of this relationship and its dimensions in humanistic paradigm is inevitably necessary . considering the dearth of studies about supportive relationship between student and teacher in clinical nursing education in iran , as well as the different experiences and perceptions of nursing students and teachers according to the iranian culture
the purpose of the study is to explore the experiences of nursing students and teachers about student - teacher relationship in clinical nursing education .
this paper presents part of a larger study that served as the corresponding author 's doctoral dissertation .
the larger study focused on the process of student - teacher relationship in clinical nursing education in iran . to achieve the aim of the study , conventional content analysis approach was used .
participants included six bachelor nursing students and six clinical educators in school of nursing and midwifery who were selected through purposive sampling in 2010 - 2011 [ table 1 ] .
summary of participants profile data were collected through semi - structured interviews and participant observation .
based on the participants choice , interviews were conducted either in classroom or teachers office .
each student or teacher participated in a semi - structured interview which lasted approximately 45 - 90 min . at first
, some questions were asked about the experiences of student - teacher relationship as an introduction and then the interview process was guided through the participant 's answer .
unstructured observation ( observer as participant ) was carried out in the cardiac and surgery wards in imam reza ( al ) hospital . because the primary reason for using observational methods is to verify whether what people say is the same as what they actually do , it is ideal that interviews and observations are conducted for the same people .
thus in this study , in some cases that it was possible , observation and interview were done as complementary data for the same participants and in other cases were carried out separately from each other .
the role of the researcher was watching what participants did , listening to what they said and recording the way they acted .
the behaviours of students and teachers and their relationship were observed for 3 - 4 h in each day .
the study was designed to minimize observer effect , as the researcher 's presence could affect teachers and students behaviours . to minimize this effect , it was tried to give a general awareness of research aim to the participants , whereas they did not know exactly about the time of observation and the way that they were observed .
collected data from observation of participants whom were both interviewed and observed were analysed as complementary for interviews data with regard to this point that do they confirm each other ? however , in remained cases they were analysed as separated data .
this approach is usually appropriate when existing theory or research literature on a phenomenon is limited .
researchers used inductive category development , i.e. , avoided using preconceived categories , instead allowed the categories flow from the data .
researchers also immersed themselves in the data to allow new insights to emerge . according to content analysis process , at first each interview or field note
was read again and again carefully in order to gain a universal and primary understanding of the important under - lined statements .
then , meaning units about participants experiences of relationship existent in the interview text were determined . in the next phase , the meaning units were abstracted through condensation and were labelled as codes .
codes were compared for similarities and differences within the same interview and in different interviews and compared with field notes , and then categorization of codes was done accordingly . in the next stage ,
categories and sub - categories were examined under supervision of expert supervisors who were experienced in qualitative analysis .
credibility was established through prolonged engagement with participants , triangulation of methods and data sources , member checking , peer debriefing and review of data analysis with supervisors .
for dependability , renewed coding of the interviews was carried out by colleagues who had experience in coding qualitative data . moreover
, the researchers documented research details in order to provide the possibility of external review .
this research was carried out after getting ethical approval from research ethics committee , university of medical sciences and with the permission and consent of the dean of nursing and midwifery school .
informed consent was obtained from all participants and they were ensured of their anonymity and were allowed to repudiate to answer any question or withdraw from the study at any time without prejudice to their clinical education .
three major categories emerged based on data analysis . those were including : ( 1 ) educational support , ( 2 ) emotional support , and ( 3 ) social support . based on the participants experiences in this study , educational support was one of the most important aspects in the context of student - teacher relationship .
they believed that the importance is such that the relationship in clinical education becomes meaningless without supporting the student . educational support in this relationship was accessible through two methods including getting support for the accomplishment of clinical skills and also through creating learning situations .
receiving support for doing clinical skills seemed very essential in relationship in clinical education from the experiences of both students and teachers , because they experienced that supporting students in doing clinical skills is a major factor in a successful clinical learning .
nursing students commented that they need to presence of clinical educator in a supportive atmosphere in clinical environments for better learning of skills .
they experienced that if this support is available , the students will have enough self - confidence for skill accomplishment . at the same time , student 's ability to carry out the skills correctly will be increased and clinical learning goals will be achieved .
if we do an iv catheter insertion or some skills which we are not able to do alone , the teacher comes and helps us .
a accompanies me during the insertion of iv catheter or blood taking , because her speech and behaviour increases my self - confidence ( s1 ) .
one major aspect of supportive relationship was accompanying students in clinical setting to perform the skills until the procedure is completed .
this issue was one of the main requests of students in establishing relationship with clinical teachers .
nursing students expected their teachers to be supportive and in occurrence of a problem in clinical setting , they attend and support students and do nt leave them alone .
they pointed out if the teacher does not accompany them in doing clinical skill , they will receive assistance and support from their peers and/or ward staff .
one of them commented : in the apprenticeship that we had the previous semester , we did nt know how to do the uterine massage .
so on the 1 day , i asked my teacher several times to train me in this relation , but i eventually had no chance to be trained by my teacher until the end of the clinical course .
so , i asked the other students and staff to help me with that . ( s6 ) .
clinical teachers in this study experienced that educational support in their relationship with students can be provided through creating good learning and teaching situations in clinical setting as well as establishing supportive relationship with students through having suitable clinical planning with the aim of decreasing the theory - practice gap and maximizing the usage of educational situations for students learning .
spending time with students and helping them to use of optimal learning situations was salient aspect of these supportive actions that led to more trust to teacher in mutual relationship .
i try to select the best cases for my students in the ward , so that they ll have the opportunity to see various types of disease in practice . in this way , they trust me , because they know that i do my best to support them , and they do nt miss the practical situations in the clinical settings ( t3 ) .
students were also aware of the support of their teachers for helping them to learn better and this caused a pleasant experience of their relationship in clinical education .
one of them commented : when our teacher supports us in learning situations , we trust him / her in our relationship . for example
one of our teachers in cardiac ward tried to teach us the procedures according to the text books and supported us not to practice just routinely ; you know , so we experienced a good relationship with her based on these supporting behaviors
this aspect of supportive relationship was carried out through various methods including calming down students , helping them to verbalize and correct the mistakes .
teachers experienced that support of students in student - teacher relationship could be occurred by calming them down in stressful situations .
they believed that giving confidence and calmness to the students and decreasing their stress during performing the skills can be considered as a kind of support . in the circumstances that students show low self - confidence and endure much stress while carrying out clinical skills , calming down and reassuring them seems to be a salient issue . according to the clinical teachers in these cases
, they could provide suitable situations to increase students self - confidence which itself creates a sense of serenity and consequently improved performance .
when one of the students with low self - confidence performs a skill , her hands tremble ; i take her hand in my hands and tell her : now i take your hands , how do you feel ?
i do nt feel that you are standing over my head and i am reassured now .
i told the student to go to the patient 's bedside with confidence so that you demonstrate yourself as a skilled nurse ; i will give you hints and help you there .
another participant stated : in some cases that students are stressful or hesitant , i practice with them before starting the clinical work and ask them to do this work with my help , you know , i want to decrease their stress in patients bedside when they want to do the clinical skills
clinical teachers emphasized on helping students to verbalize their mistakes as a kind of support .
they experienced that teachers should accept that students may have committing error and mistake in doing clinical procedures .
so providing a warm atmosphere for students help them not to show fear or panic to verbalize the errors , as timely expression of mistakes can protect the patient 's safety .
additionally , the teacher can support students to truly verbalize their mistakes in the clinical settings through reassuring them about expression of mistake without any fear .
meanwhile , providing a ground for convenient and fearless expression of mistakes can cause a pleasant feeling in teacher .
i say to my students that when you make a mistake i wo nt punish you .
you should tell me immediately , because if i know what you did wrong , i may be able to resolve it to prevent deterioration of the patient 's conditions .
i m very glad when a student who has made a mistake tells it me without any fear
( t2 ) . according to data , providing emotional support through helping students to correct their mistakes was another issue that was highlighted by both groups of participants .
they experienced that the teacher should preferably manage the student 's mistakes so that there is neither a danger for patient nor the student to incur a lot of stress .
when a problem occurs , the teacher not only should remind the student about the mistake but also should help him / her to solve that problem . a teacher in the clinical setting having observed a student 's mistake
i always tell my students that you should be careful , but as soon as you did something wrong you must tell me about it , i try to resolve the problem .
i think in case i am too strict towards them , they may refrain from revealing their mistakes ( t2 ) .
one of the students commented if we do wrong in a procedure , some of the teachers do not tell us in presence of patient and do nt impair our personality , you know , they try to remind and advise us in another place regarding our wrong doing .
they say : look ; now i m going to do it again and you try to learn it .
another category in this study was social support in which the teacher tried to support the students in presence of patients or staff in essential cases .
clinical teachers experienced that to support the students socially they should not leave the students alone in difficult clinical situations .
students were also willing themselves to get more support in presence of patients or staff and assume that this is their right .
they experienced that through granting this support , proper learning situations are provided for them .
the reality is that in the majority of cases , nursing students and teachers were considered as foreign members of clinical wards , the existence of such support will create a sense of belonging to clinical environments and a feeling of having a supporter for students .
one of the teachers commented what the students mostly expect from the clinical teachers is their support in the clinical settings , because when we go to the clinical wards , we and our students are like foreign members or stranger , so i feel that i need to provide a situation in which my students can work with the staff ; for example if a problem occurs , i do nt give the priority to the staff and also do nt leave the students alone
( t5 ) . above participant commented most often , the staffs are trying to find faults with the students efforts .
students are the only ones who are usually blamed for any problem which occurs in the ward .
what the students care mostly about is that their teacher stands beside them and defends them in sticky situations .
there have been cases when such problems have occurred , and i have reprimanded the staff for that . ( t5 ) . moreover , the teacher 's powerful support in the presence of staff is also important from the viewpoint of students . students must be able to lean on the teacher as a strong supporter in clinical setting .
one of the students commented : this is very effective in establishing the relationship that the student can rely on the teacher . ( s2 ) . according to experiences of students , another type of support which can be provided by the clinical teachers is supporting them in the presence of patients .
they experienced that this support has a vital role in the prospective relationship between student and teacher and also the quality and depth of their relationship .
the degree of support in presence of patient or staff influence on the establishment of our relationship with the teacher , but when we observe that our teacher may despise a student in front of a patient in the clinical setting at any time , we try to avoid relationship with that teacher ( s3 ) .
based on the participants experiences in this study , educational support was one of the most important aspects in the context of student - teacher relationship .
they believed that the importance is such that the relationship in clinical education becomes meaningless without supporting the student . educational support in this relationship was accessible through two methods including getting support for the accomplishment of clinical skills and also through creating learning situations .
receiving support for doing clinical skills seemed very essential in relationship in clinical education from the experiences of both students and teachers , because they experienced that supporting students in doing clinical skills is a major factor in a successful clinical learning .
nursing students commented that they need to presence of clinical educator in a supportive atmosphere in clinical environments for better learning of skills .
they experienced that if this support is available , the students will have enough self - confidence for skill accomplishment . at the same time , student 's ability to carry out the skills correctly will be increased and clinical learning goals will be achieved .
if we do an iv catheter insertion or some skills which we are not able to do alone , the teacher comes and helps us .
a accompanies me during the insertion of iv catheter or blood taking , because her speech and behaviour increases my self - confidence ( s1 ) .
one major aspect of supportive relationship was accompanying students in clinical setting to perform the skills until the procedure is completed .
this issue was one of the main requests of students in establishing relationship with clinical teachers .
nursing students expected their teachers to be supportive and in occurrence of a problem in clinical setting , they attend and support students and do nt leave them alone .
they pointed out if the teacher does not accompany them in doing clinical skill , they will receive assistance and support from their peers and/or ward staff .
one of them commented : in the apprenticeship that we had the previous semester , we did nt know how to do the uterine massage .
so on the 1 day , i asked my teacher several times to train me in this relation , but i eventually had no chance to be trained by my teacher until the end of the clinical course .
so , i asked the other students and staff to help me with that . ( s6 ) .
clinical teachers in this study experienced that educational support in their relationship with students can be provided through creating good learning and teaching situations in clinical setting as well as establishing supportive relationship with students through having suitable clinical planning with the aim of decreasing the theory - practice gap and maximizing the usage of educational situations for students learning .
spending time with students and helping them to use of optimal learning situations was salient aspect of these supportive actions that led to more trust to teacher in mutual relationship .
i try to select the best cases for my students in the ward , so that they ll have the opportunity to see various types of disease in practice . in this way , they trust me , because they know that i do my best to support them , and they do nt miss the practical situations in the clinical settings ( t3 ) .
students were also aware of the support of their teachers for helping them to learn better and this caused a pleasant experience of their relationship in clinical education .
when our teacher supports us in learning situations , we trust him / her in our relationship . for example
one of our teachers in cardiac ward tried to teach us the procedures according to the text books and supported us not to practice just routinely ; you know , so we experienced a good relationship with her based on these supporting behaviors
this aspect of supportive relationship was carried out through various methods including calming down students , helping them to verbalize and correct the mistakes .
teachers experienced that support of students in student - teacher relationship could be occurred by calming them down in stressful situations .
they believed that giving confidence and calmness to the students and decreasing their stress during performing the skills can be considered as a kind of support . in the circumstances that students show low self - confidence and endure much stress while carrying out clinical skills , calming down and reassuring them seems to be a salient issue . according to the clinical teachers in these cases
, they could provide suitable situations to increase students self - confidence which itself creates a sense of serenity and consequently improved performance .
when one of the students with low self - confidence performs a skill , her hands tremble ; i take her hand in my hands and tell her : now i take your hands , how do you feel ?
i do nt feel that you are standing over my head and i am reassured now .
i told the student to go to the patient 's bedside with confidence so that you demonstrate yourself as a skilled nurse ; i will give you hints and help you there .
another participant stated : in some cases that students are stressful or hesitant , i practice with them before starting the clinical work and ask them to do this work with my help , you know , i want to decrease their stress in patients bedside when they want to do the clinical skills
clinical teachers emphasized on helping students to verbalize their mistakes as a kind of support .
they experienced that teachers should accept that students may have committing error and mistake in doing clinical procedures . so providing a warm atmosphere for students help them not to show fear or panic to verbalize the errors , as timely expression of mistakes can protect the patient 's safety .
additionally , the teacher can support students to truly verbalize their mistakes in the clinical settings through reassuring them about expression of mistake without any fear .
meanwhile , providing a ground for convenient and fearless expression of mistakes can cause a pleasant feeling in teacher .
i say to my students that when you make a mistake i wo nt punish you .
you should tell me immediately , because if i know what you did wrong , i may be able to resolve it to prevent deterioration of the patient 's conditions .
i m very glad when a student who has made a mistake tells it me without any fear
( t2 ) . according to data , providing emotional support through helping students to correct their mistakes was another issue that was highlighted by both groups of participants .
they experienced that the teacher should preferably manage the student 's mistakes so that there is neither a danger for patient nor the student to incur a lot of stress .
when a problem occurs , the teacher not only should remind the student about the mistake but also should help him / her to solve that problem . a teacher in the clinical setting having observed a student 's mistake
i always tell my students that you should be careful , but as soon as you did something wrong you must tell me about it , i try to resolve the problem .
i think in case i am too strict towards them , they may refrain from revealing their mistakes ( t2 ) .
one of the students commented if we do wrong in a procedure , some of the teachers do not tell us in presence of patient and do nt impair our personality , you know , they try to remind and advise us in another place regarding our wrong doing .
they say : look ; now i m going to do it again and you try to learn it .
another category in this study was social support in which the teacher tried to support the students in presence of patients or staff in essential cases .
clinical teachers experienced that to support the students socially they should not leave the students alone in difficult clinical situations .
students were also willing themselves to get more support in presence of patients or staff and assume that this is their right .
they experienced that through granting this support , proper learning situations are provided for them .
the reality is that in the majority of cases , nursing students and teachers were considered as foreign members of clinical wards , the existence of such support will create a sense of belonging to clinical environments and a feeling of having a supporter for students .
one of the teachers commented what the students mostly expect from the clinical teachers is their support in the clinical settings , because when we go to the clinical wards , we and our students are like foreign members or stranger , so i feel that i need to provide a situation in which my students can work with the staff ; for example if a problem occurs , i do nt give the priority to the staff and also do nt leave the students alone
( t5 ) . above participant commented most often , the staffs are trying to find faults with the students efforts .
students are the only ones who are usually blamed for any problem which occurs in the ward .
what the students care mostly about is that their teacher stands beside them and defends them in sticky situations .
there have been cases when such problems have occurred , and i have reprimanded the staff for that . ( t5 ) . moreover ,
the teacher 's powerful support in the presence of staff is also important from the viewpoint of students .
students must be able to lean on the teacher as a strong supporter in clinical setting .
one of the students commented : this is very effective in establishing the relationship that the student can rely on the teacher .
according to experiences of students , another type of support which can be provided by the clinical teachers is supporting them in the presence of patients .
they experienced that this support has a vital role in the prospective relationship between student and teacher and also the quality and depth of their relationship . the degree of support in presence of patient or staff influence on the establishment of our relationship with the teacher , but when we observe that our teacher may despise a student in front of a patient in the clinical setting at any time , we try to avoid relationship with that teacher ( s3 ) .
based on the study purpose that was to explore the experience of student - teacher relationship in clinical education in this study , findings revealed that participants experienced and described a kind of relationship in which the teachers were support providers and students were who received the support .
the most important finding in this study was the necessity of establishing supportive relationship through various routes in clinical nursing education . in some cases similar findings
have been reported in previous studies but we found some other findings which are more specific to iranian socio - cultural context of clinical settings .
three major categories that emerged from the data analysis in this study included : educational , emotional and social support . in aspect of educational support
, the findings of current study demonstrated the potential benefits that the supportive relationship can provide to achieve goals in clinical settings , increasing student 's self - confidence and existence more trust in their relationship .
these findings showed that in iranian socio - cultural context , educational support in relationship between student and teacher is a valuable and helpful issue in clinical settings . with respect to support for the accomplishment of clinical skills , according to quantitative study by craige ( 1991 ) , students stated that they learn better and more in a supportive environment and they will have more confidence in their ability to do the clinical practice .
crick and mccombs ( 2004 ) stated that : if supportive relationship is established with learners , they will learn more and better . in craige study ,
when students receive no support from teachers , they have to depend on each other for support that the finding of current study is congruent with this study . although campbell et al .
( 1994 ) found that peer support facilitate learning through the sharing of knowledge and experience and support in the completion of tasks , but the request of help from teacher can strengthen the relationship between them .
also , in croxon 's and maginnis study ( 2008 ) , the most important aspect of students experiences was receiving support from teachers in relationship with them . in mc laughlin study , students stated that their support has been associated with enhancement of their self - confidence , which is consistent with the findings of the current study .
therefore dependence on the clinical teacher for support is a necessity and teachers should have a moral responsibility for supporting students .
ideally , these processes will foster student learning by a facilitative , supportive and humanistic approach rather than the traditional , autocratic , teacher - oriented , and one - way approach to teaching .
another important aspect of educational support in this study was supporting through improving learning situation .
clinical teachers are responsible for preparing the situation in order to provide optimal level of clinical learning for students .
johansson ( 2006 ) cites ekerbergh ( 2001 ) who indicates that : nursing students need support from their clinical teachers to integrate theory and practice .
also , the results of lord 's study ( 2002 ) showed that clinical teachers in relationship with their students support them through determining their educational needs , better planning for learning and assessment and giving appropriate feedback .
clinical teacher 's responsibility is supporting students through providing opportunities for practical experiences . in current study , in some cases that supportive relationship was established , the pleasant experiences of clinical education and relationship were created for students .
also , the results of andrew 's study ( 2006 ) demonstrated that students will report positive clinical experiences if the clinical teachers support them through facilitating their teaching .
this finding is consistent with finding of the current study and introduces the necessity of clinical teachers attention to an appropriate educational planning for the maximum provision of learning situation in the clinical settings . in current study , emotional support in relationship was done through several activities .
findings showed that this kind of supportive relationship lead to decrease stress of student , increase students self - confidence and sense of calm and consequently improve of clinical performance .
studies by cooke ( 1996 ) , lopez ( 2003 ) and hsu ( 2006 ) also corroborated the results of this study . according to lopez ( 2003 ) in jordan , nursing students stated that supporting role of clinical teachers was very important in reducing their anxiety . lopez ( 2003 ) cited krichbaum ( 1994 ) that a sense of trust , competence , and mastery can come from a successful relationship thereby ; developing a sense of confidence and adequacy in students . also in the study by cooke ( 1996 ) , students described their teachers as supportive in their relationship .
one aspect of this support was reduce anxiety and calming down students by preparing them before experience of clinical tasks .
that is consistent with findings of current study . in study by lord ( 2002 )
, the teachers also increased the students confidence in doing their tasks . in study by mc laughlin ( 1998 ) , finding revealed that students appreciated that the emotional support led to an increase in their self - confidence .
also , in a study by ling ( 2006 ) , clinical teachers provided emotional support for students in stressful clinical conditions .
also , parsell and bligh ( 2001 ) stated that the responsibility of nursing clinical teachers is providing necessary explanations and being supportive in difficult clinical situations .
similar findings have been reported in research studies regarding to aspect of verbalize and correct the student 's mistakes .
results of mc kee 's study ( 2005 ) showed that when students encounter with any problem , clinical teachers should be available to support them .
moreover , in a study by kube ( 2010 ) , one of the proper behaviours of teachers was correcting the students mistakes without belittling them .
teachers , who are non - supportive in relationship with their students , have much negative effect on students learning , because of increasing their anxiety .
social support was the third aspect of supportive relationship and in socio - cultural context of iran was accessible through support of students in presence of others in clinical setting that seemed important and valuable . with applying supportive actions in this aspect ,
some outcomes were occurred such as create a sense of belongingness to clinical settings for students , more relying on teacher and tendency for future relationship with teacher .
the findings of orelly - kenapp ( 1994 ) similarly demonstrated that students are willing to receive social support in relationship with teachers in clinical settings .
also , based on the findings of lopez ( 2003 ) in jordan , nursing students commented that clinical teachers support them in their relationship by advocating their performance or behaviours in presence of patients and personnel . in current study , junior students were willing to receive more support .
this is noteworthy that novice students had more emphasis on positive relationship behaviours of teachers which led to their calmness and comfort in clinical environments .
congruence of findings of current study with the findings of other studies is important , because it proved that clinical teachers should provide proper social support in relationship with students in clinical settings .
although the researchers tried to recruit male teachers for the study , the most teachers were female , which may affect the results and act as a limitation .
also , with attention to this point that the results of current study are the portions of a large study , the results can be impressed and were richer if the current study was done inflectionally with concentration of supportive relationship .
the experiences of nursing teachers and students in iran demonstrated the presence of supportive relationship in clinical nursing education .
the results revealed that this type of relationship in clinical education is achieved through educational , emotional and social support .
the value of support is evident in student - teacher relationship through the meaning that exists in their experiences of supportive relationship in clinical education .
it is obvious that the role of clinical teachers is critical in facilitating such relationship and consequently improving the students clinical learning .
students positive experiences of supportive relationship are related to how their clinical teachers communicate with them .
the above findings demonstrate that teachers need to know more about the influence of their interpersonal relationship skills and also the comprehensive impact of supportive relationship on the student in clinical settings . | background : student - teacher relationship is a salient issue in nursing education and has long - lasting implication in professional development of nursing students .
nowadays , this relationship in clinical settings is different from the past due to changing in nursing education paradigm .
the purpose of this qualitative study was to explore the experiences of students and teachers about student - teacher relationship in the context of clinical nursing education in iran.materials and methods : in this qualitative study that has been carried out adopting conventional qualitative content analysis approach , six bachelor nursing students and six clinical teachers in school of nursing and midwifery , were selected through purposive sampling .
semi - structured interview and participant observation were used for data collection .
interviews transcribed verbatim and analyzed using conventional content analysis through the process of data reduction and condensation , coding and also generating the categories and themes.results:results of the study showed the existence of a type of relationship in clinical education in which supportive actions of clinical teachers were prominent .
these supportive actions appeared as three major categories including educational support , emotional support and social support which emerged from data.conclusion:the results of this study explicit the ways that support could be provided for students in their relationship with clinical teachers .
it also determines the teachers need to know more about the influence of their supportive relationship on students learning and the best possible outcomes of their education in clinical settings . |
malaria remains a significant , debilitating and often lethal disease in many parts of south america , although its incidence , severity and impact is highly regional - dependent [ 1 , 2 ] . in lowland colombia , for example , malaria has been rated by focus groups as one of the most important health problems facing communities . in colombian and ecuadorian communities ,
the cost of malaria prevention is much less expensive than the sum of direct costs ( e.g. , treatment , travel ) and indirect costs ( e.g. , days of work lost ) . in both countries ,
national malaria control programs monitor the disease and carry out mosquito control programs in malaria endemic areas [ 5 , 6 ] . due to the success of these programs , statistics in ecuador indicate that overall malaria incidence has declined over the last fifteen years , with occasional regional - scale epidemics at lower elevations ( ca .
< 1500 m ) . at higher elevations , malaria was epidemic in the inter - andean valleys of ecuador ( > 1500 m ) prior to the mid-1940s , when large - scale eradication efforts prevented ongoing malaria transmission in these regions , mainly through the reduction and possible elimination of local populations of anopheles pseudopunctipennis .
since that time , malaria has only caused illness in highland regions among migrants and travelers returning from low altitudes [ 1 , 7 ] .
several reviews of the effects of various forms of global change on the incidence of insect - borne disease have stressed that malaria might move into higher - altitude regions as highland habitats become increasingly suitable for anopheles mosquitoes [ 914 ] .
indeed , highland malaria has recently been observed in higher - altitude regions of kenya , rwanda , and uganda [ 1517 ] , as well as in andean bolivia [ 18 , 19 ] .
plasmodium vivax has been incriminated as the parasite in highland malaria epidemics in both 1940s ecuador and recently in bolivia [ 8 , 18 ] .
additionally , recent widespread highland records of three coastal malaria vectors : anopheles albimanus , an .
, it is believed that in higher - altitude regions , residents and health care practitioners may lack experiential knowledge of the disease and therefore do not know how to recognize malaria or protect themselves from mosquito bites .
as well , a person 's experience with malaria could potentially raise a greater perception of malaria risk .
therefore , highland communities may not rate malaria risk strongly or recognize symptoms in order to seek treatment .
residents may also not approach mosquito habitat elimination and personal mosquito bite protection as seriously as residents of malaria - endemic lower altitudes .
although there is essentially no data regarding the perceptions of malaria in the south american andes , there have been several studies conducted in lower - altitude regions .
the knowledge of a connection between mosquito bites and malaria is regionally variable , for example , 56% of respondents in a rural community in northern coastal ecuador versus 85% of respondents in an urban community in pacific coastal colombia [ 3 , 22 ] . in colombia
, malaria has been inaccurately attributed to strenuous activities , ingesting hot foods , poor nutrition , drinking contaminated water , and/or proximity to garbage .
individual differences in knowledge and perceptions have also been attributed to gender , with women having limited access to malaria - relevant information and treatment , among other factors .
malaria prevention in colombia incorporates both modern techniques ( e.g. , bed nets and anopheline mosquito larval habitat elimination ) as well as traditional techniques more commonly used in rural areas [ 3 , 23 , 25 ] .
urban residents in amazonian colombia also have been reported to feel less at risk of malaria than rural residents .
malaria diagnosis by national bodies in south america is often based on passive reporting , where misdiagnosis of malaria could lead to a general underreporting of the disease over the landscape .
the actual risk and occurrence of malaria in lowlands is complicated by human migration patterns between endemic and nonendemic regions , as well as by proximity to mosquito breeding sites .
landowners and land managers , rather than lower - status workers , have the capability to eliminate anopheline larval habitats on their property and to promote and enforce the use of malaria control measures such as bed nets among their families and employees . in this study , a quantitative kap ( knowledge , attitudes and practices )
approach was used to assess the degree of knowledge , concerns and beliefs of land managers and landowners in both lowland and highland regions of ecuador for topics relevant to malaria prevention .
the larval habitat management practices used and motivations for eliminating larval habitat were also examined .
finally , the study assessed mosquito bite protection that is currently in use in both lowland and highland regions and compared it to protection that would be used by respondents for hypothetical travel to a known malaria - endemic area .
to assess the knowledge , beliefs , concerns , land management practices , and forms of bite protection used by ecuadorian land owners and/or managers , researchers conducted a series of 262 structured , questionnaire - based interviews of these individuals throughout all road - accessible regions of ecuador between 2008 and 2010 ( figure 1 ) .
most questionnaires were administered during the period from july to december in 2009 and 2010 . although the regions are endemic for malaria , we did not assess respondents in northern esmeraldas , sucumbos , and orellana provinces due to travel safety regulations ( figure 1 ) .
we attempted to eliminate clustering of respondents by traveling along as many different roads as possible .
however , some clusters of respondents occurred ( e.g. , in pichincha and santo domingo provinces ) due to a higher number of potential respondents present outside their homes at the time of visiting , as well as a greater population and road density in that particular lowland - highland transition area , which may have led to a regional bias in our analysis ( figure 1 ) . to permit statistical inference , we interviewed a pre - established number of land owners and/or managers from each of five generalized land uses : cattle pasture ( whether for meat or dairy ) , fish farms , plantations , human residences , and nature reserves .
fish farm and nature reserve managers were given fewer interviews than the other categories since they were encountered less frequently ( table 1 ) .
no more than one person was interviewed on the same property for the same land use .
although all five land use categories are present in all general regions of the country , there is some clustering of land uses in particular regions which may have caused small levels of spatial bias due to availability ( figure 1 ) .
land owners and managers were stratified on a second level by gender since women and men are expected to have different access to information and women may more readily apply knowledge toward prevention and treatment than men ( table 1 ) . although we attempted to interview an equal number of men and women , we were not able to interview many female owners or managers of nature reserves due to the scarcity of women encountered in these positions ( table 1 ) .
we further stratified interviews by interviewing an approximately equal number of landowners and/or managers above and below 1500 m ( table 1 ) , which travel medical literature suggests as an appropriate altitudinal division for malaria endemism in ecuador .
current statistics from the sistema nacional de eradicacin de malaria ( snem ) indicate that almost all areas below 1500 m have been affected by at least occasional cases of malaria during the last 10 years ( figure 1 ) , which we assume should have provided residents of the below 1500 m category with some experiential knowledge of malaria .
entomological data further supports this altitudinal risk division , since densities of anopheles mosquitoes ( malaria vectors ) are more prevalent below 1500 m , although a few larvae have been collected up to an elevation of nearly 2000 m .
this altitudinal division was used rather than actual reported malaria rates , since the distribution of malaria varies substantially from year to year , data comes from urban reporting centers rather than rural communities , and we expect rural parts of the country to underreport malaria cases due to poorer access to health centres .
therefore , for the purposes of this study we also assume that all respondents below this altitude are potentially at risk for malaria . to locate possible respondents , researchers working in groups of two or three traveled along all roads deemed suitable for travel and potential respondents
were approached when visible from the road or other public domain . to introduce randomization , after spotting a potential respondent for a given land use , a coin toss was used to determine if the person would be approached . however , due to the scarcity of fish farm and nature reserve managers / owners , all of these possible respondents were approached when observed . often ,
the land owner and/or manager was not present and the researchers did not interview any person on the site . to avoid a possible feeling of deference toward the interviewer due to any perceived class differences , for example , , questionnaires were conducted by l. pinault as well as one or two ecuadorian interviewers who spoke the local dialects ( although all field researchers spoke fluent spanish ) and a few contradictory statements were placed into the interview to identify respondents who might not be answering truthfully or might not understand the questions ( e.g. , all affirmative answers ) .
questions were asked directly in spanish by both interviewers to respondents , rather than through translation to english .
voluntary participation and oral consent were received after researchers explained the study , including rights to withdraw at any stage during the interview process .
all research was approved brock university 's ethics in human research committee , file number 07 - 336 .
aspects of the interview included : ( 1 ) knowledge of where malaria occurs in ecuador , ( 2 ) knowledge of the ecological relationship among standing water and mosquitoes , and malaria , ( 3 ) belief in climate change , and belief that climate change could potentially cause malaria vectors to move into higher altitudes , ( 4 ) concern about insect - borne disease , the presence of mosquitoes on their property , and the presence of malaria on their property , ( 5 ) land management practices for standing water ( mosquito habitat ) reduction , and ( 6 ) mosquito bite protection used and bite protection that respondents would use to travel to an area known to have malaria .
researchers also recorded the geographic coordinates and elevation of the site using a handheld gps unit ( garmin gps e - trex summit ) .
respondents answered most questions with the options : yes ( 1 ) or no ( 0 ) . to determine the role of altitudinal category and gender on the frequency of positive answers , a binary logistic regression ( logit ) was used .
those who answered do not know or maybe were considered uncertain answers and were eliminated from analysis when they were reported less than 10% of the time , although the percentage of uncertain answers is summarized in the text when provided more frequently .
since multiple binary logistic regressions were used in some cases , significance levels were modified using a bonferroni correction , where /n ( n = number of tests ) are used to decrease the probability of false positive results .
some questions , including land management practices and bite protection methods , asked respondents to list all possible answers .
chi - square tests ( of equal variances among groups of gender and altitude class , unless otherwise indicated ) were used to compare count data from responses between land use and elevation categories .
all statistical analyses were conducted in minitab v.15 software ( 2007 ) . at the end of the interview , we asked respondents to list all sources of water on their property , with researchers prompting the respondents with a long list of options that included streams , ponds , and laundry washing areas , as well as any answers beyond the list that may occur to them .
all potentially suitable habitats listed by respondents were considered by researchers for comparison to standing water
reported from earlier in the interview to assess the relationship between reported potential habitat and actual potential habitat .
we also judged the approximate socioeconomic status of respondents based on housing type and signs of disposable income .
according to the ecuadorian census , approximately 38% of households lack sufficient income to provide basic necessities of life and 46% of citizens are rated under the nbi income - poverty index based on statistics collected in 2006 , therefore , signs of disposable income such as working televisions indicated above - average wealth .
all respondents were land owners , or in the cases of larger properties , land managers ; therefore , respondents in our study were expected to have a higher average economic status and live in rural settings more often than most ecuadorian citizens .
only one residential respondent lived in an urban area ( quito ) , while all other respondents lived in rural regions or small communities .
although we did not formally evaluate the socioeconomic status of respondents , approximately 56% of our respondents were judged to be of a higher - than - average economic status ( i.e. , living in concrete houses with completed roofing and doors , and some signs of disposable income such as televisions , radios , and cars ) , 23% of our respondents were judged to be of a lower socioeconomic status ( i.e. , living in basic housing sometimes lacking doors and sufficient roofing , with no signs of disposable income ) , and 20% of our respondents were judged to be very wealthy ( i.e. , living in large , finished homes with some degree of landscaping , on expansive properties ) .
greater than 90% of our respondents were ethnically identifiable as mestizo ( a mixture of indigenous , african , and spanish ancestry found in all regions of ecuador ) , while fewer than 10 respondents were each of indigenous , black , or white .
five people who were approached by interviewers declined to participate in the study , therefore , the interviews did not proceed .
eleven percent of respondents answering certainly above 1500 m stated that they could become ill with malaria on their property at present , whereas only 52.6% of respondents below 1500 m provided the same answer ( table 2 ) .
however , 96.4% of respondents correctly agreed with the statement that one could get malaria on the coast and/or in the amazon region of ecuador , with the responses not differing among genders or altitudinal categories .
agreement with the statement that one could get malaria above 3000 m was affected by both gender and altitude category of the respondent ( table 2 ) : women and low - altitude dwellers were more likely to erroneously agree than men and high - altitude dwellers ( women : above 1500 m : 29.7% , below 1500 m : 55.9% ; men above 1500 m : 18.5% , below 1500 m : 30.6% ) .
thirty - three percent of respondents were uncertain if malaria could occur above 3000 m. most respondents answering certainly ( 89.9% ) agreed that eliminating standing water reduces the local population of mosquitoes , thereby acknowledging that they understand where larvae live . a slightly lesser percentage ( 83.8% ) agreed that eliminating standing water reduces the risk of malaria , thereby demonstrating an understanding that mosquito adults arise from larval stages in standing water , and the resulting adults can transmit malaria .
there was no effect of gender or altitude category on responses to either statement .
most respondents ( 93.3% of those answering certainly , 74.8% total ) believed that climate change was occurring , although 19.4% of were uncertain .
fewer ( 75.6% of those answering certainly , 54.9% total ) believed that it may be possible for malaria to move into highland regions with climate change , although 26.1% were uncertain about the possibility of this phenomenon .
a degree of concern regarding insect - borne diseases was reported significantly more often at elevations below 1500 m ( 83.8% ) than above 1500 m ( 57.7% ) , but did not differ between genders ( table 2 ) .
concern about the presence of mosquitoes on a person 's property was greater for respondents below 1500 m ( 87.2% ) than above 1500 m ( 61.4% ) , although there were insignificant differences among genders after the bonferroni correction ( table 2 ) .
concern about the presence of malaria on a person 's property was reported more often below 1500 m ( 82.3% ) than above 1500 m ( 60.0% ) , regardless of gender ( table 2 ) .
different numbers of respondents reported the presence of some type of standing water on their property depending on their land use ( = 14.82 ; df = 4 ; p = 0.005 ) .
this significant difference was due mainly to cattle farmers that reported more standing water , and fish farmers that reported less standing water than expected ( figure 2 ) .
there was no difference in the reporting of standing water presence above and below 1500 m ( = 0.05 ; df = 1 ; p = 0.816 ) . after being asked
generally if there was some type of standing water present on the property , all sources of water on the property ( standing water or otherwise ) were enumerated by respondents and later evaluated by the researchers as potentially suitable larval habitat or not .
figure 2 compares the presence of potentially suitable habitat ( enumerated by respondents ) to the rate at which the presence of some type of standing water was reported , for each land use .
there was a significant association between the land use type and the proportion that reported standing water elimination versus the proportion with standing water reported in the list at the end of the interview ( two - way chi - square contingency table : = 12.9 , df = 4 , p = 0.012 ) , with strongest statistical effects due to the strong initial underreporting of some type of standing water by fish farmers ( figure 2 ) .
all other respondents under - reported the actual presence of standing water by about half ( figure 2 ) .
it is also possible that respondents are reporting the elimination of some standing water , though not all sources of water .
the types of standing water observed on each of the different land uses are summarized in figure 3 .
approximately 77.5% of fish farms had at least one fish pond or tank , with an area of stable water surface that could potentially support mosquito larvae ( figure 3(a ) ) .
ponds were the most common type of standing water for nature reserves ( figure 3(c ) ) , while irrigation canals or inland ditches were the most common for cattle farms ( figure 3(b ) ) , residences ( figure 3(d ) ) , and plantations ( figure 3(e ) ) .
it should be noted that not all standing water types listed would be suitable for anopheles mosquito larvae , although they may be suitable for other species of mosquitoes .
the percentage of respondents that presently eliminate their standing water differed significantly among land uses ( = 16.1 ; df = 4 ; p = 0.003 ) , with the strongest effect due to a smaller number of cattle farmers than expected reporting that they eliminate standing water ( figure 4(a ) ) .
however , the proportions of respondents that eliminate standing water for each land use did not differ between altitude categories ( above / below 1500 m ) ( = 2.4 ; df = 4 ; p = 0.655 ) ( figure 4(a ) ) .
reasons provided for not eliminating standing water were enumerated ( figure 4(b ) ) , and approximately twice the number of reasons were given by respondents below 1500 m than above ( figure 4(b ) ) .
the top three reasons provided were logistical difficulty ( 6.2% ) , lacking human assistance ( 3.5% ) , and lacking time to eliminate the water ( 3.5% ) .
water being natural was only provided as a reason for not eliminating water by nature reserves . not being able to eliminate standing water due to economic use of the water body
was reported for rice farms and fish ponds below 1500 m. four possible motivations that might encourage respondents to eliminate standing water were compared and all four appear to be strong motivators to eliminate water : ( 1 ) the presence of malaria nearby ( 95.3% ) , ( 2 ) financial support to eliminate water ( 93.9% ) , ( 3 ) increased frequency of mosquito bites ( 93.3% ) , and ( 4 ) recommendations by government or medical authorities ( 89.9% ) .
there was no statistical difference in agreement with motivational sources between genders or altitude categories .
respondents were asked if they currently use any form of mosquito bite protection , and if they would use protection if they visited an area known to have malaria .
use of some type of malaria protection differed between altitude categories as well as current use versus use for a known malaria - endemic region , although bite prevention did not differ between genders ( table 3 ) .
current use of protection was higher below than above 1500 m ( figure 5 ; table 3 ) .
respondents reported that they would use malaria protection more often in an area with known malaria than what they currently use , even if they already live in a possibly malaria - endemic area ( below 1500 m ) ( figure 5 ; table 3 ) .
the most commonly used prevention methods above 1500 m are ( 1 ) insecticides for the home , ( 2 ) long sleeves , and ( 3 ) chemical repellent ( figure 6(a ) ) .
if those respondents visited an area with known malaria , ( 1 ) bed nets , ( 2 ) long sleeves , and ( 3 ) insecticides for the home would be the most commonly used ( figure 6(b ) ) .
the most common types of prevention methods used below 1500 m are ( 1 ) bed nets , ( 2 ) insecticides in the home , ( 3 ) chemical repellent , ( 4 ) long sleeves , ( 5 ) screen or curtain on doors and windows , and ( 6 ) chlorine or chemicals in standing water ( figure 6(c ) ) .
the same top six prevention methods are listed by respondents for hypothetical travel to an area with known cases of malaria , although the order in which they are listed is different ( figure 6(d ) ) .
the less - often reported prevention methods for respondents living below 1500 m include calling the sistema nacional de eradicacin de malaria ( government agency ) , use of a fan or air conditioning , or a community effort to eliminate mosquito habitat , all of which have the potential to prevent mosquito bites .
since the present study focuses on land owners and land managers who are of a higher socioeconomic status than the average ecuadorian citizen , it is also likely that respondents had received more education than average , which we expect to relate to a better understanding of the topics under discussion .
therefore , the present study should not be extrapolated to the knowledge , attitudes , and beliefs of all ecuadorians , but rather should provide a baseline of these factors among land owners and/or land managers , that is , the decision makers for larval habitat modification in the overall landscape . although most participants in this study correctly identified general regions where malaria does and does not occur in their country , it is troubling that 52.6% of respondents below 1500 m did not feel that they were at risk of contracting malaria on their property .
we did not formally discuss personal experiences with malaria with respondents , which would likely explain some of the variation in this response , and is one of the shortcomings of our study .
although a small percentage of these are probably not at immediate risk , several respondents appeared to deny personal risk despite anecdotally providing us with examples of neighbours or nearby towns with recent malaria cases .
another explanation might be that respondents feel that malaria is a controllable risk , and they might not be susceptible to infection because they use mosquito bite protection , for example , . the current findings might indicate that lowland residents would benefit from an educational program in their community that provides updated data concerning their immediate and potential risk of malaria . generally , most respondents understood that standing water could increase mosquito populations , and thereby malaria incidence .
along the same lines , a large number were also receptive to the possibility of highland regions becoming affected by malaria in the future with climate change .
such a widespread open - mindedness would be useful for rapid identification of malaria cases and acceptance of risk should malaria spread to previously - unaffected highland regions due to meteorological or other causes . as we would predict from experience , those at altitudes lower than 1500 m reported being more concerned by the presence of mosquitoes , malaria , and other types of insect - borne disease . substantially , fewer respondents reported the presence of standing water on their property early in the interview than those reported later in the interview .
although part of this could be due to denial , or to a lack of awareness of their own property , a more likely reason for many respondents to deny the presence of water could be the too - specific definition of standing water for mosquito habitat .
many respondents did not likely consider often - flowing water types such as irrigation canals to be standing water .
anecdotally , several respondents talked about the necessity of swamps and marshes for mosquito habitat , whereas the most commonly observed standing water types on properties were ponds , reservoirs , and irrigation canals .
therefore , we suggest that the malaria education programs that target standing water elimination need to define potential larval habitat as any water where the surface of the water is not moving or moving slowly , rather than the more typical standing water , or aguas estancadas .
since the type of standing water available is dependent on land use , it is also important for future studies to more extensively define suitable versus unsuitable habitat types for anopheles larvae to include those that could be considered to flow , such as irrigation canals and roadside ditches .
although ecuadorian land owners / managers could be motivated to eliminate standing water through direct observation or advice from authorities , the largest reason provided for not eliminating the water is difficulty ( usually due to topography)a practical problem that would be expensive to address . a large number of respondents below 1500 m used some type of mosquito protection ( 84.0% ) .
bed nets were the most prevalent mosquito bite prevention method in low - altitude regions ( 66.4% ) , and were more commonly used than in a community in northern coastal ecuador ( 23% ) , lowland colombia ( 59% ) , or rural uganda ( 26% ) , possibly due to greater socioeconomic standing among our interviewed landowners / managers than other community members [ 3 , 22 , 31 ] .
we did not personally assess whether or not the respondents ' bed nets were in good condition , or if they were treated with insecticide , which is important for bite prevention or might help to reduce resting populations of adult mosquitoes within homes [ 32 , 33 ] .
the numbers might have also been inflated due to the common use of bed nets to prevent the annoyance of mosquitoes rather than malaria .
insecticides within the home , including sprays , were also often employed in low - altitude areas ( 57.5% ) , similar to communities in the colombian pacific coast ( 57% ) .
their common usage is positive , since one study in the highlands of kenya reports that indoor residual house - spraying actually reduces infection rates more than bed nets .
interestingly , mosquito spirals were not presently used by ecuadorian respondents , despite 60% usage in the colombian pacific coast .
mosquito protection methods that would be employed by ecuadorian land owners / managers during travel to a malaria - endemic area differ slightly from those reported in the travel medical literature .
although 8090% of our respondents state that they would use a bed net in a malaria - endemic area of their own country , in one study only 58% of foreign travelers to a malaria - endemic area would do the same .
however , a higher proportion of travelers in the same paper would use long sleeves ( 93% ) and chemical repellent ( 90% ) than our respondents ( approximately 5060% ) .
travelers from developed countries also rely heavily on chemical prophylaxis for malaria prevention [ 3638 ] , while fewer than 5% of our respondents would use it for travel into a region with known cases of malaria .
we had expected more folklore - based and traditional medicine and mosquito prevention methods from our respondents , since it is known that plant - based malaria prevention and treatments are still quite common in parts of ecuador , for example , [ 4 , 22 , 39 ] and in colombia [ 3 , 23 ] , as well as in rural parts of africa [ 31 , 40 , 41 ] .
this may have been in part due to the higher average socioeconomic status of our respondents , who may have been more able to purchase nontraditional mosquito bite prevention methods , such as deet - based chemical repellent .
some of the plant - based repellents that were mentioned by respondents included citrus fruits ( e.g. , lemon juice ) and menthol . the use of smoke as a mosquito repellent included the traditional burning of termite nests in amazonian regions , as well as burning
further studies are required to evaluate the malaria kap of ecuadorian citizens who do not own properties in ecuador , as well as citizens residing elsewhere in the andes , and to elucidate cultural and educational differences among perceptions of malaria risk .
the present study focused on individual responses , although community approaches to larval source reduction can also play an important role in habitat elimination and highland malaria prevention .
a large proportion of land owners / managers presented a good understanding of the occurrence of malaria on the ecuadorian coast or in amazonia and were able to form a basic causative link between standing water , mosquitoes , and malaria .
however , about half of the landowners / managers in potential risk areas ( i.e. , altitudes < 1500 m ) did not believe that it was possible to become ill from malaria on their own property .
most respondents expressed a belief in climate change and were open - minded to the possibility of anopheline mosquitoes moving into higher - altitude regions . different generalized land uses provided different types of standing water , which may vary in suitability for anopheline larvae .
most respondents reported eliminating standing water on their property . when it was not reported to be eliminated ,
respondents were most often unable to eliminate water due to logistical difficulties . in many cases , respondents reported eliminating standing water despite later reporting sources of standing water that might provide potentially suitable anopheline habitat .
this may have been due to partial , but not thorough , elimination of standing water .
most respondents in lowland ( at - risk ) regions use some type of mosquito bite prevention , most commonly bed nets . during hypothetical travel to a known malaria - endemic region ,
therefore , residents of malaria - endemic regions might be expected to increase their use of prevention if informed that their property lies in a malaria - endemic area .
generally , it is hoped that the findings of this study will assist malaria control organizations by providing useful data to improve the efficiency of potential educational programs .
while knowledge of malaria 's occurrence patterns is strong in ecuador , even in highland regions , it might be necessary to inform residents of actual malaria risk , so that risk does not continue to be underestimated .
although most respondents understand that there is a link between standing water and malaria , the definition of standing water is often too restrictive and does not encompass all potential mosquito habitats on a person 's property .
ecuadorian land owners / managers appear to be strongly motivated to eliminate mosquito habitat on their property , and when they do not eliminate standing water , it is usually due to factors beyond their control ( difficulty or limited financial resources ) .
most at - risk ecuadorian land owners / managers currently use mosquito bite protection methods , although the use of protection might increase if they were informed that they live in a potentially malarious area . | to control malaria effectively , it is essential to understand the current knowledge , beliefs , concerns , land management practices , and mosquito bite protection methods in use by citizens .
this study presents a comparative , quantitative , interview - based study of land owners and/or managers ( n = 262 ) in the ecuadorian lowlands ( presently considered malarious ) ( n = 131 ) and highlands ( potentially malarious in the future ) ( n = 131 ) .
although respondents had a strong understanding of where the disease occurs in their own country and of the basic relationship among standing water , mosquitoes , and malaria , about half of respondents in potential risk areas denied the current possibility of malaria infection on their own property . as well , about half of respondents with potential anopheline larval habitat did not report its presence , likely due to a highly specific definition of suitable mosquito habitat .
most respondents who are considered at risk of malaria currently use at least one type of mosquito bite prevention , most commonly bed nets . |
cerebral palsy ( cp ) is a chronic disorder of movement and posture caused by a non - progressive brain lesion .
cp can manifest itself in many ways , causing spastic , dyskinetic , dystonic , ataxic and mixed palsies .
the prevalence of cp is 2 2.5 of every 1000 live - born children in the western world , making it one of the most common causes of motor disability in children .
cp has important comorbidities , such as visual impairments , learning difficulties and epilepsy ( 5 ) .
epilepsy is one of the most common neuroimpairments in childhood , with a prevalence of 5 7% in the general population ( 5 ) .
although there is some conflict in the literature , it appears that the prevalence of epilepsy in children and adults with cp is between 15 55% ( 5 ) .
in addition , if learning difficulties and cp coexist , the risk of epilepsy in children with cp rises to 71% ( 5 ) .
furthermore , robinson found that 21% of children with language impairment had seizures ( 6 ) .
these are children who can not understand or express themselves ( either verbally or non - verbally ) in the normal way expected of age - matched peers within the general population ( 6 ) . utilizing an audit of clinical notes for children at two schools in one city ,
this study investigated whether a higher incidence of epilepsy existed in children with cp , learning difficulties ( ld ) or language and communication difficulties ( lacd ) compared to children without these conditions . in addition , data was studied to ascertain whether an earlier age of onset , low birth weight or gestational age is associated with increased incidence of cp , ld or lacd .
conclusions made from local data could then allow for the possibility of adequate planning of services for these children .
school a and school b are special primary schools in the city of newcastle upon tyne , uk . school a enrolls children with a variety of developmental difficulties , whereas pupils at b , a more specialist school , either have cp or lacd .
medical records for current pupils in 2004 were obtained and carefully studied over seven weeks ( january to february ) to collate epidemiological information .
details of the child s birth history , diagnosis ( as cp , lacd and/or ld ) , past history of syndromes , past history of epilepsy , age of onset , presence of neonatal encephalopathy , infantile spasms and febrile convulsions , type of epilepsy , frequency of fits were recorded .
epilepsy was recorded as active if the child was currently on anti - epileptic medication .
medical records for all 59 of the pupils at school b were obtained and studied . medical records for 83 ( 77% of pupils ) at school a were collected .
the mean age of children at school b was 8.20 years ( s.d . 2.48 ) .
children at school b were older ( t = 2.17 , p = 0.03 ) table 1 shows the number of children with each diagnosis by gender .
many children had a combination of diagnoses , therefore totals in each gender row exceed 100% .
epilepsy , both active and inactive , was more common in females than males ( x = 9.07 , p < 0.01 ) .
although there were more females with cp and more males with lacd and ld , the differences between groups were not statistically significant .
at each school there were three children that had a combination of cp , ld and lacd .
there were 62 cases of epilepsy ( seizures at any time ) in the combined school population , representing 43% of the total .
separate school data shows that more children at school b had cp than school a ( x = 9.56 , p < 0.01 ) or language and communication problems ( x 4.25 , p = 0.04 ) .
more children at school a had learning difficulties ( x = 32.41 , p < 0.01 ) or active epilepsy ( x = 3.03 , p = 0.08 ) compared to school b. table 3 reports the association between active epilepsy or cp , lacd , or ld .
the results show that active epilepsy was significantly more common in those children with cp when compared to children without cp ( x = 7.58 , p = 0.01 ) .
there was no association between active epilepsy and the presence of ld however , all children with cp and ld had epilepsy ( n = 6 ) .
interestingly , epilepsy was more likely to occur in children without lacd ( x = 8.45 , p < 0.01 ) . using combined data from both schools ,
females had an earlier age of onset of epilepsy than males ( t = 2.27 , p = 0.03 , data not shown ) .
the average age of onset in children with cp was earlier at 21.50 months compared to 37.15 in children without cp , although this was not statistically significant ( t = 1.40 , p = 0.17 ) .
there was no difference in age of onset of epilepsy in those with or without lacd ( t = 0.51 , p = 0.62 ) or ld ( t = 0.10 , p = 0.92 ) .
although not statistically significant , the data also showed a trend that those children who developed epilepsy within the first 24 hours of life were more likely to have cp than those who developed epilepsy later in life ( x = 3.10 , p = 0.08 ) .
finally , children with neonatal encephalopathy are more likely to have cp than those without neonatal encephalopathy ( x = 8.74 , p < 0.01 , data not shown ) . table 4 shows the mean gestational age and birth weight in children with cp , lacd , and ld .
the results showed that those with cp tended to have a lower birth weight ( t = 3.15 , p < 0.01 ) and a shorter gestation ( t = 3.17 , p < 0.01 ) than children without cp . although not statistically significant , children with lacd tended to be heavier at birth than children without lacd ( t = 1.79 , p = 0.08 ) .
those with learning difficulties have a longer gestation compared to those without ( t = 2.00 , p = 0.047 ) there were three anomalous results for birth weight and gestation age . these results ,
although collected from the notes , seemed unlikely to be correct therefore were excluded from the analysis ( see discussion for details ) .
medical records for all 59 of the pupils at school b were obtained and studied . medical records for 83 ( 77% of pupils ) at school a were collected .
the mean age of children at school b was 8.20 years ( s.d . 2.48 ) .
children at school b were older ( t = 2.17 , p = 0.03 ) table 1 shows the number of children with each diagnosis by gender .
many children had a combination of diagnoses , therefore totals in each gender row exceed 100% .
epilepsy , both active and inactive , was more common in females than males ( x = 9.07 , p < 0.01 ) .
although there were more females with cp and more males with lacd and ld , the differences between groups were not statistically significant .
at each school there were three children that had a combination of cp , ld and lacd .
there were 62 cases of epilepsy ( seizures at any time ) in the combined school population , representing 43% of the total .
separate school data shows that more children at school b had cp than school a ( x = 9.56 , p < 0.01 ) or language and communication problems ( x 4.25 , p = 0.04 ) .
more children at school a had learning difficulties ( x = 32.41 , p < 0.01 ) or active epilepsy ( x = 3.03 , p = 0.08 ) compared to school b.
table 3 reports the association between active epilepsy or cp , lacd , or ld .
the results show that active epilepsy was significantly more common in those children with cp when compared to children without cp ( x = 7.58 , p = 0.01 ) .
there was no association between active epilepsy and the presence of ld however , all children with cp and ld had epilepsy ( n = 6 ) .
interestingly , epilepsy was more likely to occur in children without lacd ( x = 8.45 , p < 0.01 ) .
using combined data from both schools , females had an earlier age of onset of epilepsy than males ( t = 2.27 , p = 0.03 , data not shown ) .
the average age of onset in children with cp was earlier at 21.50 months compared to 37.15 in children without cp , although this was not statistically significant ( t = 1.40 , p = 0.17 ) .
there was no difference in age of onset of epilepsy in those with or without lacd ( t = 0.51 , p = 0.62 ) or ld ( t = 0.10 , p = 0.92 ) .
although not statistically significant , the data also showed a trend that those children who developed epilepsy within the first 24 hours of life were more likely to have cp than those who developed epilepsy later in life ( x = 3.10 , p = 0.08 ) .
finally , children with neonatal encephalopathy are more likely to have cp than those without neonatal encephalopathy ( x = 8.74 , p < 0.01 , data not shown ) .
table 4 shows the mean gestational age and birth weight in children with cp , lacd , and ld .
the results showed that those with cp tended to have a lower birth weight ( t = 3.15 , p < 0.01 ) and a shorter gestation ( t = 3.17 , p < 0.01 ) than children without cp . although not statistically significant , children with lacd tended to be heavier at birth than children without lacd ( t = 1.79 , p = 0.08 ) .
those with learning difficulties have a longer gestation compared to those without ( t = 2.00 , p = 0.047 ) there were three anomalous results for birth weight and gestation age . these results ,
although collected from the notes , seemed unlikely to be correct therefore were excluded from the analysis ( see discussion for details ) .
the data from the schools shows that the percentage of children with epilepsy was high compared with the general population ( 43% compared to 7% ) .
school a takes children with multiple and more complex disabilities , illustrated by the tendency of more children to have active epilepsy and learning difficulties at this school .
school b takes children with cp or lacd , also shown by the data , and is therefore considered more specialised .
the data demonstrates that 40% of children with cp alone and 100% of children with a combined diagnosis of cp and ld also had epilepsy ( x = 4.44 , p = 0.035 ) .
this is comparable with the literature , in which epilepsy occurs in 15 55% of children and adults with cp alone , rising to 71% in children also suffering from ld ( 5,710 ) .
the proportion of children with epilepsy and lacd ( 14% ) was comparable to that of the literature ( 21% ) , and is greater than that of the general population ( 6 ) .
we found that epilepsy occured more in children without lacd , but this may be due to the fact that these children suffered from other , predisposing conditions .
the average age of onset of 21.5 months in children with cp was higher when compared to children without cp . furthermore , this age of onset was greater than data in the literature [ 18.9 months ( 10 ) ] .
we also found that children who developed epilepsy within the first 24 hours of life were more likely to develop cp than those with a later onset ( p = 0.08 ) , a fact supported by existing literature ( 7 ) .
finally , we found that neonatal encephalopathy , characterised by disturbed neurological function including seizure activity occurring in the first days of life ( 7,11 ) , was associated with cp ( p < 0.01 ) .
the bw and ga of children with cp from both schools was lower when compared to children without cp ( p < 0.01 ) , supporting theories that the etiology of cp is due to prenatal events ( 2,7 ) .
this data also supports conclusions made by an epidemiological study of cp performed in the north east of england from 1964 to 1993 ( 3 ) .
this study concluded that there was a substantial increase in the overall rate of cp over time , caused by increased rate of cp in those children born weighing less than 2.5 kg .
it is conceivable that many children with cp who would have died in the past could now survive because of improvements in the perinatal care of premature and low birth weight babies .
the small study cohort ( only 37 children had active epilepsy ) is a considerable limitation and it can not be determined whether the schools studied were representative of special schools as a whole .
many of the medical records for pupils in school a were inaccessible due to the amalgamation of hospital files across three different sites .
this issue limited data collection , and as a result , also limits potential conclusions that could be drawn .
three pupils were left out of the analysis of bw and ga due to improbable data - two were noted to be above 4 kg at birth , likely reflecting an older weight , and one recording was illegible .
in addition , the lack of documentation of certain details such as association with family history , type of epilepsy and frequency of seizure activity meant that this information could not be included in formal analyses .
in addition , definite negatives , such as the absence of seizure activity and family history , are rarely recorded in medical records .
the results are at risk of type two error , which indicates that statistical significance could have been found by chance .
this is possible due to the fact that there was no hypothesis stated prior to the beginning of the study , and also to the fact that the the data was collected by
data dredge and analysed looking for statistically significant results ( 12 ) . this study could be improved by carrying out a prospective study using questionnaires and consultations thereby directly asking patients and their families about their medical history .
in addition , investigations such as eeg , video recording and ct could give useful information about the type of epilepsy and its underlying pathology .
standardised scores for ld versus the relatively crude manner in which data was documented would refine the methodology in a future study .
. the comparison group could be children with epilepsy and no cp at a mainstream school , or a random sample taken from the general population .
it would be interesting to broaden the study questions to include a detailed look at the type and the severity of cp , as some studies have found that epilepsy occurs in a higher rate in certain types of cp ( 7 ) .
in addition , it would be of benefit to look at behaviour and educational attainment in children with epilepsy and cp as opposed to no epilepsy and cp .
the data in this study builds on conclusions made by existing studies , reinforcing the fact that epilepsy commonly accompanies cp .
the association between low bw and ga , and early age of onset of seizures in children with cp , illustrates the importance of past medical history from birth to determine any risk factors for epilepsy in later life .
diagnosis and treatment of these children require skills in addition to those needed for non - complicated cp .
therefore , members of the multidisciplinary team managing children in special schools like a and b need to be aware of this association and be educated to adequately manage these children . | purpose of the study : to determine in children the proportion and characteristics of epilepsy associated with cerebral palsy , learning difficulties and language and communication difficulties in a specific population of two special schools.basic procedures : retrospective review of case notes for 142 children in two special schools ( school a and school b ) in newcastle , ukmain findings : school a had more children with learning difficulties ( x2 = 32.41 , p < 0.01 ) and active epilepsy ( x2 = 3.03 , p=0.08 ) than school b. there were more children with cerebral palsy ( x2 = 9.56 , p < 0.01 ) and language and communication problems ( x2 = 4.25 , p = 0.03 ) at school b compared to school a. active epilepsy is significantly more common in children with cerebral palsy ( x2 = 7.58 , p = 0.01 ) .
all children with cerebral palsy and learning difficulties had epilepsy ( n = 6 ) .
although not statistically significant , those children who developed epilepsy within the first 24 hours of life were more likely to have cerebral palsy than those who developed epilepsy later in life ( x2 = 3.10 , p = 0.08 ) .
those children with cerebral palsy tended to have a lower birth weight ( t = 3.15 , p < 0.01 ) and a shorter gestation ( t = 3.17 , p < 0.01 ) than children without cerebral palsy.principal conclusions : the data supports evidence from previous studies , demonstrating that epilepsy commonly accompanies cerebral palsy , thus complicating this difficult chronic condition .
we show an association between both low birth weight and gestational age , and early age of onset of seizures , in children with cerebral palsy .
this illustrates the importance , in these children , of past medical history from birth to determine risk factors for epilepsy later in life . |
ocular symptoms can represent first signs of potentially life - threatening systemic disorders like severe anemia in this case .
we report a case of severe vitamin b12 deficiency anemia presenting with white centered retinal hemorrhages .
a 40-year - old man , general practitioner himself , presented with a 1-day history of diminished left visual acuity and a drop - shaped central scotoma .
he reported dyspnea seemingly caused by a respiratory tract infection , but an antibiotic therapy with amoxycillin / clavulanic acid 875/125 mg b.i.d .
, which he had prescribed himself 14 days before , had not induced any recovery so far .
the corrected visual acuities were 20/20 , od and 20/100 , os . ophthalmic examination revealed bilaterally pale tarsal conjunctiva , discretely icteric bulbar conjunctiva and disseminated white centered intraretinal hemorrhages with foveal involvement ( fig .
oct imaging through these lesions revealed a retinal thickening caused by a sub - ilm accumulation of hyperreflective and inhomogeneous deposits within the nerve fiber layer ( nfl ) ( fig .
immediate laboratory work - up showed : megaloblastic anemia caused by vitamin b12 deficiency , with erythrocytes 0.95 10/l , hb
4 g / dl , hct 12.3% , mch 42.1 pg , mcv 129 fl , thrombocytes 33 10/l , vitamin b12 40 pmol , folate 4.9 g / l , bilirubin 2.5 mg / dl , -gt 340 u / l , d - dimer 2,715 g / l , crp and esr normal .
because of vital threat , the patient was admitted to the department of internal medicine and consecutively administered four erythrocyte and two thrombocyte concentrates .
the subsequent causal therapy with intravenous vitamin b12 supplements resulted in resolution of the retinal changes ( fig .
most reports of white centered retinal hemorrhages have been described in patients with leukemic retinopathy and bacterial endocarditis .
the retinal findings in the latter disease were first described by moritz roth ( swiss pathologist , 18391914 ) .
roth 's spots were considered as pathognomonic for subacute bacterial endocarditis but a wide spectrum of pathologies like leukemia , anemia , diabetes , sickle cell disease , scurvy and connective tissue disorders with increased capillary fragility can act as causative factors . in leukemia
the retina is involved more often than any other ocular tissue and the proposed histological correlate is capillary rupture accompanied by hemorrhage and an extravasal concentration of leukocytes .
they may be related to endothelial ischemia secondary to anemia , direct occlusion by leukemic cells , occlusion by platelet fibrin aggregates , or sludging resulting from hyperviscosity .
these lesions are found at all levels of the retina , but especially in the inner layers with focal destruction [ 2 , 3 ] .
it is interesting that this case of vitamin b12 deficiency anemia retinopathy has a clinically indistinguishable fundus appearance .
this is probably due to the common pathology of capillary disruption and subsequent hemostatic fibrin plug formation . in megaloblastic anemia
hence it was shown that retinal lesions can be observed more frequently in anemic patients with concomitant thrombocytopenia and the same coincidence was also observed in leukemic patients . in this case , the patient presented with low platelet counts ( 33 10/l ) too .
this association may substantiate the pathophysiologic hypothesis of disruption of vascular stability in anemia by disturbance of the balance between damaging endothelial factors and its reparative sequences .
the loss of impermeability allows extrusion of whole blood and subsequent diffusion from the disrupted site throughout and above the nfl .
this is confirmed by histologic serial sections of white centered retinal hemorrhages showing a symmetric distribution of fibrin , platelets and infiltrating red blood cells arising from the point of damage to the capillary wall .
accordingly , oct cross sections through a white centered lesion depict hyperreflective deposits below the internal limiting membrane and thickening of the nfl most likely representing the fibrin - platelet hemostatic complex ( fig .
therefore the biomicroscopic pattern of white centered hemorrhages observed in anemic retinopathy seems to be due to the clot formation as the reparative sequence after capillary rupture .
our case illustrates that vitamin b12 deficiency anemia must be considered as a cause for white centered retinal hemorrhages . in our patient
megaloblastic anemia was caused by vitamin b12 deficiency , resulting from inadequate diet and alcohol abuse .
prompt assessment of blood samples is obligatory because of possible vital threat and the need for consecutive therapy .
the authors indicate no financial relationship of any kind and state to have full control of all primary data and agree to allow case reports in ophthalmology to review their data if requested . | background : to report a case of severe vitamin b12 deficiency anemia presenting with white centered retinal hemorrhages . methods : interventional case report .
results : a 40-year - old man , general practitioner himself , presented with a 1-day history of diminished left visual acuity and a drop - shaped central scotoma .
the corrected visual acuities were 20/20 , od and 20/100 , os .
ophthalmic examination revealed bilaterally pale tarsal conjunctiva , discretely icteric bulbar conjunctiva and disseminated white centered intraretinal hemorrhages with foveal involvement .
oct imaging through these lesions revealed a retinal thickening caused by a sub - ilm accumulation of hyperreflective and inhomogeneous deposits within the nerve fiber layer .
immediate laboratory work - up showed severe megaloblastic anemia caused by vitamin b12 deficiency requiring erythrocyte transfusions .
discussion : most reports of white centered retinal hemorrhages have been described in patients with leukemic retinopathy and bacterial endocarditis .
it is interesting that this case of vitamin b12 deficiency anemia retinopathy has a clinically indistinguishable fundus appearance .
this is probably due to the common pathology of capillary disruption and subsequent hemostatic fibrin plug formation . in megaloblastic anemia ,
direct anoxia results in endothelial dysfunction .
the loss of impermeability allows extrusion of whole blood and subsequent diffusion from the disrupted site throughout and above the nerve fiber layer .
therefore the biomicroscopic pattern of white centered hemorrhages observed in anemic retinopathy is most likely due to the clot formation as the reparative sequence after capillary rupture . |
subjects were enrolled by assuring verbal understanding and obtaining written informed consents , which , along with the study protocol , were reviewed by an ethical review board in accordance with the declaration of helsinki .
an independent clinical research organization monitored the study progress and results , and the study was registered through clinicaltrials.gov , identifier nct02330523 .
forty subjects , 1870 years of age , intended for extraction and subsequent implant placement , were enrolled with 10 study sites . only posterior ( first premolar to first molar ) extraction sockets , with intact adjacent teeth or dental implants ( for measurement stent indexing ) and with substantial buccal wall dehiscences of at least 1/3 the overall socket height and width were included ( class ii / iii , elian et al .
subjects were excluded if they had a history of tobacco use within the last 6 months , healing disorders , i.e. diabetes mellitus , cancer , hiv , bone metabolic diseases , or had received systemic corticosteroids , immunosuppressive agents , radiation therapy , and/or chemotherapy within the past 2 months .
subjects taking intramuscular or intravenous bisphosphonates or who had allergies or sensitivity to alginate , latex , collagen or acrylic were also excluded , as were pregnant , lactating or intending to become pregnant women , or those participating in other clinical intervention studies .
the ridge preservation control therapy was demineralized allograft ( oragraft dgc , lifenet health , inc .
, virginia beach , va , usa ) plus reconstituted and crosslinked bovine collagen membrane ( biomend extend ; zimmer dental , inc . ,
the ridge preservation test therapy was deproteinized bovine bone mineral with collagen binder plus extracted , native porcine , bilayer collagen membrane ( biooss collagen plus biogide ; geistlich pharma ag , wolhusen , switzerland )
subjects were randomly assigned to either the control or test therapy in a block 1:1 ratio , first by the 10 investigators and then overall for all 40 subjects , so that each centre might have equal numbers of test and control subjects , with a minimum of one and a maximum of three test and control patients per investigator .
investigators received randomization instructions only after enrolling a subject and immediately prior to surgery .
primary and secondary endpoints and exploratory measures were as follows :
primary outcome variable
bone ridge dimension preservation horizontal change from baseline to 6 months , as measured using indexed stents , from the outside of the stent , at the apical extent of the buccal dehiscence , buccolingually to the interior bony wall .
secondary outcome variables
bone ridge dimension preservation vertical changes from baseline to 6 months , apicocoronally from the outside of the stent to the apical extent of the lingual and buccal walls .
exploratory variables
histomorphometric % new bone , % graft and % connective tissue / bone marrow components from midsection bone core biopsies at 6 months , including :
% new bone in contact with graft% graft in contact with new bone
wound closure ( incision line gap ) at all time points.soft tissue inflammationbaseline bone measurements extraction socket morphology measured directly at baseline surgery
baseline extraction socket dimensions , measured as the mesialdistal distance of the dehiscence at the crest and the coronalapical distance from the crest to most apical extent of the buccal dehiscence , and also as the overall socket dimensions , mesialdistal at crest and coronalapical from crest to the apical extent of the extraction socket.bony wall thickness , measured horizontally at the coronal tip of the most apical extent of the dehiscences of the buccal and , if present , lingual bony walls .
primary outcome variable
bone ridge dimension preservation horizontal change from baseline to 6 months , as measured using indexed stents , from the outside of the stent , at the apical extent of the buccal dehiscence , buccolingually to the interior bony wall .
bone ridge dimension preservation horizontal change from baseline to 6 months , as measured using indexed stents , from the outside of the stent , at the apical extent of the buccal dehiscence , buccolingually to the interior bony wall .
secondary outcome variables
bone ridge dimension preservation vertical changes from baseline to 6 months , apicocoronally from the outside of the stent to the apical extent of the lingual and buccal walls .
bone ridge dimension preservation vertical changes from baseline to 6 months , apicocoronally from the outside of the stent to the apical extent of the lingual and buccal walls .
exploratory variables
histomorphometric % new bone , % graft and % connective tissue / bone marrow components from midsection bone core biopsies at 6 months , including :
% new bone in contact with graft% graft in contact with new bone
wound closure ( incision line gap ) at all time points.soft tissue inflammationbaseline bone measurements extraction socket morphology measured directly at baseline surgery
baseline extraction socket dimensions , measured as the mesialdistal distance of the dehiscence at the crest and the coronalapical distance from the crest to most apical extent of the buccal dehiscence , and also as the overall socket dimensions , mesialdistal at crest and coronalapical from crest to the apical extent of the extraction socket.bony wall thickness , measured horizontally at the coronal tip of the most apical extent of the dehiscences of the buccal and , if present , lingual bony walls .
histomorphometric % new bone , % graft and % connective tissue / bone marrow components from midsection bone core biopsies at 6 months , including :
% new bone in contact with graft% graft in contact with new bone
% new bone in contact with graft % graft in contact with new bone wound closure ( incision line gap ) at all time points . soft tissue inflammation baseline bone measurements extraction socket morphology measured directly at baseline surgery
baseline extraction socket dimensions , measured as the mesialdistal distance of the dehiscence at the crest and the coronalapical distance from the crest to most apical extent of the buccal dehiscence , and also as the overall socket dimensions , mesialdistal at crest and coronalapical from crest to the apical extent of the extraction socket.bony wall thickness , measured horizontally at the coronal tip of the most apical extent of the dehiscences of the buccal and , if present , lingual bony walls .
baseline extraction socket dimensions , measured as the mesialdistal distance of the dehiscence at the crest and the coronalapical distance from the crest to most apical extent of the buccal dehiscence , and also as the overall socket dimensions , mesialdistal at crest and coronalapical from crest to the apical extent of the extraction socket .
bony wall thickness , measured horizontally at the coronal tip of the most apical extent of the dehiscences of the buccal and , if present , lingual bony walls .
prior to surgery , alginate impressions were obtained for fabrication of indexed measuring stents ( see fig
medical and dental histories and demographic data were recorded ; oral exams and dental cleanings were performed .
( a ) baseline extraction socket measures insured that buccal wall mesialdistal and vertical dehiscences were at least 1/3 of the overall extraction socket dimensions .
( b & c ) measuring stents were fabricated from 0.020 thermoplastic and registered on adjacent teeth .
the stents included three indexing holes for measuring ridge buccallingual width , vertical height to the lingual wall and vertical height to the buccal wall .
following administration of antibiotics , extractions were performed with flap reflection , periotomes , elevators and forceps . to ensure that appropriate biopsy specimens were obtained , i.e. new bone and graft and not old host bone in the cores ,
extraction socket morphology dimensions were recorded through direct measures , and baseline extraction socket buccallingual and vertical dimensions were recorded using indexed stents , as described above and shown in fig .
co. , chicago , il , usa ) , depending on the dimension of the defect .
the measurement methods were similar to those employed by wood and mealey ( wood & mealey 2012 ) .
bone defects were filled with particulate bone grafts confined to the existing alveolar ridge dimensions , making no attempt to go outside the confines of ridge , and then covered with absorbable collagen membranes placed to cover the grafts and extend slightly 23 mm beyond bone defect margins .
periosteal releasing incisions were not employed , and incision lines were not required to be closed primarily so that mucogingival junction dimensions might be preserved . remaining soft tissue incision gaps , if any , were recorded both mesiodistally and buccallingually .
subjects were instructed to take ibuprofen 800 mg or hydrocodone / paracetamol up to three times a day for pain , as needed .
subjects were instructed to use chlorhexidine ( 0.12% ) mouth rinse for 30 s twice daily and to avoid excessive muscle tractioning or trauma and not to brush the study areas for the first 2 weeks .
amoxicillin was provided 875 mg bid for 1014 days ( with clindamycin 300 mg qid for 710 days for subjects with penicillin allergies ) .
after 2 weeks of healing , subjects were instructed in a brushing technique creating minimal apically directed trauma to the treatment area . at 4 weeks , the subjects were instructed to stop chlorhexidine rinses and resume normal oral hygiene practices . at 1week postsurgery
, photos of the test sites were taken , and clinical measurements of inflammation and remaining incision line gaps ( if any ) were assessed .
categorical inflammation scores were :
0 normal , ( absence of inflammation )
1 mild inflammation of any portion of the marginal unit , e.g. , slight change of colour
2 mild inflammation of entire gingival unit ( but no oedema )
3 moderate inflammation ( moderate glazing , redness , oedema and/or hypertrophy )
4 severe inflammation ( marked redness and oedema / hypertrophy , spontaneous bleeding or ulceration )
0 normal , ( absence of inflammation )
1 mild inflammation of any portion of the marginal unit , e.g. , slight change of colour
2 mild inflammation of entire gingival unit ( but no oedema )
3 moderate inflammation ( moderate glazing , redness , oedema and/or hypertrophy )
4 severe inflammation ( marked redness and oedema / hypertrophy , spontaneous bleeding or ulceration ) further followup evaluations occurred at 4weeks , 3months and 6months postsurgery .
photos of the test sites and clinical measurements of remaining incision line gaps ( if any ) were obtained , and inflammation was assessed .
oral hygiene instructions were reviewed throughout the study . at 1 , 3 and 6 months ,
to implant placement , vertical and horizontal ridge dimensions were recorded using the indexed measuring stents . to retrieve biopsies approximately 810 mm in length , 3 mm trephines ( ace surgical supply , bur trephine 35 tl 2id 2.8od ti 18.5cl ca marks 810131518 ) were used .
biopsies were retrieved from the implant sites , unless an implant was placed in original , nongrafted bone , in which case the biopsy was retrieved from the grafted area immediately adjacent to the implant , as indicated by the indexed stent .
biopsies were retained within their respective trephines , wrapped with lintfree gauze ( the open ends of the trephines serving as the apical orientation reference ) and preserved in 10% buffered formalin solution .
trephines together with biopsies were fixed in 4% formalin for 57 days , dehydrated in serial steps of ethanol ( 70% , 80% , 90% , 100% ) , remaining for 1 day in each concentration , and degreased for 1 day in xylene .
specimens were then infiltrated , embedded and polymerized ( technovit 9100heraeus kulzer , wehrheim , germany ) .
after polymerization , samples were hemisected and cut in 500 m sections using a precision cutting machine ( secotom 50 , stuers , ballerup , denmark ) , so that two sections were obtained per biopsy .
the sections were mounted onto opacified acrylicslides and ground to a final thickness of approximately 60 m on a rotating grinding plate ( stuers , ballerup , denmark ) .
specimens were subsequently stained with azure ii and pararosaniline ( axio imager m1 and axiocam hrc , carl zeiss , gttingen , germany ) .
histologic evaluation was performed with a microscope equipped with a digital analyzer ( axio imager m1 and axiocam hrc , carl zeiss , gttingen , germany ) .
histomorphometric analyses were performed with imaging software ( analysis five soft imaging system , mnster , germany ) on composite overview scans .
the area of new healing ( versus old / original bony tissues ) was demarcated in each section . within this area ,
prior to initiation of the study , using an indexed stent , stone cast model of an extraction socket exhibiting a substantial buccal dehiscence and a unc 15 periodontal probe ( hufriedy mfg .
co. , chicago , il , usa ) , all investigators were calibrated ( 1 mm ) to the principal investigator ( ets ) .
the first step in the analysis was to determine if the randomization resulted in balanced groups .
age , gender , race , ethnicity , bmi and all baseline clinical variables were tested to determine if the two groups were balanced .
the analyses were based on the variable types , with dichotomous variables tested using fisher exact test and categorical variables using chisquare test and ttests for continuous variables .
the primary hypothesis was that dbbmc + nbcm ( test ) was not inferior to dfdba + recxc ( control ) in the preservation of ridge volume as measured by the difference in the horizontal distance from stents , buccolingually to the interior ( lingual ) bony wall .
the noninferiority margin was 1.5 mm , as obtained from the approximate deviation in ridge dimension observed by wood and mealey .
twosample ttests were used to evaluate noninferiority , such that :
h0 :
ck
ob
o
ha :
ck
ob >
o
ck = the mean change in ridge volume for dbbmc + nbcm from baseline to 6 months
ob = the mean change in ridge volume for dfdba + recxc from baseline to 6 months
o = hypothesized mean difference = 0.0 mm
= noninferiority margin = 1.5 mm
= 0.05 , onesided1 = 0.90
ck = the mean change in ridge volume for dbbmc + nbcm from baseline to 6 months
ob = the mean change in ridge volume for dfdba + recxc from baseline to 6 months
o = hypothesized mean difference = 0.0 mm
= noninferiority margin = 1.5 mm if the test for noninferiority was statistically significant , then a twosample ttest for superiority was subsequently conducted , as detailed in the methodology of morikawa & yoshida ( 1995 ) .
treatment differences in the vertical ridge preservation changes ( baseline to 6 months ) , measured apicocoronally from the outside of the stent to the apical extent of the lingual and buccal walls were also evaluated using the noninferiority method listed above .
differences in histomorphometric variables were tested with twosample ttest for superiority due to the exploit nature of the variables at 6 months . due to multiple testing for the five histomorphometric variables , the significance level had to be less than 0.05/6 = 0.008 ( bonferroni ) . for histomorphometric differences to be clinically significant , a margin of at least 15.0% was needed based on the work of wood and mealey .
the behaviour of incision line closure over time was an additional goal of the analysis , with evaluations at 1 week , 1 month and at 3 months .
inferiority testing could not be done due to a lack of historical data and the potential time dependence of the data .
in addition to mean differences tested with ttests , the frequency of closed incisions was tested at each time point with fisher 's exact test .
sample size was predicated on assuring that the primary study objective had adequate power to assess the noninferiority hypothesis . under these assumptions ,
proc power ( sas version 9.2 , sas institute , inc , cary , north carolina , usa ) required a sample size of 18 evaluable subjects in each treatment group .
therefore , a sample size of 40 subjects was considered sufficient to meet the primary objective of this study . although the study was powered for the primary outcome variable , the secondary outcome variables each retained 90% power to demonstrate noninferiority , assuming that the treatments were , in truth , equal
subjects were enrolled by assuring verbal understanding and obtaining written informed consents , which , along with the study protocol , were reviewed by an ethical review board in accordance with the declaration of helsinki .
an independent clinical research organization monitored the study progress and results , and the study was registered through clinicaltrials.gov , identifier nct02330523 .
forty subjects , 1870 years of age , intended for extraction and subsequent implant placement , were enrolled with 10 study sites .
only posterior ( first premolar to first molar ) extraction sockets , with intact adjacent teeth or dental implants ( for measurement stent indexing ) and with substantial buccal wall dehiscences of at least 1/3 the overall socket height and width were included ( class ii / iii , elian et al .
subjects were excluded if they had a history of tobacco use within the last 6 months , healing disorders , i.e. diabetes mellitus , cancer , hiv , bone metabolic diseases , or had received systemic corticosteroids , immunosuppressive agents , radiation therapy , and/or chemotherapy within the past 2 months .
subjects taking intramuscular or intravenous bisphosphonates or who had allergies or sensitivity to alginate , latex , collagen or acrylic were also excluded , as were pregnant , lactating or intending to become pregnant women , or those participating in other clinical intervention studies .
the ridge preservation control therapy was demineralized allograft ( oragraft dgc , lifenet health , inc . , virginia beach , va , usa ) plus reconstituted and crosslinked bovine collagen membrane ( biomend extend ; zimmer dental , inc . ,
the ridge preservation test therapy was deproteinized bovine bone mineral with collagen binder plus extracted , native porcine , bilayer collagen membrane ( biooss collagen plus biogide ; geistlich pharma ag , wolhusen , switzerland )
subjects were randomly assigned to either the control or test therapy in a block 1:1 ratio , first by the 10 investigators and then overall for all 40 subjects , so that each centre might have equal numbers of test and control subjects , with a minimum of one and a maximum of three test and control patients per investigator .
investigators received randomization instructions only after enrolling a subject and immediately prior to surgery . primary and secondary endpoints and exploratory measures were as follows :
primary outcome variable
bone ridge dimension preservation horizontal change from baseline to 6 months , as measured using indexed stents , from the outside of the stent , at the apical extent of the buccal dehiscence , buccolingually to the interior bony wall .
secondary outcome variables
bone ridge dimension preservation vertical changes from baseline to 6 months , apicocoronally from the outside of the stent to the apical extent of the lingual and buccal walls .
exploratory variables
histomorphometric % new bone , % graft and % connective tissue / bone marrow components from midsection bone core biopsies at 6 months , including :
% new bone in contact with graft% graft in contact with new bone
wound closure ( incision line gap ) at all time points.soft tissue inflammationbaseline bone measurements extraction socket morphology measured directly at baseline surgery
baseline extraction socket dimensions , measured as the mesialdistal distance of the dehiscence at the crest and the coronalapical distance from the crest to most apical extent of the buccal dehiscence , and also as the overall socket dimensions , mesialdistal at crest and coronalapical from crest to the apical extent of the extraction socket.bony wall thickness , measured horizontally at the coronal tip of the most apical extent of the dehiscences of the buccal and , if present , lingual bony walls
.
primary outcome variable
bone ridge dimension preservation horizontal change from baseline to 6 months , as measured using indexed stents , from the outside of the stent , at the apical extent of the buccal dehiscence , buccolingually to the interior bony wall .
bone ridge dimension preservation horizontal change from baseline to 6 months , as measured using indexed stents , from the outside of the stent , at the apical extent of the buccal dehiscence , buccolingually to the interior bony wall .
secondary outcome variables
bone ridge dimension preservation vertical changes from baseline to 6 months , apicocoronally from the outside of the stent to the apical extent of the lingual and buccal walls .
bone ridge dimension preservation vertical changes from baseline to 6 months , apicocoronally from the outside of the stent to the apical extent of the lingual and buccal walls .
exploratory variables
histomorphometric % new bone , % graft and % connective tissue / bone marrow components from midsection bone core biopsies at 6 months , including :
% new bone in contact with graft% graft in contact with new bone
wound closure ( incision line gap ) at all time points.soft tissue inflammationbaseline bone measurements extraction socket morphology measured directly at baseline surgery
baseline extraction socket dimensions , measured as the mesialdistal distance of the dehiscence at the crest and the coronalapical distance from the crest to most apical extent of the buccal dehiscence , and also as the overall socket dimensions , mesialdistal at crest and coronalapical from crest to the apical extent of the extraction socket.bony wall thickness , measured horizontally at the coronal tip of the most apical extent of the dehiscences of the buccal and , if present , lingual bony walls .
histomorphometric % new bone , % graft and % connective tissue / bone marrow components from midsection bone core biopsies at 6 months , including :
% new bone in contact with graft% graft in contact with new bone
% new bone in contact with graft % graft in contact with new bone wound closure ( incision line gap ) at all time points .
soft tissue inflammation baseline bone measurements extraction socket morphology measured directly at baseline surgery
baseline extraction socket dimensions , measured as the mesialdistal distance of the dehiscence at the crest and the coronalapical distance from the crest to most apical extent of the buccal dehiscence , and also as the overall socket dimensions , mesialdistal at crest and coronalapical from crest to the apical extent of the extraction socket.bony wall thickness , measured horizontally at the coronal tip of the most apical extent of the dehiscences of the buccal and , if present , lingual bony walls .
baseline extraction socket dimensions , measured as the mesialdistal distance of the dehiscence at the crest and the coronalapical distance from the crest to most apical extent of the buccal dehiscence , and also as the overall socket dimensions , mesialdistal at crest and coronalapical from crest to the apical extent of the extraction socket .
bony wall thickness , measured horizontally at the coronal tip of the most apical extent of the dehiscences of the buccal and , if present , lingual bony walls .
prior to surgery , alginate impressions were obtained for fabrication of indexed measuring stents ( see fig .
medical and dental histories and demographic data were recorded ; oral exams and dental cleanings were performed .
( a ) baseline extraction socket measures insured that buccal wall mesialdistal and vertical dehiscences were at least 1/3 of the overall extraction socket dimensions .
( b & c ) measuring stents were fabricated from 0.020 thermoplastic and registered on adjacent teeth .
the stents included three indexing holes for measuring ridge buccallingual width , vertical height to the lingual wall and vertical height to the buccal wall .
following administration of antibiotics , extractions were performed with flap reflection , periotomes , elevators and forceps . to ensure that appropriate biopsy specimens were obtained , i.e. new bone and graft and not old host bone in the cores ,
extraction socket morphology dimensions were recorded through direct measures , and baseline extraction socket buccallingual and vertical dimensions were recorded using indexed stents , as described above and shown in fig .
co. , chicago , il , usa ) , depending on the dimension of the defect .
the measurement methods were similar to those employed by wood and mealey ( wood & mealey 2012 ) .
bone defects were filled with particulate bone grafts confined to the existing alveolar ridge dimensions , making no attempt to go outside the confines of ridge , and then covered with absorbable collagen membranes placed to cover the grafts and extend slightly 23 mm beyond bone defect margins .
periosteal releasing incisions were not employed , and incision lines were not required to be closed primarily so that mucogingival junction dimensions might be preserved . remaining soft tissue incision gaps , if any , were recorded both mesiodistally and buccallingually .
subjects were instructed to take ibuprofen 800 mg or hydrocodone / paracetamol up to three times a day for pain , as needed .
subjects were instructed to use chlorhexidine ( 0.12% ) mouth rinse for 30 s twice daily and to avoid excessive muscle tractioning or trauma and not to brush the study areas for the first 2 weeks .
amoxicillin was provided 875 mg bid for 1014 days ( with clindamycin 300 mg qid for 710 days for subjects with penicillin allergies ) .
after 2 weeks of healing , subjects were instructed in a brushing technique creating minimal apically directed trauma to the treatment area . at 4 weeks , the subjects were instructed to stop chlorhexidine rinses and resume normal oral hygiene practices . at 1week postsurgery
, photos of the test sites were taken , and clinical measurements of inflammation and remaining incision line gaps ( if any ) were assessed .
categorical inflammation scores were :
0 normal , ( absence of inflammation )
1 mild inflammation of any portion of the marginal unit , e.g. , slight change of colour
2 mild inflammation of entire gingival unit ( but no oedema )
3 moderate inflammation ( moderate glazing , redness , oedema and/or hypertrophy )
4 severe inflammation ( marked redness and oedema / hypertrophy , spontaneous bleeding or ulceration )
0 normal , ( absence of inflammation )
1 mild inflammation of any portion of the marginal unit , e.g. , slight change of colour
2 mild inflammation of entire gingival unit ( but no oedema )
3 moderate inflammation ( moderate glazing , redness , oedema and/or hypertrophy )
4 severe inflammation ( marked redness and oedema / hypertrophy , spontaneous bleeding or ulceration ) further followup evaluations occurred at 4weeks , 3months and 6months postsurgery .
photos of the test sites and clinical measurements of remaining incision line gaps ( if any ) were obtained , and inflammation was assessed .
oral hygiene instructions were reviewed throughout the study . at 1 , 3 and 6
months , dental cleanings were performed . throughout the study , any changes in medications or adverse events were noted . at 6months postsurgery
, the test sites were reentered for implant placement . prior to implant placement , vertical and horizontal ridge dimensions were recorded using the indexed measuring stents . to retrieve biopsies approximately 810 mm in length , 3 mm trephines ( ace surgical supply , bur trephine 35 tl 2id 2.8od ti 18.5cl
biopsies were retrieved from the implant sites , unless an implant was placed in original , nongrafted bone , in which case the biopsy was retrieved from the grafted area immediately adjacent to the implant , as indicated by the indexed stent .
biopsies were retained within their respective trephines , wrapped with lintfree gauze ( the open ends of the trephines serving as the apical orientation reference ) and preserved in 10% buffered formalin solution .
trephines together with biopsies were fixed in 4% formalin for 57 days , dehydrated in serial steps of ethanol ( 70% , 80% , 90% , 100% ) , remaining for 1 day in each concentration , and degreased for 1 day in xylene .
specimens were then infiltrated , embedded and polymerized ( technovit 9100heraeus kulzer , wehrheim , germany ) .
after polymerization , samples were hemisected and cut in 500 m sections using a precision cutting machine ( secotom 50 , stuers , ballerup , denmark ) , so that two sections were obtained per biopsy .
the sections were mounted onto opacified acrylicslides and ground to a final thickness of approximately 60 m on a rotating grinding plate ( stuers , ballerup , denmark ) .
specimens were subsequently stained with azure ii and pararosaniline ( axio imager m1 and axiocam hrc , carl zeiss , gttingen , germany ) .
histologic evaluation was performed with a microscope equipped with a digital analyzer ( axio imager m1 and axiocam hrc , carl zeiss , gttingen , germany ) .
histomorphometric analyses were performed with imaging software ( analysis five soft imaging system , mnster , germany ) on composite overview scans .
the area of new healing ( versus old / original bony tissues ) was demarcated in each section . within this area ,
prior to initiation of the study , using an indexed stent , stone cast model of an extraction socket exhibiting a substantial buccal dehiscence and a unc 15 periodontal probe ( hufriedy mfg .
co. , chicago , il , usa ) , all investigators were calibrated ( 1 mm ) to the principal investigator ( ets ) .
the first step in the analysis was to determine if the randomization resulted in balanced groups .
age , gender , race , ethnicity , bmi and all baseline clinical variables were tested to determine if the two groups were balanced .
the analyses were based on the variable types , with dichotomous variables tested using fisher exact test and categorical variables using chisquare test and ttests for continuous variables .
the primary hypothesis was that dbbmc + nbcm ( test ) was not inferior to dfdba + recxc ( control ) in the preservation of ridge volume as measured by the difference in the horizontal distance from stents , buccolingually to the interior ( lingual ) bony wall .
the noninferiority margin was 1.5 mm , as obtained from the approximate deviation in ridge dimension observed by wood and mealey .
twosample ttests were used to evaluate noninferiority , such that :
h0 :
ck
ob
o
ha :
ck
ob >
o
ck = the mean change in ridge volume for dbbmc + nbcm from baseline to 6 months
ob = the mean change in ridge volume for dfdba + recxc from baseline to 6 months
o = hypothesized mean difference = 0.0 mm
= noninferiority margin = 1.5 mm
= 0.05 , onesided1 = 0.90
ck = the mean change in ridge volume for dbbmc + nbcm from baseline to 6 months
ob = the mean change in ridge volume for dfdba + recxc from baseline to 6 months
o = hypothesized mean difference = 0.0 mm
= noninferiority margin = 1.5 mm if the test for noninferiority was statistically significant , then a twosample ttest for superiority was subsequently conducted , as detailed in the methodology of morikawa & yoshida ( 1995 ) .
treatment differences in the vertical ridge preservation changes ( baseline to 6 months ) , measured apicocoronally from the outside of the stent to the apical extent of the lingual and buccal walls were also evaluated using the noninferiority method listed above .
differences in histomorphometric variables were tested with twosample ttest for superiority due to the exploit nature of the variables at 6 months .
due to multiple testing for the five histomorphometric variables , the significance level had to be less than 0.05/6 = 0.008 ( bonferroni ) .
for histomorphometric differences to be clinically significant , a margin of at least 15.0% was needed based on the work of wood and mealey .
the behaviour of incision line closure over time was an additional goal of the analysis , with evaluations at 1 week , 1 month and at 3 months .
inferiority testing could not be done due to a lack of historical data and the potential time dependence of the data .
in addition to mean differences tested with ttests , the frequency of closed incisions was tested at each time point with fisher 's exact test .
sample size was predicated on assuring that the primary study objective had adequate power to assess the noninferiority hypothesis . under these assumptions , proc power ( sas version 9.2 , sas institute , inc , cary , north
carolina , usa ) required a sample size of 18 evaluable subjects in each treatment group .
therefore , a sample size of 40 subjects was considered sufficient to meet the primary objective of this study . although the study was powered for the primary outcome variable , the secondary outcome variables each retained 90% power to demonstrate noninferiority , assuming that the treatments were , in truth , equal
forty subjects were enrolled and treated as per protocol , though one randomization code was misinterpreted so that there were 21 dfdba + recxc and 19 dbbmc + nbcm cases .
the misinterpreted randomization code patient results were within the range of results reported . in all cases , grafting and soft tissue management were accomplished as directed by protocol ( fig . 2 ) .
left column dfdba + recxc and right column dbbmc + nbcm .
top to bottom ; original extraction socket illustrating extent of vertical and mesialdistal dehiscences , graft placement , membrane coverage , closure , soft tissue appearance at 6 months , ridge preservation at 6 months . at 6 months , of the 40 sites treated , all yielded areas that could be biopsied .
thirtyseven sites were deemed , according to the treating investigators , to have sufficient ridge preservation for implant placement .
the three sites with insufficient ridge volume occurred at three different investigation centres and were all from the dfdba + recxc treatment group .
baseline extraction socket defect measures were similar with no significant difference between dfdba + recxc and dbbmc + nbcm treatment sites .
soft tissue was approximated only , which left 36 of 40 sites open for secondary healing .
note : investigator centre effects were also examined and did not influence results or change significance levels .
a list of teeth treated according to investigation centre is provided in table 1 . as required by protocol ,
all extraction sites included buccal dehiscences greater than the vertical and mesialdistal extraction socket dimensions .
in fact , average dehiscences were approximately of the extraction socket vertical and mesialdistal dimensions .
investigator centres and tooth sites evaluated baseline defect measures were comparable ( not significantly different ) between the two therapies .
vertical ridge preservation changes from baseline to 6 months were also not significantly different ; however , horizontal changes were significantly different .
boxplots reveal the trend in bony ridge preservation differences between therapies , with dbbmc + nbcm providing median , first and third quartile values 13 mm greater in all dimensions .
however , given the wide range of results obtained ( see whiskers ) , only buccallingual ridge preservation results were statistically different age , gender , race , ethnicity and bmi were also comparable , though the dbbmc + nbcm treatment group had more lifetime tobacco use ( 10 of 19 subjects versus 4 of 21 dfdba + recxc subjects ) .
there was no difference in dental histories , though one of the dfdba + recxc subjects had a history of diabetes , which was deemed controlled .
there was no significant difference between treatment group subject oral hygiene compliance at any time point , though two dfdba + recxc subjects were recorded as not compliant at 1 week and one at 1 month , with all dbbmc + nbcm subjects compliant throughout the study .
horizontal changes from baseline to 6 months , as measured using stents , were significantly different between the two modalities , with dbbmc + nbcm sites , on average , providing more bony width ( 1.76 mm ) . from baseline to 6 months , vertical bone changes , measured at both buccal and lingual walls , were not significantly different between the two treatment modalities , although dbbmc + nbcm sites , on average , achieved more vertical ridge preservation ( fig .
complete trephine biopsy sections ( original magnification 50x , azure ii and pararosaniline ) for dfdba + recxc ( left pair ) and dbbmc + nbcm ( right pair ) , showing both original staining and digital labelling for histomorphometry .
( note that the split in the dbbmc + nbcm section was artifactual . )
yellow lines in the lateral regions delineate old bone ( ob ) from new bone ( nb ) and define the healing area of the defects , which were further labelled for the following tissue types : ( 1 ) red for nb
not in contact with graft , ( 2 ) pink for nb in contact with graft .
dark blue for dfdba
not in contact with nb , and light blue for dfdba in contact with nb .
light green for dbbmc
not in contact with nb , and dark green for dbbmc in contact with nb ( composite overview scans , individual microphotographs original magnification x50 ) .
there was no significant difference in inflammation between treatment modalities at any postoperative time point .
however , inflammation was higher at 1 week compared with later time points , and , on average , was higher for dfdba + recxc , with more 2 and 3 inflammation levels recorded ( 13 dfdba + recxc sties versus 8 dbbmc + nbcm sites ) . at 1 month , though overall inflammation scores decreased for both therapies , dfdba + recxc still involved more mild , localized inflammation than dbbmc + nbcm ( 13 versus 7 ) . by 3 months , inflammation was ranked as
inflammation at 1 week was nearly significantly correlated ( spearman correlation 0.0573 pvalue ) with horizontal ridge change over 6 months , in an inverse relationship , i.e. more inflammation tended to produce poorer ridge preservation results . at baseline ( surgical closure ) ,
incision line gaps were not significantly different between the two treatments , but at 1 week , the buccallingual gaps were , on average , 1.82 mm greater for dfdba + recxc sites . by 1 month , gap differences were statistically and significantly different ( approximately 1.5 mm difference
table 3 ) . additionally , at 1 month , the frequency of closed incision gaps was significantly higher for the dbbmc
+ nbcm group ( 14 out of 19 ) versus the dfdba + recxc ( 8 out 21 ) p = 0.0309 .
the frequency of closed incision lines was not different at either 1 week ( most still open in both groups ) or 3 months ( most closed in both groups ) .
though starting out with no significant difference ( baseline , following surgical closure ) , incision line gaps were significantly greater by 1 month for dfdba + recxc .
median values were approximately 1.52 mm greater for dfdba + recxc in both mesialdistal and buccallingual dimensions , though there was a wide range ( boxplot whiskers ) of gap measures for both therapies all biopsies ( n = 40 ) were successfully retrieved , processed , sectioned and digitally labelled for histomorphometry .
graft remnants were embedded either in bone marrow / connective tissue or juxtaposed with new bone trabeculae . in the lower ( apical ) portions of the sections ,
graft remnants were most often directly encompassed with or in contact with new bone . in the coronal portion of the sections , connective tissue / bone marrow tended to be more prevalent .
dfdba grafting biomaterial observed prior to implantation appeared to be a combination of both mineralized and demineralized bone , including possible nuclear material .
after 6 months , dfdba biopsies tended to show signs of remineralization emanating from the mineralized portions of the grafts , and around this remineralization zone , osteocyte lacunae sometimes appeared to be the nidi for remineralization islands .
occasionally , osteoclasts could be seen resorbing the graft mineral a phenomenon not observed with dbbmc grafts .
dbbmc granules , which were more prevalent than dfdba remnants , tended to form a dense trabecular network with new bone ( fig . 4 ) .
circular and elliptical outlines indicated endothelial structures of vascularization in both the apical and coronal portions of the sections , with the coronal portions also containing osteoid and osteoblasts .
osteonlike structures were sometimes , but rarely , seen within the dbbmc granules . ( a ) dfdba graft prior to implantation showing different degrees of mineralization within the virgin grafting material : fully mineralized bone ( mdb ) , partially demineralized bone ( pdb ) , and almost completely demineralized bone ( ddb ) , including osteocyte lacunae ( ol ) empty or filled with organic material .
( b ) six month biopsy showing remineralization of dfdba : demineralized dfdba ( ddb ) , mineralized dfdba ( mdb ) , remineralized dfdba ( rdb ) , and islandlike calcified structures ( i ) in the remineralization zone .
( c ) resorption ( r ) of dfdba by osteoclast ( oc ) a phenomenon not observed with dbbmc .
( d ) original , native bone ( ob ) with dbbmc ( bb ) embedded in connective tissue ( ct ) or in newly formed bone ( nb ) .
( e ) a possible vascular channel in dbbmc within the coronal portion of the biopsy .
( f ) woven new bone ( wnb ) with tightly integrated dbbmc ( bb ) granules forms a dense trabecular network ; loose connective tissue is free of inflammation and densely vascularized .
mean and standard deviation values for the percentages of each tissue type and graft remnants are presented in table 4
. only tissues ( and graft ) within the extraction socket defect were taken into account ; older original bone was excluded .
accordingly , the sum of new bone , connective tissue / bone marrow and graft equalled the entire area measured , or 100% .
relative area of biopsy section tissue components ( % ) , n = 40 the percentage of new bone formed was not significantly different between treatment modalities , though there were significantly more graft remnants within the dbbmc sites , and new bone in contact with graft remnants was , accordingly , greater for dfdba .
overall , there were no statistical differences between the two therapies either in the percentage of graft remnants in contact with new bone or in the overall percentage of trabecular network formed by the combination of new bone and grafts .
there was no statistical difference in baseline bony wall thicknesses between test and control therapies , but both the buccal and lingual baseline wall thicknesses were related to the amount of ridge preserved vertically and horizontally at 6 months ; inversely , smaller bony wall thicknesses were related to better ridge preservation results .
both were significant ( buccal p 0.02 ; lingual p 0.01 ) .
there was no significant relationship found between the size of the bony wall dehiscences at baseline and the ridge preservation achieved at 6 months . however , the baseline extraction socket measurement crest to apical extent of socket ( which was also not statistically different between test and control therapies ) was related to the 6 month ridge area changes , pvalue 0.03 .
the relationship was positive , i.e. deeper baseline defects were associated with better ridge preservation at 6 months .
horizontal changes from baseline to 6 months , as measured using stents , were significantly different between the two modalities , with dbbmc + nbcm sites , on average , providing more bony width ( 1.76 mm ) .
from baseline to 6 months , vertical bone changes , measured at both buccal and lingual walls , were not significantly different between the two treatment modalities , although dbbmc + nbcm sites , on average , achieved more vertical ridge preservation ( fig . 3 ) .
complete trephine biopsy sections ( original magnification 50x , azure ii and pararosaniline ) for dfdba + recxc ( left pair ) and dbbmc + nbcm ( right pair ) , showing both original staining and digital labelling for histomorphometry .
( note that the split in the dbbmc + nbcm section was artifactual . )
yellow lines in the lateral regions delineate old bone ( ob ) from new bone ( nb ) and define the healing area of the defects , which were further labelled for the following tissue types : ( 1 ) red for nb
not in contact with graft , ( 2 ) pink for nb in contact with graft .
dark blue for dfdba
not in contact with nb , and light blue for dfdba in contact with nb .
light green for dbbmc
not in contact with nb , and dark green for dbbmc in contact with nb ( composite overview scans , individual microphotographs original magnification x50 ) .
there was no significant difference in inflammation between treatment modalities at any postoperative time point . however , inflammation was higher at 1 week compared with later time points , and , on average , was higher for dfdba + recxc , with more
2 and 3 inflammation levels recorded ( 13 dfdba + recxc sties versus 8 dbbmc + nbcm sites ) . at 1 month , though overall inflammation scores decreased for both therapies , dfdba + recxc still involved more mild , localized inflammation than dbbmc + nbcm ( 13 versus 7 ) . by 3 months
inflammation at 1 week was nearly significantly correlated ( spearman correlation 0.0573 pvalue ) with horizontal ridge change over 6 months , in an inverse relationship , i.e. more inflammation tended to produce poorer ridge preservation results . at baseline ( surgical closure ) ,
incision line gaps were not significantly different between the two treatments , but at 1 week , the buccallingual gaps were , on average , 1.82 mm greater for dfdba + recxc sites . by 1 month , gap differences were statistically and significantly different ( approximately 1.5 mm difference
additionally , at 1 month , the frequency of closed incision gaps was significantly higher for the dbbmc + nbcm group ( 14 out of 19 ) versus the dfdba + recxc ( 8 out 21 ) p = 0.0309 .
the frequency of closed incision lines was not different at either 1 week ( most still open in both groups ) or 3 months ( most closed in both groups ) .
though starting out with no significant difference ( baseline , following surgical closure ) , incision line gaps were significantly greater by 1 month for dfdba + recxc .
median values were approximately 1.52 mm greater for dfdba + recxc in both mesialdistal and buccallingual dimensions , though there was a wide range ( boxplot whiskers ) of gap measures for both therapies
all biopsies ( n = 40 ) were successfully retrieved , processed , sectioned and digitally labelled for histomorphometry .
graft remnants were embedded either in bone marrow / connective tissue or juxtaposed with new bone trabeculae . in the lower ( apical ) portions of the sections ,
graft remnants were most often directly encompassed with or in contact with new bone . in the coronal portion of the sections , connective tissue / bone marrow tended to be more prevalent .
dfdba grafting biomaterial observed prior to implantation appeared to be a combination of both mineralized and demineralized bone , including possible nuclear material .
after 6 months , dfdba biopsies tended to show signs of remineralization emanating from the mineralized portions of the grafts , and around this remineralization zone , osteocyte lacunae sometimes appeared to be the nidi for remineralization islands .
occasionally , osteoclasts could be seen resorbing the graft mineral a phenomenon not observed with dbbmc grafts .
dbbmc granules , which were more prevalent than dfdba remnants , tended to form a dense trabecular network with new bone ( fig .
circular and elliptical outlines indicated endothelial structures of vascularization in both the apical and coronal portions of the sections , with the coronal portions also containing osteoid and osteoblasts .
osteonlike structures were sometimes , but rarely , seen within the dbbmc granules . ( a ) dfdba graft prior to implantation showing different degrees of mineralization within the virgin grafting material : fully mineralized bone ( mdb ) , partially demineralized bone ( pdb ) , and almost completely demineralized bone ( ddb ) , including osteocyte lacunae ( ol ) empty or filled with organic material .
( b ) six month biopsy showing remineralization of dfdba : demineralized dfdba ( ddb ) , mineralized dfdba ( mdb ) , remineralized dfdba ( rdb ) , and islandlike calcified structures ( i ) in the remineralization zone .
( c ) resorption ( r ) of dfdba by osteoclast ( oc ) a phenomenon not observed with dbbmc .
( d ) original , native bone ( ob ) with dbbmc ( bb ) embedded in connective tissue ( ct ) or in newly formed bone ( nb ) .
( e ) a possible vascular channel in dbbmc within the coronal portion of the biopsy .
( f ) woven new bone ( wnb ) with tightly integrated dbbmc ( bb ) granules forms a dense trabecular network ; loose connective tissue is free of inflammation and densely vascularized . mean and standard deviation values for the percentages of each tissue type and graft remnants are presented in table 4
. only tissues ( and graft ) within the extraction socket defect were taken into account ; older original bone was excluded .
accordingly , the sum of new bone , connective tissue / bone marrow and graft equalled the entire area measured , or 100% .
relative area of biopsy section tissue components ( % ) , n = 40 the percentage of new bone formed was not significantly different between treatment modalities , though there were significantly more graft remnants within the dbbmc sites , and new bone in contact with graft remnants was , accordingly , greater for dfdba .
overall , there were no statistical differences between the two therapies either in the percentage of graft remnants in contact with new bone or in the overall percentage of trabecular network formed by the combination of new bone and grafts .
there was no statistical difference in baseline bony wall thicknesses between test and control therapies , but both the buccal and lingual baseline wall thicknesses were related to the amount of ridge preserved vertically and horizontally at 6 months ; inversely , smaller bony wall thicknesses were related to better ridge preservation results .
both were significant ( buccal p 0.02 ; lingual p 0.01 ) .
there was no significant relationship found between the size of the bony wall dehiscences at baseline and the ridge preservation achieved at 6 months .
however , the baseline extraction socket measurement crest to apical extent of socket ( which was also not statistically different between test and control therapies ) was related to the 6 month ridge area changes , pvalue 0.03 .
the relationship was positive , i.e. deeper baseline defects were associated with better ridge preservation at 6 months .
this investigation compared two ridge preservation methodologies in substantial buccal wall dehiscence defects . since bone remodelling and soft tissue collapse and their interference with bony healing are common in these defects , they provide a critical methodological test .
one technique ( dfdba + rexc ) was derived from the history of demineralized allograft use and the belief that such allografts might be osteoinductive and produce bone formation .
dfdba was combined with a reconstituted and crosslinked collagen membrane thought to better preserve ( through stiffness ) the volume intended for regeneration , and for a longer time than noncrosslinked collagen membranes .
the other technique ( dbbmc + nbcm ) was derived from a history of mineralized xenogeneic graft use and the belief that such grafts might be osteoconductive , giving rise to bone integration , and yet resorb slowly to preserve the volume intended for regeneration .
dbbmc was combined with an extracted and noncrosslinked collagen membrane thought to encourage transmembrane vascularity and rapid tissue integration , with the membrane degrading over a time period thought sufficient for bone regeneration . for the primary outcome of ridge preservation , horizontal ridge preservation was significantly greater for dbbmc + nbcm .
there was only a trend , and not statistically significant , for more vertical bone preservation with dbbmc + nbcm .
as a clinical consequence , implants could be placed in all but three treatment sites , which were dfdba+ recxc sites .
( 2015 ) , he postulated that , following tooth extraction , ( i ) up to 50% reduction in the original ridge width will occur ; ( ii ) the amount of bone resorption will be greater at the buccal aspect than at its lingual / palatal counterpart ; and ( iii ) a larger amount of alveolar bone reduction will take place in the molar regions . in this review of clinical studies with various biomaterials , it was concluded that ridge contraction following tooth extraction can be minimized with socket grafts and/or the use of mechanical barriers .
while grafts alone might be sufficient for spacemaintaining sockets , others have found grafts plus membranes to be advantageous , particularly for limited spacemaintaining defects like the dehiscence defects studied herein ( kim et al .
the secondary outcomes , based on histomorphometry , incision line gap closure and degree of inflammation , were included in the hope they might help explain the primary outcome of ridge preservation .
indeed , the percentage of new bone seen in the biopsy sections was not statistically different between the two therapies studied .
this , in turn , may support the premise that minimal dbbmc resorption provided spacemaintenance for improved ridge volume ( buser et al .
2014 ) . moreover , there was no difference in the percentage of trabecular structure ( new bone plus graft ) observed between the two therapies .
dbbmc biopsies showed little to no signs of osteoclastic resorption and graft remodelling ; rather , dbbmc was intimately incorporated into new bone trabeculae . however , over time , particularly in the coronal regions where bone formation appeared less advanced than in apical regions , studies of dbbmc in the sinus have indicated woven bone turns into lamellar bone , along with further bone formation , integration and maturation ( sartori et al .
dfdba biopsies depicted robust bone formation with islands of new bone that might be interpreted as evidence of bone induction ; but in contrast to dbbmc , dfdba appeared to be in a more active state of turnover and replacement .
this boneremodelling phenomenon has also been observed in the maxillary sinus ( soardi et al .
2013 ) a phenomenon that might help explain the difference in ridge volume preservation observed between the two therapies in this study . examining soft tissue healing ,
the spearman correlation , pvalue of 0.0573 , just on the edge of significance , was a strong pvalue , considering inflammation was a categorical and subjective variable .
more inflammation was observed , by count , at dfdba + recxc sites .
of the 18 sites with incision line gaps still open at 1 month , 13 were dfdba + recxc .
early vascularization and soft tissue integration of nbcm membranes , as compared with crosslinked collagen membranes , may have improved soft tissue healing and underlying bony tissue regeneration results , with nbcm membranes degrading over a time period reported as sufficient for guided regeneration ( bornstein et al .
2007 , schwarz et al . 2008 ) . following the hypothesis that gbr was simply a consequence of tissue separation and that the volume created by the membranes determined the volume of bone that could be regenerated , stiffer ,
titanium reinforced and shapeable expanded polytetrafluoroethylene ( eptfe ) membranes were developed ( schenk et al .
these membranes were designed for defects in which there was an absence of residual bony walls , i.e. the defects themselves were not able to prevent membrane collapse into the area intended for gbr .
however , eptfe membrane shortcomings included soft tissue dehiscences , along with potential inflammation and infection , recognized as clinical complications that diminished regenerative therapy outcomes ( machtei et al .
, biodegradable collagen membranes were not only designed to integrate with the healing tissues but also to degrade over time , particularly when exposed to the oral environment ( zitzmann et al .
1997 ) . still , the quest for spacemaintaining membranes and the uncertainty over what might be a suitable time period for degradable membranes to remain intact led to investigations of stiffer and more slowly degrading collagen barriers ( bunyaratavej & wang 2001 ) .
despite this history , and even though the historical suggested removal time for eptfe in gbr procedures was after several months of healing , today some researchers speculate that the regenerative disposition of tissues may occur over the course of a few weeks ( susin et al .
membrane integration and duration of resorption , which could be associated with the degree of soft tissue inflammation and incision line gapping reported in the study herein , might help explain the differences observed between the two therapies compared .
regardless , given the four biomaterials tested in this study ( two grafts and two membranes ) , it was not possible to isolate cause and effect for any single biomaterial
. only the combined effects of the grafts and their respective membranes could be evaluated .
other potential design limitations included evaluation of implant integration and survival , the number of investigators and the variety of defect morphologies tested .
success of implant integration and longterm survival were not reported herein but will be provided in a subsequent report . despite the potential variability inherent with a large group of investigators
, the number of investigators may have provided a more poignant finding , with results that might better represent the clinical community at large .
baseline extraction bony wall thickness and extraction socket depth ( crest to apex ) were related to ridge preservation outcomes , and case photos appeared to indicate both of these phenomena involved the regenerative potential of the baseline defects , i.e. remaining bony walls ( measured as thin because of their coronal extension and deep in extent ) were more spacemaintaining .
in future studies , extraction defect morphologies should be further apportioned for more definitive analyses .
in addition , studies should be performed in the anterior region , where aesthetics , labial bone loss and soft tissue management are more critical .
finally , we recommend , as reported herein , that future ridge preservation studies include substantial , buccal wall dehiscence defects as critical tests of biomaterials , surgical techniques and , ultimately , patient outcomes . | abstractaimto compare the effectiveness of tworidge preservation treatments.materials and methodsforty subjects with extraction sockets exhibiting substantial buccal dehiscences were enrolled and randomized across 10 standardized centres .
treatments were demineralized allograft plus reconstituted and crosslinked collagen membrane ( dfdba + recxc ) or deproteinized bovine bone mineral with collagen plus native , bilayer collagen membrane ( dbbmc + nbcm ) .
socket dimensions were recorded at baseline and 6 months .
wound closure and soft tissue inflammation were followed postoperatively , and biopsies were retrieved for histomorphometric analysis at 6 months.resultsprimary endpoint : at 6 months , extraction socket horizontal measures were significantly greater for dbbmc + nbcm ( average 1.76 mm greater , p = 0.0256 ) .
secondary and exploratory endpoints : ( 1 ) lingual and buccal vertical bone changes were not significantly different between the two treatment modalities , ( 2 ) histomorphometric % new bone and % new bone + graft were not significantly different , but significantly more graft remnants remained for dbbmc ; ( 3 ) at 1 month , incision line gaps were significantly greater and more incision lines remained open for dfdba + recxc ; ( 4 ) higher inflammation at 1 week tended to correlate with lower ridge preservation results ; and ( 5 ) deeper socket morphologies with thinner bony walls correlated with better ridge preservation .
thirtyseven of 40 sites had sufficient ridge dimension for implant placement at 6 months ; the remainder were dfdba + recxc sites.conclusion
dbbmc + nbcm provided better soft tissue healing and ridge preservation for implant placement .
deeper extraction sockets with higher and more intact bony walls responded more favourably to ridge preservation therapy . |
keratosis follicularis spinulosa decalvans ( kfsd ) is a hereditary disorder of the hair follicle which presents with scarring alopecia and follicular papules affecting the scalp and other areas of the body .
it usually follows an x - linked pattern of inheritance , but sporadic and autosomal dominant cases can also be seen .
our first case was an 18-year - old female who complained of progressive hair loss over the scalp since 10 years and over the eyebrows since 5 years .
on examination of the scalp , she had multiple patches of cicatricial alopecia with sparse hair in the frontal , parietal , and temporal areas of the scalp .
sparse hair over the eyebrows , eyelashes , axillae , and pubic area were noted [ figure 1 ] . she also had generalized dry skin and multiple follicular papules over upper and lower limbs and buttocks [ figure 2 ] .
follicular papules and alopecia seen over the scalp along with sparse eyebrows and eyelashes follicular keratotic papules over dorsum of hand our second case was a 26-year - old male with decreased hair growth over scalp and body .
he had no family history of similar complaints . on examination , he had diffuse alopecia of the scalp with sparse hair over eyebrows , eyelashes , and beard area .
multiple keratotic papules were noted over the scalp , upper limbs , and abdomen [ figures 3 and 4 ] . sparse hair and papular lesions noted over the scalp follicular papules seen over abdomen the third case was a 23-year - old male with complaints of decreased hair over scalp and body with skin lesions over the trunk .
he gave a history of similar but milder complaints being present in his younger brother suggesting x - linked inheritance .
on examination , he had alopecia over the crown area of the scalp [ figure 5 ] , with sparse hair over the eyebrows and axillae , multiple follicular papules over the trunk and all four limbs .
scarring alopecia seen over scalp plantar keratoderma seen over the foot scalp biopsies from all three cases showed variable amounts of acanthosis and papillomatosis and dilated hair follicles filled with keratin plugs and entrapped hair shafts [ figures 7 and 8 ] .
dilated follicular infundibulum filled with keratin plug ( h and e , 40 ) entrapped hair shafts within hair follicle ( h and e , 40 )
keratosis pilaris atrophicans ( kpa ) is a group of cutaneous disorders which are characterized by follicular hyperkeratosis along with scarring .
kfsd ( omim 308800 ) is a rare x - linked disorder of the hair follicle .
the term was first used by siemens in 1926 when he reported the disorder in a bavarian family .
the candidate gene suggested is the membrane - bound transcription factor protease site 2 ( mbtps2 ) gene which is required for cleavage of sterol regulatory element - binding proteins ( srebps ) .
altered srebp cleavage impairs cholesterol and lipid homeostasis in the skin causing defective epidermal differentiation .
males are more commonly and severely affected than female heterozygotes in families having an x - linked inheritance pattern .
the age of onset is early in life with keratotic papules appearing initially on the face and later on the trunk and limbs .
as the disease progresses , cicatricial alopecia ensues on the scalp , eyebrows , and eyelashes along with the loss of hair in axillary and pubic areas .
the most important differential diagnoses include ichthyosis follicularis alopecia photophobia ( ifap ) syndrome and graham little piccardi lasseur syndrome ( glpls ) .
however , the alopecia in ifap syndrome is of the nonscarring type while that in kfsd is scarring .
furthermore , ocular features like photophobia and corneal dystrophy are essential for the diagnosis of ifap .
glpls is a variant of lichen planopilaris which presents with scarring alopecia of the scalp , nonscarring alopecia of axillae and pubic region , and keratotic follicular spinous papules over a body .
the distinction from kfsd is made mainly on histopathology which reveals changes suggestive of lichen planus including vacuolar degeneration and interface dermatitis .
systemic retinoids like isotretinoin and etretinate are beneficial in the early stages of the disease as they downregulate follicular hyperkeratosis and inflammation .
dapsone was found to be useful due to its inhibition of leukocyte chemotaxis and stabilization of lysosomal enzymes .
the authors certify that they have obtained all appropriate patient consent forms . in the form the patient(s )
has / have given his / her / their consent for his / her / their images and other clinical information to be reported in the journal .
the patients understand that their names and initials will not be published and due efforts will be made to conceal their identity , but anonymity can not be guaranteed .
the authors certify that they have obtained all appropriate patient consent forms . in the form the patient(s )
has / have given his / her / their consent for his / her / their images and other clinical information to be reported in the journal .
the patients understand that their names and initials will not be published and due efforts will be made to conceal their identity , but anonymity can not be guaranteed . | keratosis follicularis spinulosa decalvans is a disorder affecting the hair follicles characterized by scarring alopecia of the scalp , eyebrows , and axillae , sometimes associated with photophobia and keratoderma .
being x - linked , it is more commonly seen in males but can be rarely seen in females also .
we report three cases of this rare disorder including one in a female . |
a 6-year - old female spayed birman cat presented with a history of weight loss , stiff and short - strided gait in the pelvic limbs and reluctance to jump , progressing to non - ambulatory tetraparesis over 6 weeks .
neurological examination revealed mildly depressed mental status , non - ambulatory flaccid tetraparesis and severely decreased proprioception and spinal reflexes in all four limbs .
haematology , feline immunodeficiency virus / feline leukaemia virus serology , serum biochemistry , including creatine kinase and thyroxine , thoracic radiographs and abdominal ultrasound did not reveal significant abnormalities .
electromyography revealed fibrillation potentials and positive sharp waves in axial and appendicular muscles . decreased motor conduction velocities and compound muscle action potential amplitudes were detected in ulnar and sciatic tibial nerves .
histologically , several intramuscular nerve branches were depleted of myelinated fibres and a few showed mononuclear infiltrations . toxoplasma gondii serology titres were compatible with active toxoplasmosis .
four days after treatment initiation with oral clindamycin the cat recovered the ability to walk .
t gondii serology titres and neurological examination were normal after 11 and 16 weeks , respectively .
one year after presentation the cat showed mild relapse of clinical signs and seroconversion , which again resolved following treatment with clindamycin .
to our knowledge , this is the first report of distal polyneuropathy associated with toxoplasmosis in a cat .
this case suggests the inclusion of toxoplasmosis as a possible differential diagnosis for acquired polyneuropathies in cats .
t gondii can potentially affect all warm - blooded animals , including humans , with cats and other felidae being definitive hosts .
congenital infection and ingestion of infected tissue from intermediate hosts or of food and water contaminated with sporulated oocysts are the primary modes of transmission in cats . neurological signs in cats with serologically or histologically confirmed toxoplasmosis are rare , with reports of 1/15 and 7/100 cases .
non - suppurative myelitis / meningomyelitis and intracranial granuloma with histological identification of t gondii or toxoplasma - like parasites have been previously documented in cats with neurological signs including seizures , circling , altered behaviour , blindness , anisocoria , ataxia , para-/hemi-/tetraparesis or paraplegia .
considering the association between toxoplasmosis and polyradiculoneuritis in dogs , the potential for t gondii to affect the peripheral nervous system in cats has always been suspected ; however , to our knowledge , no clinical report confirming this hypothesis has been published previously .
herein we describe clinical , serological , electrodiagnostic , histological findings and treatment in an adult birman cat presented for progressive neuromuscular signs associated with distal polyneuropathy and serologically confirmed toxoplasmosis .
a 6-year - old spayed female birman cat presented with a history of weight loss , stiff and short - strided gait in the pelvic limbs and reluctance to jump , progressing to non - ambulatory tetraparesis over 6 weeks .
the cat had always lived in the uk and was rehomed 2 months before the onset of neurological signs .
when initially presented to the primary veterinarian , lumbosacral pain was suspected and 0.1 mg / kg meloxicam ( metacam ; boehringer ingelheim ) po q24h was initiated .
the treatment was discontinued after 48 h because of vomiting , diarrhoea and lack of clinical improvement .
the neurological signs slowly progressed for 5 weeks then rapidly deteriorated 1 week before referral , when the cat became non - ambulatory paraparetic and , finally , non - ambulatory tetraparetic . at that stage diarrhoea also recurred .
the owner reported a weight loss of 600 g , despite the cat having a normal appetite . at presentation , the cat s body weight was 2.05 kg and body condition score was 3/9 .
the neurological examination revealed mildly obtunded mental status , non - ambulatory flaccid tetraparesis ( almost plegia in the pelvic limbs ) and severely decreased to absent general proprioception and spinal reflexes in all four limbs .
differential diagnoses included infectious , immune - mediated , metabolic , idiopathic , neoplastic , paraneoplastic and degenerative polyneuropathies .
twelve hours after presentation , the cat was sedated with 0.2 mg / kg butorphanol ( alvegesic vet ; dechra ) iv and 0.25 mg / kg midazolam ( hypnovel ; roche ) iv .
anaesthesia was induced with propofol ( vetofol ; norbrook ) to effect and maintained with isofluorane in oxygen .
electromyography revealed fibrillation potentials and positive sharp waves in axial and appendicular muscles ( figure 1 ) .
the motor nerve conduction study is summarised in table 1 ( figure 2a , b ) .
biopsies were obtained from the right biceps femoris and tibialis cranialis muscles and from the right common peroneal nerve .
stains employed included haematoxylin and eosin , modified gomori trichrome , periodic acid schiff , myofibrillar adenosine triphosphatases at ph 9.8 and ph 4.3 , esterase , nicotinamide adenine dinucleotide tetrazolium reductase , acid phosphatase , alkaline phosphatase , oil red o and staphylococcal protein a conjugated to horseradish peroxidase .
there was a marked variability in myofibre size with numerous large and small groups of atrophic fibres having an angular - to - anguloid shape .
a few of the depleted nerve branches showed an excessive mononuclear cellularity ( figure 3a , b ) .
a fixed biopsy from the right common peroneal nerve was plastic embedded and evaluated in 1 m sections .
the density of myelinated fibres was subjectively appropriate without obvious axonal degeneration , demyelination or abnormalities of the supporting structures .
electromyography of the left biceps femoris muscle showing fibrillation potentials and positive sharp waves motor nerves conduction study mncv = motor nerve conduction velocity ; cmap = compound muscle action potential ( a ) ulnar nerve conduction study showing decreased amplitude , temporal dispersion and polyphasia .
( b ) sciatic tibial nerve conduction study showing decreased amplitude and temporal dispersion ( a ) periodic acid
schiff ( pas ) stain of a cryosection from the cranial tibial muscle showing an intramuscular nerve branch with partial depletion of myelinated fibres and mild mononuclear cell infiltrations . with the pas stain , myelin stains dark purple .
( b ) immunofluorescence staining of cryosections from the cranial tibial muscle using an antibody against feline major histocompatibility complex ii .
( a , b ) scale bar = 50 m after obtaining the surgical biopsies , clindamycin ( clindacyl ; vetoquinol ) was started at 18.3 mg / kg orally q12h .
the cat started improving within 24 h and was ambulatory , with occasional plantigrade posture , 4 days after treatment initiation .
igg and igm t gondii serology titres measured with indirect immunofluorescence were 1:200 and 1:160 , respectively .
the owner reported that the cat had been inappetent during the previous week and clindamycin administration was challenging . on neurological examination the cat was again non - ambulatory tetraparetic ; however , when adequately supported , the motor function was greater than at initial presentation . repeated t gondii serology titres revealed 1:50 igg and 1:40 igm .
after 3 days of appropriate administration of clindamycin the cat was again ambulatory but only for short distances , with occasional plantigrade posture .
muscle size and withdrawal reflexes returned to normal in all four limbs 11 and 16 weeks after treatment initiation , respectively .
clindamycin was discontinued after 16 weeks of treatment , when the cat recovered the ability to jump onto house furniture .
one year after initial presentation , the owner noticed that the cat again became reluctant to jump . at that stage ,
clindamycin was restarted at the same total dose ( at that stage equivalent to 14.4 mg / kg ) with improvement and normalisation of the ability to jump within 1 and 3 weeks , respectively .
t gondii serology titres were rechecked 2 and 4 weeks after the suspected relapse and revealed negative igg with igm 1:40 , and igg 1:200 with igm 1:40 , respectively .
two months after the relapse , the serology titres showed again negative igg and 1:40 igm .
clindamycin was then discontinued and the cat has remained clinically normal for the 4 months until the time of writing of this report .
differential diagnoses for acquired polyneuropathies in cats include infectious , immune - mediated , traumatic , toxic , metabolic , nutritional , idiopathic , neoplastic and degenerative diseases .
reports that associate specific infectious agents with neuropathies in cats are limited to an experimentally induced polyneuropathy / polymyopathy in a cat inoculated with fiv , one case of mycobacterial neuritis , and one case of polyneuritis / polymyositis with associated fever , anaemia and icterus in which numerous intramuscular sarcosporidia species were detected . in our case ,
the clinical presentation , diagnostic investigations and outcome were suggestive of t gondii as the possible causative agent of the polyneuropathy .
the current guidelines for ante - mortem diagnosis of toxoplasmosis include : ( 1 ) serum igm titres > 1:64 or fourfold increase in igg titres over 23 weeks ; ( 2 ) presence of appropriate clinical signs ; ( c ) exclusion of other possible causes ; and ( d ) positive response to appropriate medications . the case we reported fulfilled at least three of the above criteria . regarding the presence of appropriate clinical signs , as no other case of polyneuropathy associated with toxoplasmosis in cats
was previously described it may be difficult to support the fulfilling of this criterion ; however , as reported above , toxoplasmosis is a recognised differential diagnosis of polyneuropathy in dogs , and it has been previously discussed by other authors as a possible cause of similar signs in cats . despite the absence of direct parasite identification on histopathology , a causal relation between t gondii and the clinical signs in this case is suggested by the serology titres compatible with active infection at both initial presentation and relapse ( significant increase of igg in 4 weeks from clinical recurrence ) and by the rapid response to clindamycin in three different occasions .
further tests to detect the presence of t gondii in the muscle samples , including pcr , could have been performed ; however , a positive result would have not necessarily supported the causal relation between the parasite and the clinical signs as detectable parasitic dna does not necessarily differentiate between chronic and acute forms of the disease . considering the lack of exposing factors after being rehomed , reactivation of a previous infection was hypothesised .
the electrodiagnostic findings were suggestive of an axonopathy with a demyelinating component , similar to findings reported in bengal and other young cats with recurrent demyelinating remyelinating polyneuropathy ( rdrp ) .
a distal localisation of the disease was suspected based on the mildly increased residual latency in the sciatic
a limitation in this report is that f - wave , sensory nerve conduction and repetitive nerve stimulation studies were not performed , which could have further characterised the distribution of the disease .
cerebrospinal fluid analysis would also have helped in excluding a radicular and/or central involvement ; however , it was declined by the owner .
nevertheless , even if not confirmed with electrodiagnostic investigations , the clinically preserved sensory function together with the severely affected motor function suggested a predominantly motor polyneuropathy , and the normal histological appearance of the nerve biopsy with associated significant distal pathology made the concurrent possibility of a radicular component of the disease unlikely .
our histological findings indeed revealed a moderately severe neuropathy characterised by distal nerve fibre loss and mononuclear infiltrates .
the pathogenic mechanism causing distal nerve fibre depletion and inflammation in the absence of direct identification of t gondii remains unclear ; however , it is possible that parasitic cysts were present in other areas of the sampled muscles or in unsampled muscles .
an immune - mediated process triggered by the protozoal infection could not be excluded ; however , the clinical response to clindamycin was considered too rapid to be compatible with this option .
the possibility of the positive serology being an incidental / unrelated finding and the response to antibiotic therapy coincidental owing to a self - limiting disease was also considered ; however , this was deemed improbable considering the serology titres suggestive of active infection at both presentation and recurrence of clinical signs and the consistently rapid response to clindamycin .
similar histological findings affecting the distal nerve fibres were previously described in young bengal cats with self - resolving rdrp . however , in adjunction to the different signalment , in our case no pathological changes were present in the nerve biopsy that would suggest demyelination or remyelination .
history and histopathological findings in our cat also differ from previously reported cases of self - resolving acute idiopathic polyneuropathy that were characterised by acute onset , rapid progression and moderate - to - severe axonal degeneration and demyelination in ventral nerve roots and nerves .
chronic inflammatory demyelinating polyneuropathy ( cidp ) is an adult - onset , chronic , slowly progressive inflammatory polyneuropathy with some clinical similarities to the case we described .
however , histological findings in cidp differs from our case as characterised by primary paranodal demyelination and mononuclear endoneurial infiltrates sparing the intramuscular nerve branches .
in addition , 88% cats with cidp have been reported to improve following steroid treatment , which was not administered to our cat .
a steroid - responsive chronic , slowly progressive neuropathy has also been reported in two cats with pronounced depletion of intramuscular nerve fibres but in the absence of inflammatory infiltrates . in birman cats ,
this condition is characterised by an onset of signs at 810 weeks of age , slow deterioration and histopathological findings of axonal degeneration , and therefore was considered an unlikely differential diagnosis in this birman cat .
clindamycin has been described as the drug of choice in the treatment of toxoplasmosis . of the cats with
previously reported neurological toxoplasmosis , two cases with myelitis received clindamycin with no clinical improvement , and two cases with encephalic granuloma underwent decompressive surgery and clindamycin treatment with recurrence of clinical signs at 8 and 21 months , respectively . in our case ,
the initial response to clindamycin was extremely rapid ; however , 4 months were required to obtain clinical remission .
considering the severity of the clinical signs , the immediate relapse associated with inappropriate treatment administration and the absence of previously described successfully treated similar cases , we elected to continue the antiprotozoal therapy until resolution of the clinical signs .
apart from the possibly related inappetance that developed after 2 weeks of treatment , the drug was well tolerated .
it is possible that prolonging the treatment until clinical remission was an unnecessary precaution as the active infection may have already subsided , with the persistent clinical signs being simply associated with gradual recovery of anatomical and functional integrity of the affected structures ; however , this can not be proven .
given the absence of direct association between serology titres and clinical signs , basing the duration of treatment on normalisation of the titres was not recommended .
the episode of suspected relapse was supported by repeated serology showing positive igm , significant increase of the igg within 4 weeks ( therefore compatible with active toxoplasmosis according to the reported guidelines ) and again rapid clinical response to clindamycin .
here we describe a case of serologically confirmed toxoplasmosis in a cat with distal polyneuropathy characterised by intramuscular nerve fibre depletion and mononuclear infiltrates .
long - term treatment with clindamycin was efficacious in achieving remission of the clinical signs .
an episode of mild relapse occurred 1 year after diagnosis and responded to reintroduction of the treatment .
this case suggests the inclusion of toxoplasmosis as a possible differential diagnosis for acquired polyneuropathies in cats . | case summarya 6-year - old female spayed birman cat presented with a history of weight loss , stiff and short - strided gait in the pelvic limbs and reluctance to jump , progressing to non - ambulatory tetraparesis over 6 weeks .
poor body condition , dehydration and generalised muscle wastage were evident on general examination .
neurological examination revealed mildly depressed mental status , non - ambulatory flaccid tetraparesis and severely decreased proprioception and spinal reflexes in all four limbs .
the neuroanatomical localisation was to the peripheral nervous system .
haematology , feline immunodeficiency virus / feline leukaemia virus serology , serum biochemistry , including creatine kinase and thyroxine , thoracic radiographs and abdominal ultrasound did not reveal significant abnormalities .
electromyography revealed fibrillation potentials and positive sharp waves in axial and appendicular muscles . decreased motor conduction velocities and compound muscle action potential amplitudes
were detected in ulnar and sciatic tibial nerves .
residual latency was increased in the sciatic
tibial nerve .
histologically , several intramuscular nerve branches were depleted of myelinated fibres and a few showed mononuclear infiltrations .
toxoplasma gondii serology titres were compatible with active toxoplasmosis .
four days after treatment initiation with oral clindamycin the cat recovered the ability to walk .
t gondii serology titres and neurological examination were normal after 11 and 16 weeks , respectively .
clindamycin was discontinued after 16 weeks .
one year after presentation the cat showed mild relapse of clinical signs and seroconversion , which again resolved following treatment with clindamycin.relevance and novel informationto our knowledge , this is the first report of distal polyneuropathy associated with toxoplasmosis in a cat .
this case suggests the inclusion of toxoplasmosis as a possible differential diagnosis for acquired polyneuropathies in cats . |
severe skin damage and loss of the protective layer exposes underlying tissue to secondary infections [ 1 , 2 ] . in the united states , treatment of fire and
burn wound infections amount to more than 7.5 billion us dollars per annum . from one million patients , an estimated 10
000 die from secondary microbial infections [ 2 , 49 ] . if wounds are not treated effectively , pathogens form biofilms , rendering them resistant to antibiotics [ 1013 ] . in severe cases ,
staphylococcus aureus , including methicillin resistant strains ( mrsa ) , are the most prevalent in skin infections . pseudomonas aeruginosa , escherichia coli , acinetobacter spp . , and coagulase - negative staphylococci have also been isolated from skin lesions [ 1519 ] .
more than 95% of s. aureus strains are resistant to penicillin and 6070% are resistant to methicillin [ 2022 ] .
methicillin resistance is attributed to the mec a gene encoding penicillin binding proteins [ 20 , 23 ] .
these proteins occur in the cell wall and play a role in the synthesis of peptidoglycan and are usually inactivated by beta - lactam antibiotics
. however , in mrsa the mec a gene encodes a low - antibiotic affinity penicillin binding protein , known as pbp2a , conferring methicillin resistance to the cells .
some mrsa strains are also resistant to tetracyclines , sulphonamides , trimethoprim , macrolides , aminoglycosides , mupirocin , mafenide acetate , silver sulphadiazine , bacitracin , ciprofloxacin , and vancomycin [ 2429 ] . vancomycin - resistant enterococci and p. aeruginosa strains resistant to several antibiotics have also been reported .
this review focuses on the treatment of skin infections and open wounds and the biomedical application of electrospun nanofibers , with specific emphasis on antimicrobial delivery systems .
silver sulfadiazine , a combination of silver nitrate and sodium sulfadiazine , has been used to treat invasive burn wound sepsis [ 6 , 14 , 32 , 33 ] . with prolonged use ,
silver ions may be toxic , as it binds to dna and prevents replication [ 34 , 35 ] .
mafenide acetate and chlorexidine digluconate creams have been used to treat burn wound infections [ 14 , 39 ] .
the disadvantage of these topical creams is that they have to be applied twice daily .
if incorporated in wound dressings , the bandages have to be changed daily , which may expose the wound to further infection . as in the case of many other antimicrobials , pathogens with resistance to mafenide acetate
nanotechnology offers the latest alternatives to wound dressings , of which acticoat a.b , silverlon , and silvasorb with nanoparticles are good examples .
supporters of this technology claim that the nanocrystalline silver particles are released in a controlled manner , inhibiting the growth of a broad spectrum of pathogens [ 4345 ] .
entrance of the nanoparticles into cells and their mode of action is summarized in figure 1 .
endosomes , filled with silver nanoparticles , lysosomes , and silver nanoparticles in the nucleus of treated cells , have been observed .
some concerns exist regarding the medical use of silver particles , as they form reactive oxygen species ( ros ) , reduce atp , and damage mitochondria and dna [ 31 , 46 ] .
silver nanoparticles are , however , less toxic compared to gold nanoparticles , as observed in experiments with j774 a1 murine macrophages .
nanofibers are produced from polymers treated in a specific manner to form threads of a few micrometers to nanometers in diameter .
the large surface to volume ratio , and manipulation of surface properties , renders nanofibers the ideal matrix to develop super fine structures [ 4951 ] .
the possibility to immobilize antibiotics , enzymes , antimicrobial peptides , and growth hormones to nanofibers , or encapsulation into fiber matrixes , opens a new field in biomedical engineering [ 5260 ] .
several methods are used to produce ultra fine fibers , for example , self assembly of polymers , template synthesis , phase separation , and electrospinning [ 49 , 6266 ] .
electrospinning , schematically presented in figure 2 , is the most cost effective and easiest way to produce large volumes of nanofibers .
one electrode is placed in a polymer solution and the other electrode is linked to a collector , which is usually a stationary or rotating metal screen , plate , or wheel .
the electrically charged polymer forms a taylor cone at the tip of the needle and is ejected at a specific charge . as the polymer solution accelerates , the solvent evaporates and nanofibers are formed .
fibers are aligned by using a rotating collector , an auxiliary electrical field , or a rotating collector with a sharp edge and a rapidly oscillating frame [ 6771 ] .
coaxial electrospinning ( figure 3(a ) ) is used to produce nanofibers with a core - shell structure ( figure 4(a ) ) , which is ideal for encapsulating hydrophilic molecules .
emulsion electrospinning is also used to produce core - shell - structured nanofibers ( figures 3(b ) and 4(b ) ) .
an emulsion is prepared by emulsifying an aqueous phase , which contains a hydrophilic polymer or molecule to be encapsulated , into an organic phase containing a polymer that forms the shell [ 42 , 61 ] .
the emulsion is then electrospun into core - shell - structured nanofibers ( figure 4(b ) ) .
the quality and characteristics of the final product are determined by the temperature , viscosity , elasticity , conductivity , and surface tension of the solution , strength of the electric field , distance between the needle tip and collector , and humidity [ 49 , 73 , 74 ] . larger fibers ( bigger diameter )
polyvinylpyrrolidone ( pvp ) at 4% ( w / w ) in a 50 : 50 ( w / w ) dimethylformamide : ethanol solution is used to produces fibers of 20 nm in diameter
. however , pvp at 8% ( w / w ) in the same solution produces fibers of 50 nm in diameter , and pvp at 10% ( w / w ) produce fibers of 300 nm in diameter .
electrospinning different concentrations of poly l - lactic acid ( plla ) in a chloroform solution produce nanofibers with different morphologies ( figure 5 ) .
plla of 1% ( w / w ) produces a bead on a string structure whereas 3% ( w / w ) plla forms nanofibers with a smooth structure .
solvents influence the surface tension and viscosity of the solution and affect the morphology of fibers [ 75 , 76 ] .
pvp ( 4% , w / w ) dissolved in dichloromethane ( mc ) forms fibers with spindle - like beads and hollow or solid structures whereas the same concentration pvp in n , n - dimethylformamide ( dmf ) forms sphere - like beads with solid structures .
pvp dissolved in ethanol yields smooth fibers with a diameter ranging from 100 to 625 nm .
the pvp / dmf and pvp / mc solutions have a high surface tension ( 47.1 and 38.7 centipoise , resp . ) and low viscosity ( 9.8 and 13.0 centipoise , resp . ) .
a pvp / ethanol solution , on the other hand , has a low surface tension ( 29.3 centipoise ) and high viscosity ( 17.3 centipoise ) .
changes in current may also affect the morphology of fibers , as observed with polyethylene oxide ( peo ) .
an increase from 5.5 kv to 9.0 kv changed the morphology of the peo fibers from smooth to a
the importance of processing variables that influence fiber morphology during electrospinning is reviewed by deitzel et al . .
natural and synthetic polymers have been spun into nanofibers for potential use in biomedical engineering [ 53 , 54 , 61 , 7679 ] .
chitin , a structural polysaccharide from arthropods , yielded fibers ranging from 40 to 600 nm in diameter .
a combination of water - soluble carboxyethyl chitosan and poly - vinyl alcohol ( pva ) was electrospun to produce a wound dressing .
the nanofibers revealed no toxicity when tested with a mouse fibroblast l929 cell line , and promoted cell attachment and proliferation .
chitosan acetate bandages proved effective as an antimicrobial dressing when tested on balb / c mice with burn wounds that have been infected with p. aeruginosa and proteus mirabilis .
backbone and degrades over a long period whereas the more hydrophilic polymer degrades or dissolves faster .
the choice of polymer or polymer blends play an important role in devices aimed at controlled release .
examples of using hydrophilic and/or hydrophobic polymers for the controlled release of molecules , for example , antibiotics , plasmids , growth factors , proteins , silver particles , bacteria and viruses will be discussed in more detail [ 54 , 61 , 82 , 83 , 89 , 93 ] .
rifampin , encapsulated in plla during electrospinning , and incubated in a 0.05 m tris - hcl buffer , was only released when proteinase k was added to the solution .
this suggests that the release of rifampin was initiated by the degradation of plla and not by normal diffusion . in another experiment , doxorubicin hydrochloride and paclitaxil
doxorubicin hydrochloride was detected on the surface of the nanofibers but paclitaxil remained encapsulated .
rifampin and paclitaxil were more soluble in the chloroform / acetone solvent compared to doxorubicin hydrochloride .
the solubility of the molecule to be encapsulated in the polymer solvent plays an important role in its distribution throughout the nanofibers .
tetracycline hydrochloride ( 5% , w / w ) encapsulated in poly - ethylene - co - vinyl acetate ( peva ) , or in a blend of peva and plla , has a relatively slow and consistent release rate .
the peva and peva / plla blend containing 5% ( w / w ) tetracycline hydrochloride had a similar release rate to actisite , a commercial drug delivery system , following the initial high burst release .
the antibiotic mefoxin ( cefoxitin sodium ) was encapsulated in poly - lactate - co - glycolide ( plga ) fibers and in fibers consisting of a poly - lactate - co - glycolide / polyethylene glycol - block - poly(l - lactide ) copolymer ( plga / peg - b - plla ) .
mefoxin released from the fibers inhibited the growth of s. aureus in culture and on an agar surface .
the feasibility of encapsulating plasmid dna in electrospun nanofibers and to use these nanofibers as gene delivery vehicles has also been shown .
plga and a pla - peg block copolymer was electrospun with a 7164 bp plasmid ( pcmv ) , encoding -galactosidase .
the bioactivity of pcmv was evaluated by conducting transfection experiments with preosteoblastic mc3t3 cells .
the -galactosidase gene was successfully expressed by preosteoblastic mc3t3 cells that have taken up the plasmid .
human -nerve growth factor ( ngf ) was encapsulated into fibers consisting of a copolymer of poly -caprolactone and ethyl ethylene phosphate ( pcleep ) through electrospinning .
the bioactivity of ngf was evaluated by incubating rat pheochromocytoma ( pc 12 ) cells in the supernatant of nanofibers containing encapsulated ngf and searching for differentiation into neurons .
human glialcell - derived neurotrophic factor ( gdnf ) , encapsulated into pcleep nanofibers , was released in active form for up to two months .
a 15 mm nerve lesion was made in the left sciatic nerve of 3.5 month - old sprague dawley rats .
aligned fibers with no gdnf encapsulated within served as controls . in rats that received encapsulated gdnf ,
a bridge formed across the lesion and the nerve was regenerated after three months . however , the nerve system in only half the number of rats that received control fibers was regenerated .
the stimulation of bone regeneration in nude mice that have been treated with bone morphogenetic protein-2 ( bmp-2 ) encapsulated in plga - hydroxyapatite ( hap ) nanofibers was also shown in .
the highest release of lysozyme ( 87% over 12 days ) was recorded in a peo / pcl nanofiber with a 90/10 ratio .
this was done by emulsifying an aqueous solution of cytochrome c in a chloroform solution containing plla .
controlled release was obtained when plla was blended with poly(l - lysine ) ( pll ) and poly(ethylene imine ) ( pei ) , hydrophilic polymers . a blend containing 50% pll released most of the protein ( 75% ) with a high initial burst release .
escherichia coli , staphylococcus albus , and bacteriophages t7 , t4 , and were encapsulated in pva nanofibers with water as solvent .
the encapsulated cells and bacteriophages survived the electrospinning process and remained viable for three months at 20 and 55c .
the fibers were dissolved in a tris - buffered saline solution ( ph 7.5 ) .
micrococcus luteus and e. coli were encapsulated into peo nanofibers with water as solvent .
up to 74% of the m. luteus cells , but only 0.1% of the e. coli cells , survived the electrospinning process .
we recently reported on the encapsulation of a probiotic lactic acid bacterium in electrospun peo nanofibers .
however , the cells that survived were still able to produce the antimicrobial peptide ( bacteriocin ) and inhibited the growth of e. faecium hklhs that served as target strain .
silver nanoparticles were incorporated in cellulose acetate nanofibers by electrospinning cellulose acetate with 0.5 wt% agno3 .
silver particles were generated on the surface of the nanofibers after irradiation at 245 nm .
almost all viable cells ( 99.9% ) of gram - positive bacteria , e. coli , klebsiella pneumoniae , and p. aeruginosa were killed after 18 hours of exposure to the encapsulated silver particles .
growth inhibition of up to 99.27% of s. aureus and up to 98.44% of e. coli was recorded when the strains were cultured in the presence of these nanofibers .
human dermal fibroblasts that attached to the nanofibers continued to proliferate , suggesting that the fibers may be used as wound dressings .
however , some authors have reported that silver may elicit toxic side effects on human cells as discussed elsewhere in this paper .
table 1 summarizes various molecules that have been encapsulated into synthetic and natural polymers , or blends thereof , by electrospinning to facilitate their release .
lactic acid bacteria ( lab ) are a diverse group of organisms with gras ( generally regarded as safe ) status and have been consumed over decades .
most species produce bacteriocins , that is , ribosomally synthesized proteins or protein complexes with bacteriostatic or bactericidal activity against closely related species [ 97 , 98 ] .
the peptides have a net positive charge ( cationic ) and are amphiphilic or rather more hydrophobic .
they intercalate into the cell membrane of sensitive cells , form pores and disrupt the proton motive force ( pmf ) [ 99101 ] .
class i contains the lantibiotics that are small peptides that undergo posttranslational modifications and have lanthionine or -methyllanthionine residues , for example , nisin , merscadin [ 102 , 103 ] .
class ii contains the nonlanthionine - containing bacteriocins that are small ( < 10 kda ) heat - stable peptides that do not undergo extensive post - translational modifications .
examples include pediocin pa-1/ach , plantaricin s , enterocin as-48 , and lactococcin a [ 104107 ] .
some bacteriocins , such as mersacidin , have shown activity towards mrsa that have been associated with various hospital acquired infections .
nisin f was also investigated as treatment for subcutaneous skin infections caused by s. aureus .
bacteriocins are thus attractive natural alternatives to antibiotics , which can be used in the treatment of bacterial infections .
a localized delivery system is , however , required to control the level and rate of bacteriocins delivered to the wound
. a novel approach would be to encapsulate bacteriocins into electrospun nanofibers and use this as wound dressings for burned victims .
the bacteriocin plantaricin 423 retained activity after electrospinning and inhibited the growth of e. faecium hklhs and lactobacillus sakei dsm 20017 that served as target strains .
nisin , a bacteriocin produced by lactococcus lactis , was successfully loaded in plla nanoparticles by using semicontinuous compressed co2 antisolvent precipitation .
nisin was released in the active form and exerted its antibacterial activity up to 45 days , when incubated with a culture of lactobacillus delbrueckeii .
electrospinning is a versatile and relatively easy technique to produce large amounts of nanofibers with diverse molecules encapsulated within .
the large surface to volume ratio of nanofibers allows the encapsulation of high concentrations of bacteriocins and direct delivery to sites of skin infection . the use of bacteriocins to control infections may help to prevent further increase in antibiotic resistance amongst bacteria and may prevent negative side effects some current medication has on patients .
release of bacteriocins from nanofibers can be controlled by selecting polymers of specific composition .
furthermore , specific nanofiber scaffolds can be designed that are oxygen permeable and structurally similar to the extracellular matrix ( em ) in skin .
ideally , nanofiber wound dressings should not only contain antimicrobial agents , but a combination of compounds that would accelerate the healing process and alleviate discomfort .
although anti - inflammatory drugs have been encapsulated into nanofibers , no reports have been published on the encapsulation of combined compounds .
future research has to focus on developing nanofber wound dressings containing a combination of antimicrobial compounds , anti - inflammatory drugs , and painkillers .
furthermore , the nanofiber scaffold has to be designed to allow controlled released of the drugs over an extended period to avoid frequent changes of wound dressings .
the toxicity of nanofibers needs to be researched , that is , much more in vivo studies need to be performed . | injury to the skin causes a breach in the protective layer surrounding the body .
many pathogens are resistant to antibiotics , rendering conventional treatment less effective .
this led to the use of alternative antimicrobial compounds , such as silver ions , in skin treatment . in this
review nanofibers , and the incorporation of natural antimicrobial compounds in these scaffolds , are discussed as an alternative way to control skin infections .
electrospinning as a technique to prepare nanofibers is discussed .
the possibility of using these structures as drug delivery systems is investigated . |
a primary reason for this perspective is to highlight the utility of in vivo model systems , and specifically the skin , in studies of basic cell biological regulators .
for basic cell biologists looking for in vivo systems to expand into , the skin offers a combination of advantages that other tissues do not possess .
we discuss these , along with examples of success stories in which research in skin has uncovered mechanisms of basic cell biology , most prominently in the understanding of the cell adhesion / intermediate filament systems .
in addition , we discuss how genetic studies often reveal unexpected phenotypes and roles for cell biological regulators .
these are especially enlightening and often yield insights into novel functions for core machinery and additional levels of regulation .
although we focus on the skin as a useful model , it is important to emphasize that cell biological machinery is adapted for diverse functions in different tissues .
therefore lessons learned in cultured cells and in studies of the skin may not necessarily explain the complexity of how these proteins function and interact within a different tissue context .
in addition , this is clearly a two - way street , as more cell biological studies on the skin will increase our understanding of the physiology and pathology of this essential organ .
advantages of using of the skin as a model system include the following :
accessibility for microscopy .
the medical study of the skin and its diseases has a long history because the skin , being on the body surface , could be described and catalogued before present imaging technologies and surgical procedures were commonplace .
this is also a boon to biologists , as it allows live microscopic analysis in living embryos or adults .
imaging has allowed visualization of diverse phenomena , including immune cell recruitment to invading pathogens , stages of hair follicle morphogenesis , migration of melanoblasts , and the dynamics of cell adhesion turnover , to name just a few ( peters et al . , 2008 ; mort et al . , 2010 ; rompolas et al . , 2012 ; foote et al . ,
techniques such as spinning disk microscopy are sufficient for some studies in the interfollicular epidermis , as the cells being imaged are very near the surface . in other cases , two - photon microscopy has been used to peer further into the skin .
lightsheet microscopy promises to further propel these studies and our abilities to observe subcellular phenomena in tissues .
this relies on the existence of transgenic mouse lines with fluorescent reporters an emerging field in which the skin is well positioned to play a vital role.genetic tools and fluorescent transgenic lines .
there are many genetic lines that allow the marking and lineage tracing of specific cell populations , as well as genetic fluorescent markers of signaling pathway activation .
however , a toolkit of transgenic lines for the visualization of subcellular phenomena has also been gradually developing in the mouse ( table 1 ) .
although we do not yet have the diversity of reporters found in flies and worms , the advent of genome editing technologies is expected to yield an ever - increasing set of reporters for the mouse .
this is part of a larger collection of genetic tools made by researchers and global projects such as the knockout mouse project ( komp ) that allow the tissue and cell type specific disruption or mis / overexpression of genes with existing alleles .
in addition to these more traditional approaches , lentiviral infection of epidermal progenitors by in utero electroporation allows much more rapid analysis by both knockdown and misexpression analysis ( beronja et al .
first , there is an abundance of tissue when performing histological analyses , so there is more forgiveness for beginners ( no fine dissections and limitations of materials ) . of greater importance
, the tissue abundance provides ample resources to facilitate biochemical studies . as an example , the initial identification of desmosomal cadherins was achieved through purification of desmosomes from a rich source : bovine muzzle epidermis ( drochmans et al .
. subsequent work would show that these are related to classical cadherins , that they form the adhesive core of desmosomes , and that some of them are targets for autoantibodies that disrupt desmosomes a group of diseases known as pemphigus ( kottke et al .
in addition to traditional biochemical approaches , the skin is amenable to biochemical interaction screens and proteomic analysis , promising to further spur our understanding of cell biology.cell culture systems .
the importance of the ability to culture and passage epidermal cells can not be overstated . through the pioneering work of howard green in the 1970s , we can isolate epidermal progenitors ( human and mouse ) , grow them for more detailed cell biological studies in vitro , and gain understanding of basic mechanisms .
we can then take what we find back into the mouse to understand the physiology .
although care must be taken in interpretation ( as cultured cells clearly exist in a very different environment from that in the tissue ) , such systems have proven to be invaluable tools in many studies .
cultured cells can also be used to regrow stratified epidermis , a technique that can be used to generate a differentiated tissue from human cells as well as mouse cells . in addition
, cultured cells can be engrafted back onto the mouse for analysis of genetically modified cells in a physiological environment.physiological readouts .
, the different cell types require a multitude of fundamental biological processes to generate and maintain tissue function .
in addition to readouts of tissue architecture , proliferation , and differentiation , we can also assess tissue function in a variety of ways , such as examination of the barrier activity of the epidermis .
connecting basic cell biological regulators to tissue functions often provides surprises , as discussed later.clinical insights . in some cases ,
basic cell biology can be advanced by the existence of human mutations ( see examples in the next sections ) .
the extensive descriptive work in dermatology , combined with new sequencing technologies , will allow the identification of genes and pathways that affect tissue architecture and function .
the medical study of the skin and its diseases has a long history because the skin , being on the body surface , could be described and catalogued before present imaging technologies and surgical procedures were commonplace .
this is also a boon to biologists , as it allows live microscopic analysis in living embryos or adults .
imaging has allowed visualization of diverse phenomena , including immune cell recruitment to invading pathogens , stages of hair follicle morphogenesis , migration of melanoblasts , and the dynamics of cell adhesion turnover , to name just a few ( peters et al .
, 2008 ; mort et al . , 2010 ; rompolas et al . , 2012 ; foote et al . ,
techniques such as spinning disk microscopy are sufficient for some studies in the interfollicular epidermis , as the cells being imaged are very near the surface . in other cases , two - photon microscopy has been used to peer further into the skin .
lightsheet microscopy promises to further propel these studies and our abilities to observe subcellular phenomena in tissues .
this relies on the existence of transgenic mouse lines with fluorescent reporters an emerging field in which the skin is well positioned to play a vital role .
there are many genetic lines that allow the marking and lineage tracing of specific cell populations , as well as genetic fluorescent markers of signaling pathway activation .
however , a toolkit of transgenic lines for the visualization of subcellular phenomena has also been gradually developing in the mouse ( table 1 ) .
although we do not yet have the diversity of reporters found in flies and worms , the advent of genome editing technologies is expected to yield an ever - increasing set of reporters for the mouse .
this is part of a larger collection of genetic tools made by researchers and global projects such as the knockout mouse project ( komp ) that allow the tissue and cell type specific disruption or mis / overexpression of genes with existing alleles .
in addition to these more traditional approaches , lentiviral infection of epidermal progenitors by in utero electroporation allows much more rapid analysis by both knockdown and misexpression analysis ( beronja et al . , 2010 ) .
first , there is an abundance of tissue when performing histological analyses , so there is more forgiveness for beginners ( no fine dissections and limitations of materials ) .
of greater importance , the tissue abundance provides ample resources to facilitate biochemical studies . as an example
, the initial identification of desmosomal cadherins was achieved through purification of desmosomes from a rich source : bovine muzzle epidermis ( drochmans et al . , 1978 ) .
subsequent work would show that these are related to classical cadherins , that they form the adhesive core of desmosomes , and that some of them are targets for autoantibodies that disrupt desmosomes a group of diseases known as pemphigus ( kottke et al .
in addition to traditional biochemical approaches , the skin is amenable to biochemical interaction screens and proteomic analysis , promising to further spur our understanding of cell biology .
the importance of the ability to culture and passage epidermal cells can not be overstated . through the pioneering work of howard green in the 1970s
, we can isolate epidermal progenitors ( human and mouse ) , grow them for more detailed cell biological studies in vitro , and gain understanding of basic mechanisms .
we can then take what we find back into the mouse to understand the physiology .
although care must be taken in interpretation ( as cultured cells clearly exist in a very different environment from that in the tissue ) , such systems have proven to be invaluable tools in many studies .
cultured cells can also be used to regrow stratified epidermis , a technique that can be used to generate a differentiated tissue from human cells as well as mouse cells .
in addition , cultured cells can be engrafted back onto the mouse for analysis of genetically modified cells in a physiological environment . physiological readouts .
as such , the different cell types require a multitude of fundamental biological processes to generate and maintain tissue function .
in addition to readouts of tissue architecture , proliferation , and differentiation , we can also assess tissue function in a variety of ways , such as examination of the barrier activity of the epidermis .
connecting basic cell biological regulators to tissue functions often provides surprises , as discussed later . clinical insights . in some cases ,
basic cell biology can be advanced by the existence of human mutations ( see examples in the next sections ) . the extensive descriptive work in dermatology , combined with new sequencing technologies , will allow the identification of genes and pathways that affect tissue architecture and function .
determination of the composition , functions , and pathologies associated with these cell adhesion systems are among the greatest successes of research in the skin .
reverse genetic studies were the first to demonstrate a functional role for keratins in mechanical integrity of the skin ( vassar et al . ,
1991 ) , studies that were subsequently supported by analysis of human patients with mutations in keratins ( coulombe and lee , 2012 ) .
continuing work has identified genetic lesions in many desmosomal proteins , hemidesmosomal proteins , and other keratins that lead to a spectrum of disorders not only in the skin , but also in the heart ( kottke et al . , 2006 ) .
in this case , the cell biology and the disease phenotypes have clear correlates for example , the position of blistering ( between cells , within cells , or between cells and the basement membrane ) depends on where in this network the protein is localized and functioning .
further studies on identification of components of the desmosomes , how they assemble , and how they connect to keratins have depended largely on experiments performed in cultured keratinocytes ( kowalczyk and green , 2013 ) .
although there are many questions left to answer , the combination of tools and the clinical relevance of the findings will make the process particularly exciting . as an aside
because this tissue experiences many physical insults , it is likely to continue to play an important role in understanding how cells / tissues sense and respond to force .
k14 , keratin 14 promoter ; h2b , histone h2b ; gfp , green fluorescent protein ; emtb , ensconsin microtubule - binding domain ; nmiic , nonmuscle myosin iic ; zo-1 , zonula occludens 1 ; dpi , desmoplakin i ; numa , nuclear mitotic apparatus ; erk , extracellular signal regulated kinase ; fret , forster resonance energy transfer .
one of the earliest examples of patient - directed research leading to deeper understanding of cell biological mechanisms is the study of xeroderma pigmentosum ( xp ) .
the condition is a rare hereditary disease characterized by high sensitivity to ultraviolet ( uv ) radiation and increased susceptibility to cancers of the skin ( cleaver and bootsma , 1975 ) .
cultured fibroblasts isolated from xp patients had impaired responses to the repair of double - stranded dna lesions after uv irradiation , a finding that was confirmed in vivo using tissue sections ( cleaver , 1968 ; epstein et al . , 1970 ) .
further studies revealed that these cells did not initiate the first step of nucleotide excision repair and that xp is caused by a failure to excise pyrimidine dimers after exposure of dna to uv ( setlow et al .
before these discoveries , the mechanisms that controlled dna repair had only been fully characterized in microorganisms ( cleaver , 1968 , 1978 ) .
consequently , the examination of xp patient samples provided the first functional evidence for nucleotide excision repair in humans and on how defects in this process are associated with a clinical phenotype .
mutations in other nucleotide excision repair genes lead to cockayne syndrome and trichothiodystrophy , disorders with both overlapping and distinct clinical features ( kraemer et al . , 2007 ) .
thus the genetics and cell biology continue to teach us about the roles of this pathway .
determination of the composition , functions , and pathologies associated with these cell adhesion systems are among the greatest successes of research in the skin .
reverse genetic studies were the first to demonstrate a functional role for keratins in mechanical integrity of the skin ( vassar et al . ,
1991 ) , studies that were subsequently supported by analysis of human patients with mutations in keratins ( coulombe and lee , 2012 ) .
continuing work has identified genetic lesions in many desmosomal proteins , hemidesmosomal proteins , and other keratins that lead to a spectrum of disorders not only in the skin , but also in the heart ( kottke et al . , 2006 ) .
in this case , the cell biology and the disease phenotypes have clear correlates for example , the position of blistering ( between cells , within cells , or between cells and the basement membrane ) depends on where in this network the protein is localized and functioning .
further studies on identification of components of the desmosomes , how they assemble , and how they connect to keratins have depended largely on experiments performed in cultured keratinocytes ( kowalczyk and green , 2013 ) .
although there are many questions left to answer , the combination of tools and the clinical relevance of the findings will make the process particularly exciting . as an aside
because this tissue experiences many physical insults , it is likely to continue to play an important role in understanding how cells / tissues sense and respond to force .
k14 , keratin 14 promoter ; h2b , histone h2b ; gfp , green fluorescent protein ; emtb , ensconsin microtubule - binding domain ; nmiic , nonmuscle myosin iic ; zo-1 , zonula occludens 1 ; dpi , desmoplakin i ; numa , nuclear mitotic apparatus ; erk , extracellular signal regulated kinase ; fret , forster resonance energy transfer .
one of the earliest examples of patient - directed research leading to deeper understanding of cell biological mechanisms is the study of xeroderma pigmentosum ( xp ) .
the condition is a rare hereditary disease characterized by high sensitivity to ultraviolet ( uv ) radiation and increased susceptibility to cancers of the skin ( cleaver and bootsma , 1975 ) .
cultured fibroblasts isolated from xp patients had impaired responses to the repair of double - stranded dna lesions after uv irradiation , a finding that was confirmed in vivo using tissue sections ( cleaver , 1968 ; epstein et al . , 1970 ) .
further studies revealed that these cells did not initiate the first step of nucleotide excision repair and that xp is caused by a failure to excise pyrimidine dimers after exposure of dna to uv ( setlow et al .
the mechanisms that controlled dna repair had only been fully characterized in microorganisms ( cleaver , 1968 , 1978 ) .
consequently , the examination of xp patient samples provided the first functional evidence for nucleotide excision repair in humans and on how defects in this process are associated with a clinical phenotype .
mutations in other nucleotide excision repair genes lead to cockayne syndrome and trichothiodystrophy , disorders with both overlapping and distinct clinical features ( kraemer et al . , 2007 ) .
thus the genetics and cell biology continue to teach us about the roles of this pathway .
the genetic analysis of core cell biological regulators often unveils unexpected or absent phenotypes . these studies can reveal unexpected additional functions for these proteins , uncover redundancy between different machineries , or expose additional levels of regulation that were previously unanticipated .
p120-catenin is a well - studied adherens junction protein that functions to regulate cortical levels of e - cadherin ( davis et al . , 2003 ;
analysis of mutants lacking epidermal p120 , however , revealed phenotypes not associated with cell adhesion . instead , epidermal hyperplasia and chronic inflammation were induced by activation of the rho / nf-b axis ( perez - moreno et al . ,
the arp2/3 complex generates branched actin networks that play diverse roles in cell migration , vesicle trafficking , and cell adhesion ( rotty et al . , 2013 ) .
surprisingly , loss of arp2/3 in the epidermis did not result in notable defects in cell architecture or adhesion . instead
, arp2/3-complex mutant epidermis was hyperproliferative and showed defects in expression of both early and late differentiation genes .
in addition , tight junction proteins showed decreased cortical organization in mutant tissue and did not generate robust resistance to ion flow in cell culture ( zhou et al . , 2013 ) .
for example , in intestinal epithelia , the arp2/3 complex regulates vesicle trafficking and the endolysosomal system ( zhou et al . , 2015 ) .
this highlights that different lessons will be learned by analysis of core cell biological regulators in diverse tissues .
whereas par3 is believed to be a master regulator of epithelial polarity , the epidermis develops normally in its absence , highlighting our incomplete understanding of how polarity is generated in a mammalian tissue .
loss of par3 , however , does have opposing effects on different tumor types , suppressing papillomas and promoting keratoacanthomas ( iden et al . , 2012 ) .
although the underlying mechanisms are not fully understood , this suggests that the epidermis will be useful for studying polarity signaling pathways and their role in both homeostasis and tumorigenesis .
it also demonstrates that comparative studies of simple and stratified epithelia will identify conserved and divergent regulators of epithelial function .
loss of reactive oxygen species through mutation of a transcription factor required for expression of electron transport chain components caused unexpected differentiation defects in the epidermis , presumably through defects in notch and wnt signaling , revealing additional roles for metabolic enzymes in tissue function ( hamanaka et al .
we have highlighted the utility of the skin as a model system , the different methodological approaches that can be used , and the types of information that can be gleaned from working with the skin .
together these examples demonstrate that the skin is a powerful tool in which one can examine a wide array of cell biological phenomena . through
continued collaborative efforts between cell biologists and skin biologists , we expect that additional fundamental insights into both basic cell biology and skin biology will continue to emerge . | advances in cell biology have often been driven by studies in diverse organisms and cell types .
although there are technical reasons for why different cell types are used , there are also important physiological reasons . for example , ultrastructural studies of vesicle transport were aided by the use of professional secretory cell types .
the use of tissues / primary cells has the advantage not only of using cells that are adapted to the use of certain cell biological machinery , but also of highlighting the physiological roles of this machinery .
here we discuss advantages of the skin as a model system .
we discuss both advances in cell biology that used the skin as a driving force and future prospects for use of the skin to understand basic cell biology .
a unique combination of characteristics and tools makes the skin a useful in vivo model system for many cell biologists . |
newcastle disease ( nd ) is still considered as an economically important disease which is highly contagious infection for many avian species .
nd is caused by virulent strains of avian paramyxovirus type 1 ( apmv-1 ) of the genus avulavirus belonging to the family paramyxoviridae . according to the world organization for animal health ( oie , 2012 )
newcastle disease affects a wide range of domestic and wild avian species ; however , the severity of the disease varies greatly , spanning from peracute disease with almost 100% mortality to subclinical disease with no lesions .
based on severity of clinical disease , the strains of ndv were originally classified into 3 groups based on their virulence such as lentogenic , mesogenic , and velogenic .
lentogenic strains , especially in adult chickens , may cause minimal or no clinical signs .
however , the disease produced by mesogenic strains may cause mortality that can reach 25% and those by in velogenic strains may reach up to 100% differences in virulence of the virus occur during any disease outbreak , so determining virulence is essential for the effective control of the disease .
nd was first reported in sudan in 1951 and still constituted a major problem facing the poultry industry .
assessment of the virulence of ndv is necessary in order to limit the outbreaks and to minimize their impact .
so the aim of this study was to evaluate the pathogenicity of some ndv field strains isolated 20082013 .
seven ndv viruses were isolated from different hosts , including chickens ( layers and broilers ) and pigeons from several regions in sudan during 2008 to 2013 from cases submitted for diagnosis to the department of avian diseases and diagnosis , veterinary research institute , khartoum , sudan .
the information concerning the origin of virus outbreaks , the year of isolation , breed , type , and age of birds involved is presented in table 1 .
mortality during the outbreak varied from 90% in young chickens to less than 2% in hens ; however , egg production dropped from 90 to 40% .
clinical signs of the disease were similar among most poultry farms including dyspnea , nervous , respiratory signs , diarrhea , and cyanosis of the combs .
nine - to - eleven - day - old embryonated eggs ( bovan ) and chicks ( cockerels ) were provided by the coral farm hatcheries . before use
filtrates of processed tissues from the trachea , lung , brain , spleen , and cecal tonsils from different hosts were used to inoculate 911-day embryonated eggs for virus isolation via allantoic route and the ndv was identified by means of ha and hi test using standards procedures .
volumes of 25 l of pbs were placed in each well of the plate followed by 25 l of allantoic fluid of virus isolate in each well in the first row .
after 2 fold dilutions were carried out in subsequent rows , 25 l of 1% suspension of chickens , cattle , sheep , and equine rbcs was added to each well .
after 30 minute of incubation at room temperature , the reciprocal of the highest dilution that produced positive ha was considered as positive virus titre .
the test was done following the procedures described previously [ 5 , 6 ] . a standard ha
test was performed with chicken rbcs in who haemagglutination plates using 500 l volumes .
the test was incubated at 4c for 1 hour before the ha titre was calculated .
after gentle mixing and further incubation , the ha titre was also recorded at 24 and 30 hours .
serial tenfold dilutions of allantoic fluid ( af ) of each virus isolate were prepared and 0.1 ml of the dilutions ( 10 , 10 , and 10 ) was inoculated into the allantoic cavity using 5 eggs per dilution . the highest dilution at which all embryos died soon was considered as mean lethal dose ( mld ) and the mdt / mld was calculated as described .
pathogenicity of ndv isolates was assessed by using the standard intracerebral pathogenicity index ( icpi ) test .
briefly , ten - one - day - old chicks were inoculated intracerebrally with 0.1 ml of a 1 : 10 dilution of allantoic fluid of each virus isolate .
all chicks were monitored during an eight - day observation period and scored as normal ( 0 ) , sick or paralyzed ( 1 ) , and dead ( 2 ) .
total scores were determined , and the mean daily score was calculated to obtain the icpi as described .
following inoculation of embryonated chicken eggs with suspensions from organs of diseased birds , embryo died 2 - 3 days after inoculation for the pmed/010 , bhalf/011 , bso/013 , and lkab/013 and 4 - 5 days for lny/08 , lny/09 , and pkam/09 ndv isolates .
the harvested allantoic fluids from dead eggs agglutinated red blood cells of chickens and this agglutination was inhibited by addition of specific ndv antiserum .
the pkam/09 , bso/013 , and lkab/013 agglutinated equine , sheep , and bovine rbcs while lny/09 agglutinated only the equine rbcs .
the lny/08 , pmed/010 , and bhalf/011 did not agglutinate any of the mammalian species rbcs .
the haemagglutination elution of the ndv field isolate recorded after 1 , 24 , and 30 hours was illustrated in table 3 .
there is a difference in ha titre recorded in 24 and 30 hours of all isolates .
the icpi of the lny/08 , lny/09 , and pkam/09 was 1.3 , 1.2 , and 1.3 which can be classified as mesogenic strains .
the icpi of the pmed/010 , bhalf/011 , bso/013 , and lkab/013 was 1.8 , 2.0 , 1.6 , and 1.6 , respectively , which classified as velogenic ndv strains .
newcastle disease ( nd ) is a serious and commonly fatal viral poultry disease , which is present all over the world .
viral characterization using the pathogenicity test or molecular typing is essential , as the importance and impact of a given ndv isolate are directly related to its virulence . in the present study ndv isolates were from outbreaks of different locations and five isolates were recovered from chickens while two were from pigeons .
the ability of ndv to agglutinate red blood cells ( rbcs ) is due to the binding of the hn protein to receptors on the surface of the rbcs .
chicken rbcs are usually used in ha tests but ndv will cause agglutination of all amphibian , reptilian , and avian cells . the ability of ndv strains to agglutinate cattle , goat , sheep , swine , and horse rbcs varied among various strains .
( 2009 ) applied the haemagglutinability tests using mammalian erythrocytes as a tool for strain differentiation . here among the field isolates tested , three isolates agglutinated mammalian rbcs .
it is noticed that there is a difference in hemagglutination patterns which could not be correlated to the virulence of the ndv isolates .
this result was similar to previous research and disagrees with the suggestion of others that the pattern of agglutination of some ndv strains may be related to the degree of virulence that more virulent strains appear to show less agglutinability of certain mammalian red blood cells than the less virulent ones .
elution time could form a basis for rough characterization of isolates of newcastle disease virus [ 5 , 6 , 13 , 14 ] .
previously no relationship was found between haemagglutination elution patterns and other properties of the same strain [ 5 , 6 ] .
the in vivo assessment of virus virulence is based on the icpi test , which is considered as the most sensitive and widely used test for measuring virulence . in the present study , based on icpi
, the results indicated that four ndv isolates had virulent strain characteristics and other three have mesogenic characteristics .
it was noticed that there was a mismatch in the indices of the mdt of the isolates . | the biological properties and pathogenicity of seven newcastle disease virus field isolates were studied .
these isolates were recovered from different outbreaks in sudan ( 5 from chickens and 2 from pigeons ) during 20082013 . based on intracerebral pathogenicity index ,
four ndv isolates were characterized as velogenic ( their icpi ranged 2.01.6 ) and three isolates were characterized as mesogenic ( icpi ranged 1.21.3 ) .
the mean death time for all isolates ranged from 54 to 76.8 hours .
the elution time of the viruses from chicken erythrocytes and the ability to haemagglutinate mammalian red blood cells differed considerably in their reactions . |
uric acid ( ua ) has been formerly considered as a major antioxidant in human plasma with possible beneficial antiatherosclerotic effects ; however , since the beginning of the twentieth century , uric acid has been suggested as a risk factor for cardiovascular diseases . while epidemiologic studies suggested that hyperuricemia ( hu ) is strongly correlated with cardiovascular disease ,
some clinical evidence has found a significant and specific association between serum uric acid levels and coronary atherosclerosis ; however , there is much controversy concerning this relationship , and some studies in fact came to the opposite conclusions [ 2 , 3 ] .
high uric acid levels could potentially increase the risk of cardiovascular disease via several biological mechanisms ; however , recent studies have suggested that endothelial dysfunction is a fundamental mechanism whereby uric acid may affect cardiovascular function .
series of animal experiments revealed that in rats , hyperuricemia induced by oxonic acid , a uricase inhibitor , causes hypertension and renal arteriolopathy and impairs nitric oxide generation .
moreover , oxidative stress plays an important role in the vascular endothelial dysfunction of hyperuricemia [ 4 , 5 ] .
an increase in the levels of adma is likely to be a factor connected with impaired nitric oxide production and resulting in endothelial dysfunction in cardiovascular disease , including hypertension , atherosclerosis , and metabolic syndrome .
recent studies suggested adma to be a sensitive marker and even an initiator of endothelial dysfunction .
similarly , the second form - symmetric dimethylarginine ( sdma ) has insignificant inhibitory effects on nitric oxide synthase .
however , it has recently been suggested that sdma might have an indirect effect on nitric oxide synthesis via inhibiting the transporter that mediates the intracellular uptake and absorption of l - arginine .
although previous studies suggested that serum uric acid as well as adma and sdma levels might correlate with cardiovascular risk or renal disease , the relationship between serum uric acid and adma or sdma has not yet been characterized .
the goal of this work was to investigate if in adolescents with hyperuricemia serum levels of adma and sdma are increased and if their levels correlate with serum uric acid .
additionally we would like to check if serum level of any of these dimethylarginines correlates with hs - crp .
the protocol was approved by the bioethics committee of the medical university of bialystok in accordance with the declaration of helsinki .
informed consent was obtained from parents of all participants and children older than 16 years of age .
( m35 ; f23 ) with elevated serum uric acid were eligible to participate in the study .
patients were recruited from referrals to department of pediatric nephrology , medical university of bialystok , from june 2010 until may 2012 .
reference group ( r ) consisted of 27 healthy children ( 12 boys , 15 girls ; aged median 15.8 q1q3 ( 10.817.1 ) ) with normal serum uric acid levels .
patients who meet all the following inclusion criteria were enrolled into the study : ( 1 ) age 1018 years , ( 2 ) hyperuricemia defined as serum uric acid above 5.5 mg / dl , ( 3 ) normal renal function ( normal creatinine level , egfr > 90 ml / min/1.73 m , no proteinuria ) , ( 4 ) no clinical and laboratory signs of infection , ( 5 ) not treated with antibiotics within the last 4 weeks , and ( 6 ) signed the informed consent .
patients with a history of gouty arthritis , renal stones , protein / creatinine ratio > 0.2 , and diabetes mellitus were excluded . in all adolescents , careful clinical history , underlying comorbidities , and physical examination were assessed .
body weight and height were measured using a balance beam scale and pediatric wall - mounted stadiometer .
body mass index ( bmi ) was calculated as weight ( kg ) divided by the square of height ( m ) .
bmi z - scores , which reflect the sd score for age- and gender - appropriate bmi distribution , were calculated using the following formula : z = [ x ]/ ; x is the bmi measured in the patient , whereas and represent the mean and the standard deviation for age- and gender - matched controls .
based on the international norms from the who with age- ( to the nearest one month ) and gender - specific bmi , bmi cutoffs were the following : overweight bmi > + 1sd ; obesity bmi > + 2sd .
hypertension ( ht ) on the basis of abpm was defined as mean systolic or diastolic daytime or nighttime bp levels that are 95th percentile .
ambulatory blood pressure monitoring ( abpm ) was performed using the oscillometric spacelab device ( spacelab cardionavigator ) .
the monitors were programmed to measure bp every 15 minutes during daytime ( 8:0022:00 ) and every 30 minutes during nighttime ( 22:008:00 ) ; however , the periods were corrected according to the patients ' diary .
recordings with a minimum 80% of measurement and without brakes longer than 2 hours were considered sufficient .
after an overnight fast , five milliliters of venous , peripheral blood was collected from each patient and reference participants during routine laboratory testing .
the biochemical workup included serum creatinine ( measured by jaffe reaction ) , urea , fasting plasma glucose ( fpg ) , lipid profile , and serum uric acid concentration .
the estimated glomerular filtration rate ( egfr ) was calculated from the updated schwartz formula : gfr ( ml / min/1.73 m ) = ( 0.41 height in cm)/creatinine in mg / dl . adma and sdma were measured in serum using elisa method ( immundiagnostik ag , bensheim , germany ) according to the manufacturer 's guidelines .
the intra- and interassay coefficients of variance ( cvs ) for adma were 6.57.0% and 6 - 7% and for sdma 4.87.5% and 6 - 7% , respectively .
serum hs - crp was assessed by particle - enhanced immunonephelometry using a standard cardiophase hs - crp for bnii ( dade behring diagnostics , marburg , germany ) and expressed in mg / l .
the intra - assay and interassay coefficient of variation for hs - crp were 4.2% and 6.8% , respectively .
all continuous variables were tested for normal distribution by the kolmogorov - smirnov , with lilliefors correction and shapiro - wilk tests .
as most of the studied parameters were not normally distributed , descriptive statistics were calculated as median with the interquartile range .
mann - whitney u test was used to compare continuous variables , and -test with yates correction was used to compare categorical variables between two groups .
the correlations between sdma , adma , and other studied variables were assessed using spearman correlation test .
two multiple linear regression models were created including the sdma and adma as dependent variables . statistical significance was determined at p < 0.05 level .
overall median age of all children with hyperuricemia was 16.15 q1q3 ( 1417 ) yrs .
the age and sex of studied children did not differ from healthy controls ( p > 0.05 ) .
median bmi z - score in both examined groups was 1.08 ( q1q3 : 0.361.64 ) in studied children and 0.2 ( q1q3 : 0.20.8 ) in reference group ( p < 0.01 ) .
total cholesterol values were significantly higher in studied patients ( median : 162 mg / dl ) than in reference group ( median : 142 mg / dl ; p < 0.05 ) . no significant differences between those two groups
were observed regarding fpg , triglycerides , hdl , and ldl . when compared to healthy controls , hyperuricemic patients showed increased serum sdma ( p < 0.01 ) , but not serum adma levels ( table 1 ) .
serum adma and sdma correlated positively with ua ( r = 0.34 , p < 0.01 ) ( r = 0.31 , p < 0.01 ) , respectively , and hs - crp ( r = 0.20 , p < 0.05 ) ( r = 0.36 , p < 0.01 ) , respectively ( figure 1 ) .
the positive correlation was observed between serum adma and bmi z - score ( r = 0.36 , p < 0.01 ) , tg ( r = 0.46 , p < 0.01 ) , total cholesterol level ( r = 0.28 , p < 0.01 ) , and ldl - cholesterol ( ldl - c ) ( r = 0.33 , p < 0.01 ) in hyperuricemic patients . additionally sdma correlated positively with creatinine level ( r = 0.27 , p < 0.01 ) and systolic ( r = 0.32 , p < 0.01 ) and diastolic blood pressure ( r = 0.25 , p < 0.01 ) during a day and negatively with egfr ( r = 295 , p < 0.01 ) . no correlation between serum sdma and adma was found .
the factors that were found to have a significant correlation with serum sdma and adma in the single regression analyses were used as explanatory variables to create the multiple regression models . to reduce the impact of multicollinearity we removed some correlated variables .
next we eliminated a few more nonsignificant ( in the model ) variables . in multivariable analysis ,
serum adma was associated with serum uric acid , tg , and ldl - c independent of age , gender , and parameters of renal function ( table 2 ) . on the other hand serum sdma
was associated with serum uric acid , serum creatinine , hs - crp , and systolic blood pressure .
in this cross - sectional study , we found that in adolescents with increased serum uric acid levels , serum sdma is significantly elevated .
we also confirmed positive correlation of serum sdma with uric acid , creatinine , and hs - crp and negative with egfr . additionally positive correlation between sdma and systolic and diastolic blood pressure was found .
it is interesting to note that we created multivariable linear regression model and found that serum uric acid , triglycerides , and ldl - cholesterol accounted for more than 37% of the variations in serum sdma . taken together , the results of this study demonstrate that serum sdma is an independent determinant of renal function but also takes part in cardiovascular disease in pediatric patients with hyperuricemia .
another important finding is that we have not found significant increase in serum adma between subjects with hyperuricemia and reference group ; however , serum adma correlated positively with serum uric acid , bmi z - score , triglycerides , total cholesterol , ldl - c , and hs - crp . to our best knowledge
this is the first clinical study on the association between the serum sdma , adma , ua , and hs - crp in adolescents with hyperuricemia . in many previous reports
the association of serum uric acid with endothelial dysfunction was reported by zoccali et al . in 217 individuals with mild untreated hypertension who underwent measurement of endothelium - dependent brachial artery vasodilation .
the authors reported that compared with participants whose serum ua was 3.5 mg / dl , endothelium - dependent vasodilation was 33% worse among those with serum ua levels of 5.5 mg / dl .
furthermore , experimental studies showed that hyperuricemic rats developed endothelial dysfunction , but early l - arginine supplementation could prevent both the systemic and glomerular hypertension . taken together
, these data suggest that higher levels of serum ua indicate the presence of endothelial dysfunction and might contribute to an increase in cardiovascular risk .
it was shown that reduced no bioavailability and oxidative stress contribute to endothelial dysfunction [ 21 , 22 ] .
asymmetric dimethylarginine ( adma ) is an endogenous modulator of endothelial function and oxidative stress , and increased levels of this molecule have been reported in some metabolic disorders and cardiovascular diseases . very little was found in the literature on role of adma in patients with futures of metabolic syndrome .
. found that adma levels were significantly increased in the metabolic syndrome ; however , the levels of adma were modestly correlated only with waist circumference but not with the other components of metabolic syndrome . in experimental study korandji et al . analyzed the relationship between dimethylarginine compounds and oxidative stress levels and cardiovascular function in fructose - hypertensive rats .
the authors suggested that elevated levels of adma could in part be secondary to the early development of oxidative stress associated with development of hypertension . to our best knowledge
no data concerning the relationship between the adma and uric acid in humans were published .
several clinical studies have shown that adma levels are increased in hypertensive patients , atherosclerosis , and hypercholesterolemia .
they found a positive correlation between adma and hs - crp and tg / hdl ratio in patients with metabolic syndrome .
patients in whom adma concentrations decreased at followup had lower tg / hdl and ldl / hdl ratios when compared to patients with increase / stabilization of adma .
similarly kanazawa et al . found significantly positive association between adma and bmi , blood pressure , ldl , hdl and cholesterol independent of age . in obese women studied by krzyzanowska et al .
adma correlated with high - sensitivity c - reactive protein at baseline and after weight loss , but no association with blood pressure or plasma lipids was observed . in reviewing the literature ,
median serum sdma levels in hu patients was significantly higher compared to the controls , similarly like hs - crp , wich was also confirmed in our previous study in similar population .
the results of this study showed that similarly adma and sdma correlated positively with serum hs - crp .
similarly we still do not know too much about the effect of sdma on the l - arginine - no pathway .
closs et al . showed that sdma had no effect on the inducible nos extracted from macrophages .
the results of tojo et al . experimental study suggested that sdma might be a potent competitor of l - arginine transport and thereby have an indirect inhibitory effect of no synthesis by limiting arginine availability to nos . in human studies
it was shown that sdma was associated with inflammatory markers in patients with chronic kidney disease .
sdma was independently associated with increased cardiovascular and total mortality in patients undergoing coronary angiography in studies by meinitzer et al . and schulze et al . .
in contrast to studies presented above zoccali et al . did not confirm sdma as a predictor of cardiovascular outcome in patients with chronic kidney disease .
both adma and sdma were significantly elevated in patients with chronic renal failure ; however , the increase was more pronounced for sdma
. in conclusion , increased circulating methylarginines have been linked to some features of the metabolic syndrome ; however , it is still difficult to answer the question about their role in this process .
the results of the present study have demonstrated increased sdma , but not adma levels in adolescents with hyperuricemia and their correlation with serum uric acid levels .
however , at the moment it is difficult to answer the question if it is just coexistence of these factors or any mechanism linking uric acid and methylated arginines really exists .
our study has limitations ; it is a single - center , cross - sectional study , and confirmation in other cohorts is necessary to validate our findings .
third , the study design did not allow to answer the question if sdma is a new player or an innocent bystander in chronic inflammation in patients with hyperuricemia . | the purpose of this work was to investigate if in adolescents with hyperuricemia serum levels of asymmetric and symmetric dimethylarginine ( adma , sdma ) are increased and if their levels correlate with serum uric acid ( ua ) .
patients and methods .
the study group consisted of 58 hyperuricemic patients aged median 16.15 q1q3 ( 1417 ) .
the reference group contained 27 healthy individuals with normal serum ua level .
adma and sdma were measured by immunoenzymatic elisa commercial kits and expressed in mol / l .
serum ua was measured by the colorimetric method . results . in hyperuricemic
patients serum adma values did not differ between two estimated groups ( p > 0.05 ) ; however , sdma was significantly higher than in reference group ( p < 0.01 ) .
serum adma and sdma correlated positively with ua ( r = 0.34 , p < 0.01 ) ( r = 0.31 , p < 0.01 ) and hs - crp ( r = 0.20 , p < 0.05 ) ( r = 0.36 , p < 0.01 ) , respectively . conclusion .
we demonstrated increased sdma but not adma levels in adolescents with hyperuricemia and their correlation with serum uric acid levels .
however , at the moment it is difficult to answer the question if it is just coexistence of these factors or any mechanism linking uric acid and methylated arginines really exists . |
a 46-year - old woman presented to kyungpook national university hospital with a one - year history of chest discomfort and dyspnea , as well as a history of three operations for cardiac myxoma , including biatrial cardiac tumor resection in 1984 , followed 10 years later by the resection of a recurrent left atrial and ventricular cardiac myxoma attached to the interatrial and interventricular septa . the first surgery , in 1984 , was performed in our medical center .
the myxoma and its pedicle attached to the fossa ovalis were excised via right atriotomy and left atriotomy with enclosure of the interatrial defect . the second surgery , in 1994 ,
the interatrial septum was nearly totally excised and the myxoid mass was identified and excised . also through aortotomy , a left ventricular myxoma on the interventricular septum below the right coronary cusp was identified and excised .
sixteen years later , in 2010 , the third such operation was performed in the same center .
after the dissection of severe adhesion , a myxoma and its stalk on the inferior vena cava and right atrial junction were excised through a right atriotomy .
this patient also had a history of recurrent cutaneous and mucosal tumors , including forearm and nose tumors ( diagnosed as myxomas based on excisional biopsies of tumors on her right forearm and right nostril ) , breast ductal adenoma ( fibroadenomas diagnosed in her bilateral breasts through mammotome excisional biopsies ) , and a uterine myoma , as well as a basilar artery aneurysm .
chest computed tomography revealed a large soft tissue mass in the right atrial and ventricular cavities and findings suspicious of pulmonary embolism ( fig .
two days later , with a presumptive diagnosis of recurrent cardiac myxoma , the patient underwent cardiac surgery through a median sternotomy .
aortic and venous cannulation , the latter through the superior vena cava and right femoral vein , were performed for cardiopulmonary bypass .
the large gelatinous tumor was visualized after right atriotomy ; its stalk was located in the right atrium - inferior vena cava junction ( fig .
moreover , the tumor involved part of the distal left pulmonary artery , leading to further pulmonary arteriotomy during surgery .
postoperatively , the patient s symptoms of dyspnea and chest discomfort resolved without complications , and the patient was discharged on postoperative day 9 .
after resection , cardiac myxoma , the most common benign cardiac tumor , rarely recurs , with a recurrence rate of approximately 3% observed in sporadic cases and a recurrence rate of 22% in patients with the carney complex , an uncommon autosomal dominant hereditary disorder first described by carney et al . in 1985 .
the carney complex , which manifests with cardiac , endocrine , cutaneous , and neural tumors and spotty pigmentation , is diagnosed when a patient exhibits either at least two main criteria , reflecting the manifestations of the disease , or one main criterion as well as one supplemental criterion associated with familial or hereditary features ( table 1 ) .
the patient presented in this report had a consistent history of cutaneous myxoma , breast ductal adenoma , and cardiac myxoma .
this case of four recurrences of cardiac myxoma , the first of its kind to be reported in korea , underscores the importance of performing a detailed investigation of patients with recurrent myxoma for the possibility of the carney complex in order to facilitate better management , prognostication , and patient education regarding this condition . | although cardiac myxoma is the most commonly encountered benign cardiac tumor in cardiac surgery practice , recurrent cardiac myxoma is very rare , is most commonly related to the carney complex , and usually requires multiple cardiac operations with specific requirements in terms of perioperative management . in this report
, we describe a patient who experienced the fourth recurrence of cardiac myxoma and review the diagnostic criteria of the carney complex .
this is the first report of such a case in korea . |
the technical convenience of milk as a source of dna can be expected to increase the field of application of the markerbased methods for genetic analysis of goat genome .
it has a remarkable importance for several reasons related to governmental regulation and public health .
mixture in dairy products and species substitution should be observed to be a cause of human adverse reactions ( bottero et al , 2003 ) .
the source of dna in milk could be from somatic cells ( fahr et al , 1999 ) .
goat milk is similar to cow milk with around 87 percent water , 67 percent energy , 3.3 percent protein , 4.0 percent fat , and 4.6 percent carbohydrates ( belanger and jerry , 2001 ) .
goat milk differs from cow and human milk in several ways , among them higher digestibility and lower lactose ( larson , 1978 ) .
one difference is that goat milk has smaller fat globules present due to the lack of the enzyme that aggregates the globules in the milk ( belanger and jerry , 2001 ) .
the objective of the present study was to evaluate milk as a source of goat dna and as a substrate for pcr amplification using mitochondrial cytochrome - b gene as a target dna for pcr amplification .
this was prospective study in which samples are collected throughout the study this study was conducted at the molecular biology laboratory , faculty of veterinary medicine , university of khartoum , and the national ribat university , khartoum , sudan . during the period from february to july 2010 25 nubian goats , maintained at the dairy farm of the faculty of veterinary medicine , university of khartoum , shmbat were included .
milk ( 2.5 ml ) was collected in sterile 2.5ml centrifuge tube by hand milking , samples were stored at 4c until testing for extraction of total genomic and mitochondrial cytochrome - b ( mtcyt - b ) dna .
blood samples were collected for preparation of positive control , blood samples were collected in clean sterile vacutainers , containing ethylene diamine tetra acetic acid ( edta ) , from goats ( positive controls ) , from the animals attended at the veterinary teaching hospital .
the blood samples then centrifuged in bench centrifuge ( hettich zentrifugen , d-785320 , germany ) in order to separate the buffy coat which is rich in white blood cells and used for extraction of total genomic and mitochondrial cytochrome - b ( mtcyt - b ) dna .
the extracted dna was used as a target dna for pcr amplification extraction of dna from goat milk and peripheral blood was made possible using a commercially available qiaamp blood kit ( qiagen inc.chatsworth , canada ) according to the manufacturer s instructions . in details , 200l of milk samples , 20 l of proteinase
k enzyme stock solution , and 200 l of lysing buffer ( la buffer ) were pipetted into 1.5 ml eppendorf tube and the mixture was vortexed on the vortexing machine ( janke & kunkel , gmbhu.cokg , germany ) and incubated at 60c for 10 minutes .
the mixture then was transferred to the qiaamp spin column , and was placed in a clean 2ml collection tube and centrifuged in microcentrifuge ( hettich zentrifugen , 12 - 24 , tuttiligen , germany ) at 8000 rpm for 1 minute .
the qiaspin column was washed firstly with 500 l of washing buffers 1 ( aw1 ) at the same previously mentioned centrifugation speed and rewashed using washing buffer 2 ( aw2 ) at speed 1200 rpm centrifugation speed for 3 minutes .
the qiaamp spin column was then placed in a clean 1.5 ml eppendorf tube and the dna was eluted with 200 l of double distilled water preheated at 70c .
maximum dna yield will be obtained by spinning at 1200 rpm for 1 minute after remaining for 1 minute in the room temperature .
five microliters of the suspended nucleic acid will be used in the pcr amplification for pcr amplification a pair of goat - specific primers ( gsl1&gsr2 ) was designed from the caprine mtcytb gene sequences .
gsl1 included bases 284 - 303 of the positive sense strand ( 5)- tca tac ata tcg gac gac gt . whereas gsr2 included bases 693 - 712 of the complementary strand ( 5)- caa gaa tta gta gca tgg cg . using of this pair of primers ( gsl1&gsr2 ) in pcr assay resulted in amplification of a 428-bp pcr product from caprine mtcy - b dna .
the primers were synthesized on a dna synthesizer ( milliigen / biosearch , a division of millipore burlington , ma ) and purified using oligo - pak oligonucleotide purification columns ( glen research corporation , sterling , va ) as per manufacturer s instructions . a stock buffered solution containing 250 l 10x pcr buffer , 100 l of mgcl2 ,
12.5 l of each datp , dttp , dgtp and dctp was prepared in 1.5 ml eppendorf tube , and double distilled water was added to bring the volume of the stock buffer solution to 1.5 ml .
the primers were used at a concentration of 20mole / l which appears to 2 l .
next , 5.0 l of the target dna was added to 42 l of the stock solution in 0.5 ml pcr tubes and mixed by vorttexing .
this is followed by 1.0 l of taq dna polymerase ( perkin elmer ) which was used at a concentration of 2.5 units .
the thermal cycling profiles were as follows : 2-minutes incubation at 95c , followed by 40 cycles at 94c for 1minute , 57c for 30 sec and 72c for 45 sec , and a final incubation at 72c for 10 minutes .
thermal profiles will be performed on a techne phc-2 thermal cycler ( techne , princeton , nj.usa ) .
the 10x tris borate edta ( tbe ) buffer was diluted to 1x solution which was used to prepare 1.0 % agarose gels and as running buffer in electrophoresis after it was stained with ethidium bromide as 0.5g / ml .
15 l from each pcr reaction containing amplified product was loaded onto gels of 1.0 % agarose ( fmc bioproduct , rockland me ) and was electrophoressed .
this study was conducted at the molecular biology laboratory , faculty of veterinary medicine , university of khartoum , and the national ribat university , khartoum , sudan . during the period from february to july 2010
25 nubian goats , maintained at the dairy farm of the faculty of veterinary medicine , university of khartoum , shmbat were included .
before collection , the teats were cleaned with alcohol to avoid samples contamination from skin .
milk ( 2.5 ml ) was collected in sterile 2.5ml centrifuge tube by hand milking , samples were stored at 4c until testing for extraction of total genomic and mitochondrial cytochrome - b ( mtcyt - b ) dna .
blood samples were collected for preparation of positive control , blood samples were collected in clean sterile vacutainers , containing ethylene diamine tetra acetic acid ( edta ) , from goats ( positive controls ) , from the animals attended at the veterinary teaching hospital .
the blood samples then centrifuged in bench centrifuge ( hettich zentrifugen , d-785320 , germany ) in order to separate the buffy coat which is rich in white blood cells and used for extraction of total genomic and mitochondrial cytochrome - b ( mtcyt - b ) dna .
the extracted dna was used as a target dna for pcr amplification extraction of dna from goat milk and peripheral blood was made possible using a commercially available qiaamp blood kit ( qiagen inc.chatsworth , canada ) according to the manufacturer s instructions . in details , 200l of milk samples , 20 l of proteinase
k enzyme stock solution , and 200 l of lysing buffer ( la buffer ) were pipetted into 1.5 ml eppendorf tube and the mixture was vortexed on the vortexing machine ( janke & kunkel , gmbhu.cokg , germany ) and incubated at 60c for 10 minutes .
the mixture then was transferred to the qiaamp spin column , and was placed in a clean 2ml collection tube and centrifuged in microcentrifuge ( hettich zentrifugen , 12 - 24 , tuttiligen , germany ) at 8000 rpm for 1 minute .
the qiaspin column was washed firstly with 500 l of washing buffers 1 ( aw1 ) at the same previously mentioned centrifugation speed and rewashed using washing buffer 2 ( aw2 ) at speed 1200 rpm centrifugation speed for 3 minutes .
the qiaamp spin column was then placed in a clean 1.5 ml eppendorf tube and the dna was eluted with 200 l of double distilled water preheated at 70c .
maximum dna yield will be obtained by spinning at 1200 rpm for 1 minute after remaining for 1 minute in the room temperature .
five microliters of the suspended nucleic acid will be used in the pcr amplification for pcr amplification a pair of goat - specific primers ( gsl1&gsr2 ) was designed from the caprine mtcytb gene sequences .
gsl1 included bases 284 - 303 of the positive sense strand ( 5)- tca tac ata tcg gac gac gt . whereas gsr2 included bases 693 - 712 of the complementary strand ( 5)- caa gaa tta gta gca tgg cg . using of this pair of primers ( gsl1&gsr2 ) in pcr assay resulted in amplification of a 428-bp pcr product from caprine mtcy - b dna .
the primers were synthesized on a dna synthesizer ( milliigen / biosearch , a division of millipore burlington , ma ) and purified using oligo - pak oligonucleotide purification columns ( glen research corporation , sterling , va ) as per manufacturer s instructions .
before collection , the teats were cleaned with alcohol to avoid samples contamination from skin .
milk ( 2.5 ml ) was collected in sterile 2.5ml centrifuge tube by hand milking , samples were stored at 4c until testing for extraction of total genomic and mitochondrial cytochrome - b ( mtcyt - b ) dna .
blood samples were collected for preparation of positive control , blood samples were collected in clean sterile vacutainers , containing ethylene diamine tetra acetic acid ( edta ) , from goats ( positive controls ) , from the animals attended at the veterinary teaching hospital .
the blood samples then centrifuged in bench centrifuge ( hettich zentrifugen , d-785320 , germany ) in order to separate the buffy coat which is rich in white blood cells and used for extraction of total genomic and mitochondrial cytochrome - b ( mtcyt - b ) dna .
extraction of dna from goat milk and peripheral blood was made possible using a commercially available qiaamp blood kit ( qiagen inc.chatsworth , canada ) according to the manufacturer s instructions . in details , 200l of milk samples , 20 l of proteinase
k enzyme stock solution , and 200 l of lysing buffer ( la buffer ) were pipetted into 1.5 ml eppendorf tube and the mixture was vortexed on the vortexing machine ( janke & kunkel , gmbhu.cokg , germany ) and incubated at 60c for 10 minutes .
the mixture then was transferred to the qiaamp spin column , and was placed in a clean 2ml collection tube and centrifuged in microcentrifuge ( hettich zentrifugen , 12 - 24 , tuttiligen , germany ) at 8000 rpm for 1 minute .
the qiaspin column was washed firstly with 500 l of washing buffers 1 ( aw1 ) at the same previously mentioned centrifugation speed and rewashed using washing buffer 2 ( aw2 ) at speed 1200 rpm centrifugation speed for 3 minutes .
the qiaamp spin column was then placed in a clean 1.5 ml eppendorf tube and the dna was eluted with 200 l of double distilled water preheated at 70c .
maximum dna yield will be obtained by spinning at 1200 rpm for 1 minute after remaining for 1 minute in the room temperature .
for pcr amplification a pair of goat - specific primers ( gsl1&gsr2 ) was designed from the caprine mtcytb gene sequences .
gsl1 included bases 284 - 303 of the positive sense strand ( 5)- tca tac ata tcg gac gac gt . whereas gsr2 included bases 693 - 712 of the complementary strand ( 5)- caa gaa tta gta gca tgg cg . using of this pair of primers ( gsl1&gsr2 ) in pcr assay resulted in amplification of a 428-bp pcr product from caprine mtcy - b dna .
the primers were synthesized on a dna synthesizer ( milliigen / biosearch , a division of millipore burlington , ma ) and purified using oligo - pak oligonucleotide purification columns ( glen research corporation , sterling , va ) as per manufacturer s instructions .
a stock buffered solution containing 250 l 10x pcr buffer , 100 l of mgcl2 , 12.5 l of each datp , dttp , dgtp and dctp was prepared in 1.5 ml eppendorf tube , and double distilled water was added to bring the volume of the stock buffer solution to 1.5 ml .
the primers were used at a concentration of 20mole / l which appears to 2 l .
next , 5.0 l of the target dna was added to 42 l of the stock solution in 0.5 ml pcr tubes and mixed by vorttexing .
this is followed by 1.0 l of taq dna polymerase ( perkin elmer ) which was used at a concentration of 2.5 units .
the thermal cycling profiles were as follows : 2-minutes incubation at 95c , followed by 40 cycles at 94c for 1minute , 57c for 30 sec and 72c for 45 sec , and a final incubation at 72c for 10 minutes .
thermal profiles will be performed on a techne phc-2 thermal cycler ( techne , princeton , nj.usa ) .
all pcr amplification product samples were visualized using agarose gel electrophoresis . the 10x tris borate edta ( tbe ) buffer
was diluted to 1x solution which was used to prepare 1.0 % agarose gels and as running buffer in electrophoresis after it was stained with ethidium bromide as 0.5g / ml .
15 l from each pcr reaction containing amplified product was loaded onto gels of 1.0 % agarose ( fmc bioproduct , rockland me ) and was electrophoressed .
the dna yield varies with the different stages of lactation as compared with the yield from peripheral blood leukocytes .
the pcr - based assay described in this study afforded sensitive and specific identification of goat mtcyt - b dna . using the pair of primers ( gsl1 , gsr2 ) .
the sensitivity studies indicate that , the 428bp pcr products were detected from not < 1.0 pg of goat mtcyt - b gene ( fig.1 ) .
the specificity studies for goat primers indicate that the described pcr assay failed to amplify the specific pcr product from dna extracted from other animal species including cattle , sheep , swine , camel , deer , horse , donkey , and human ( figure 2 ) . using primers gsl1 , gsr2 , amplification of the goat - specific 428 bp pcr product
was produced from different milk samples collected from goats included in this study ( figure 3 , 4 , 5 ) . in goat ,
however , obvious technical difficulties exist in the collection of blood samples from large numbers of individuals among widely separated herds , limiting the application of marker - based methods for genetic analysis and for genetic improvement of economic traits in dairy goat .
the scientific data presented in this study indicate that dna extracted from goat milk could serve as substrates for pcr amplification of the full length of the caprine mitochondrial cytochrome - b gene or a fragment of the gene .
since collection of milk is a non invasive procedure , it can often substitute for blood as a source of dna .
the technical convenience of milk as a source of dna can be expected to increase the field of application of the marker - based methods for genetic analysis of goat genome .
it has a remarkable importance for several reasons related to governmental regulation and public health .
protection against species substitution or admixture in dairy products is of significant importance ( bottero et al , 2003 ) .
it was found that most milk proteins , even at low concentration , are potential allergens ( sampson , 2003 ) .
also , cow s milk was reported as the main dairy product responsible for human adverse reaction ( rance et al , 2005 ) .
thus , the counterfeiting of goat s milk with cow s milk may be considered as a health risk making species identification an important issue in current food safety requirement .
the common fraudulent practice found in the dairy production line is the use of a cheaper type of milk in substitution of more expensive ones .
currently , different methods are used for species identification in milk and milk products including immunological ( addeo et al , 1995 ) , electrophoretic ( cartoni et al , 1998 ) , and chromatographic techniques ( pellegrino et al , 1991 ) . among these methods , capillary electrophoresis , two dimensional electrophoresis , isoelectric focusing of milk caseins which is the european community reference method for cow s milk detection ( ecr , 1996 ) ,
however these methods ca nt always distinguish milk from closely related species and not suitable for heat treated milk .
the pcr amplification technology , described in this study , provides a simple , rapid , reliable and sensitive method for species identification and differentiation .
the time required for the pcr amplification was approximately 3 hours ; this means that confirmatory diagnosis could be obtained within the same working day .
( 1993 ) reported that milk is less reliable source of dna than is blood because it requires large size of sample and high concentration of somatic cells ( e. lipkin et al , 1993 ) .
but in our study we used only 200l of milk sample for extraction of dna , and the pcr products were the same in length and approximate quantity for milk , for dna extracted from milk , and for dna extracted from blood , this difference may reflect the detection procedures described above .
the intensity of dna signal in gel electrophoresis , show that , there is a decline in the dna content in the late lactation periods .
this may indicate the milk dna content is high in the first and second lactation periods and then declines . for more significant results dna quantification by spectrophotometer
these non - nuclear targets possess several advantages over nuclear genes ( unseld et al,1995 ) .
they are generally more abundant in any given sample than single - copy nuclear genes , and , because mitochondrial dna has a relatively high mutation rate compared with the nuclear dna , they contain a greater accumulation of point mutations that can be used to better define species differences
. moreover , mitochondrial dna tends to be inherited through the maternal germ line , and the resulting lack of heterozygosity in the alleles under study simplifies analysis ( kocher et al , 1989 ) .
the mtcyt - b dna was selected in this work as the target sequence for species identification .
pcr - based assay described in this study would be advantageous in the variety of conditions including comparative genomics , species identification in milk and milk products , experimental physiology and can be recommended in the quality control departments in order to support policies and regulation of import / export of milk and milk products . | conflict of interest : none declared.introductionthis study was carried out to evaluate pcr - based method for detection of dna in goat milk .
it utilized primers targeting the mitochondrial cytochrome b ( mtcyt - b ) gene , which was used as a target dna for pcr amplification.methodsfor the specific identification of goat mtcyt - b gene , pair of primers ( gsl1 , gsr2 ) , were used , which produced a 428 base pair ( bp ) pcr product from milk samples as well as from peripheral blood .
amplification products were visualized on ethidium bromide - stained agarose gels.results and discussionamplification products were not detected when the pcr was applied to dna from animal species including cattle , sheep , swine , camel , deer , horse , donkey , and human , which indicates that the 2 pairs of primers are specific for goat.conclusiondna can be extracted from goat milk and would be advantageous in the variety of application such as species identification in milk and milk . |
we demonstrate a
strategy to trigger and finely control the assembly
of supramolecular dna nanostructures with ph .
control is achieved
via a rationally designed strand displacement circuit that responds
to ph and activates a downstream dna tile self - assembly process .
we
observe that the dna structures form under neutral / basic conditions ,
while the self - assembly process is suppressed under acidic conditions .
the strategy presented here demonstrates a modular approach toward
building systems capable of processing biochemical inputs and finely
controlling the assembly of dna - based nanostructures under isothermal
conditions .
in particular , the presented architecture is relevant
for the development of complex dna devices able to sense and respond
to molecular markers associated with abnormal metabolism . | |
trauma , a preventable condition , is the leading cause of death in the first four decades of life.1 trauma care starts from the first few minutes at the scene after an accident and continues on the way to the hospital where more definitive care is given .
mortality in the first hour depends on several factors among which are trauma severity and pre - hospital care .
the objectives of this study are :
to determine the rank of road traffic accident ( rta ) among other causes in patients who were already dead on arrival ( doa ) at the emergency department ( ed ) in our region.to determine the characteristics of populations involved in rta and seen as doa.to determine the type of injuries in the doa victims .
to determine the rank of road traffic accident ( rta ) among other causes in patients who were already dead on arrival ( doa ) at the emergency department ( ed ) in our region . to determine the characteristics of populations involved in rta and seen as doa . to determine the type of injuries in the doa victims
the information derived from this study , hopefully , will help health personnel to establish a system by which the injured are managed properly from the time of accident till arrival in hospital .
in addition , it will advise the law enforcement agency and legislators in particular in enacting and enforcing laws that will lead to the prevention of rta and/or reduction of the mortality and morbidity rates .
the data of all rta victims who were dead on arrival at the emergency department ( ed ) of assir central hospital ( ach ) , abha , saudi arabia , between the months of 02 -1410 ( august 1989 ) and 12 , 1414 ( may 1994 ) were obtained from the medical records of the emergency room .
the nationality , sex , and age of the victims , time and month of arrival in the ed and types of injuries clinically encountered , were analyzed .
the dates used in this study are according to both hegira and gregorian calendars , since the hegira calendar is the standard used in file keeping at the ach , abha , and it also a good indicator of the public holidays in saudi arabia , while , the gregorian calendar is a better guide to the seasons .
during the four and half - year period , 574 patients were doa in the ed ; 303 ( 53% ) of them were victims of rta .
of these 303 victims , 283 ( 93.4% ) were males and 20 ( 6.6% ) females giving a male to female ratio of 14:1 .
the age range was 3 months to 85 years , with a mean age of 34.25 years .
the peak age was between the third and fifth decades of life ( 21 - 49 years ) ( fig .
the commonest period of presentation at the ed , regardless of the time of accidents , was between 12:00 and 18:00 hours ( 116 patients ; 38.3% ) followed by 18:00 hours to 24:00 hours ( 88 patients ; 29% ) fig .
2 . the distribution of patients seen in various months of the hegira year is shown in fig .
the year of 1413 hegira had the highest number of the rta doa victims as shown in fig .
the summer season in the gregorian calendar has a significant rise of the rta doa compared to the winter season ( p < 0.05 ) ( fig .
head and neck injuries ( exclusive of skull fractures ) accounted for 45% of the injuries followed by chest injuries ( 39% ) .
thirty - five percent ( 35% ) of the victims had skull fractures ( table 1 ) .
age group distribution of doa victims due to rta distribution of time of arrival of d o a victims due to r t a hegira month distribution of d o a victims due to r t a year distribution of d o a victims due to r t a seasonal presentation of victims of d o a due to r t a type & rate of injuries in 303 ( n ) patients of r t a
the diagnoses were all clinically made without the benefit of certainty of pathology from autopsy examination since autopsies are not usually performed in the kingdom of saudi arabia2 .
rta is a major cause of instant death in this region since it consists of more than half of the cases brought doa into our ed .
the predominance of male victims ( 93% ) is mainly due to the exposure and the activity of the males ; females do not drive in saudi arabia . in a study from the united states of america ( usa )
there is a large number of saudis in this study because the population is mainly saudi in this part of saudi arabia .
the population of saudi arabia is about 16.9 million , 12.3 million ( 72.7% ) of whom are saudis and 4.6 million ( 27.3% ) are non - saudis4 .
assir region , in which this study was conducted , has a population of about one million .
although it has long been recognized that the mortality rate from injury is much higher in the elderly56 , elderly people are much less exposed to the types of trauma that are fatal to younger people .
the highest incidence of trauma occurs in the most active and productive age group ( 21 -49 years ) and lower in the older age group ( fig .
1 ) , the maximum number of cases were seen during the daylight hours between 12:00 and 18:00 hours ( 38% ) .
this includes the rush hour period when people are returning home from work or travelling to neighboring towns and villages .
seasonal variation was also noticed in this study . there is a significant correlation with the summer holidays and public holidays .
the population in this part of the country almost doubles when many tourists flock to this region during the summer months because of its mild weather .
the assir region is situated at a high altitude of about 2200 meters above sea level . the fog in this area which usually affects visibility , however , was not found to be a significant factor in this study since the incidence of rta doa victims during the winter period is about the lowest .
there seems to be no clear explanation for this and further investigation into this is therefore required .
all the victims were polytraumatized , and the types of injuries were severe . the majority of victims had a combination of severe head , neck , chest and lower extremities injuries typical of driving without a hasness on coupled with high speeds.7 the enormity of the impact of trauma can be estimated from the skull fracture ( 105 ; 35% ) , with brain matter extruding from the skull in 61 ( 20.3% ) patients .
there is documentation in the international studies of the fact restraining that devices plays a significant role in the reduction in mortality of drivers and passengers810 .
shawan et al showed in a local study on the attitude to the wearing of seat belts found that the commonest reason for not wearing a seat belt was that of complacency and 90% of the people interviewed thought that a legislation on seat belts would be useful12 . a similar study by bener et al in the united arab emirate had similar results13
road traffic accident is the first major cause of death among dead on arrival cases affecting the most active and productive age group ; most of the accidents occur during daylight hours and correlate well with public and summer holidays .
although we can not prevent all deaths from road traffic accident , we can reduce the number of deaths , since it was noted in previous studies that 20% - 30% of trauma related deaths are preventable.14 we , therefore , strongly recommend the following :
the introduction of legislation for the mandatory fitting and wearing of seat - belts , and the intensification of the current educational program and law enforcement especially during public and summer holidays.the implementation of pre - hospital emergency medical system.1516 this can be done by introducing the well established pre - hospital life support programs17 , like emergency medical technician- ambulance ( emt - a),18 emergency medical technician - intermediate ( emt - i)19 , emergency medical technician - paramedic ( emt - p)20
the introduction of legislation for the mandatory fitting and wearing of seat - belts , and the intensification of the current educational program and law enforcement especially during public and summer holidays . the implementation of pre - hospital emergency medical system.1516 this can be done by introducing the well established pre - hospital life support programs17 , like emergency medical technician- ambulance ( emt - a),18 emergency medical technician - intermediate ( emt - i)19 , emergency medical technician - paramedic ( emt - p)20 | background : although rood traffic accident ( rta ) is a noticeable common cause of death in saudi arabia , there is no published data showing the relative frequency of this disease as a cause of death.aim of the study : this study attempted to find out the relative frequency of rta as a cause of death . also , to identify age groups at risk as well as make some inferences from the different types of injuries seen.methodology:in a period of over a four and half years , 574 patients were seen dead on arrival at the emergency department of assir central hospital , abha , saudi arabia . of these , 303 ( 52.8% ) were victims of rta.results:the 303 victims revealed a male to female ratio of 14:1 , saudi nationals of 69% and age range of 3 months - 85 years ( mean = 34.25 years ) .
the peak age group was between 21 and 49 years and the peak period of presentation at the emergency department was between 12:00 noon and 18:00 hours .
the month of ten in hegira calendar represented the peak period ; a significant ( p<0.05 ) seasonal variations was also seen , summer being the highest .
clinical assessment of the victims revealed that head and neck injuries were the commonest followed by chest injuries.conclusion:rta is the primary cause of death among dead on arrival cases affecting the most active and productive age group .
the study recommended the implementation of pre -hospital emergency medical system . |
giant cell tumors ( gcts ) are rare lesions that develop in the epiphyses of long bones in the extremities .
they are exceptional lesions when they occur in the skull , and then are usually found in the sphenoid or temporal bones .
their adverse effects are usually the result of compression , often causing cranial nerve deficits relative to their position .
several cases with substantial follow up have described gross total resection to be adequate for cure . here
we describe the case of a patient that presented with a large gct that was invasive to the dura , temporal lobe , as well as the third division of the trigeminal nerve . to date
a 44 year - old woman initially saw her primary care physician for right ear pain and fullness in april 2010 .
, she saw an audiologist who noted mild conductive hearing loss on the right , and , subsequently , a right tympanostomy tube was placed .
after this , nearly a year passed and the patient began to notice significant hearing loss in her right ear .
she was referred to a community otolaryngologist , and , on examination , a small mass was noted in the right external auditory meatus ( fig .
computed tomography and magnetic resonance ( mr ) imaging of the brain and temporal bone were performed ( fig .
d ) , and she was then referred to both the ears , nose , and throat ( ent ) and neurosurgery departments at our institution in early july 2011 .
the patient underwent biopsy of the lesion from the external auditory meatus ( eam ) a few days later that revealed giant cell tumor .
( a ) photo of the right external auditory meatus taken during an otoscopic examination when the patient was initially seen in the ear , nose , and throat clinic , early july 2011 .
( b , c ) coronal and ( d ) axial slices of computed tomography bone windows showing temporal and petrous bone prior to resection .
( e ) anteroposterior towne and ( f ) lateral view of tumor model . at the time of surgery
, the patient had no focal motor deficits , and her only sensory deficit was decreased hearing in the right ear .
audiology reports revealed moderate conductive hearing loss on the right with a speech reception threshold of 35 db in the right ear and 25 db in the left ear .
there was no pathologic adenopathy identified , but there was fullness and tenderness over the right temporomandibular joint .
there was excursion and drift of the mandible to the left and a cross - bite deformity .
initially , a tracheostomy was performed with the patient supine and , after securing a number 6 shiley tracheostomy tube in place , the patient was positioned left lateral with the right side of the head facing upward and the vertex of the head slightly down .
the skin flap , incised to the temporalis facia , was rotated forward . to facilitate this ,
the external auditory canal was transected and oversewn , and the facial nerve was identified and dissected from the parotid gland .
the frontal branch had to be completely dissected and mobilized inferiorly with a cuff of the temporalis facia to gain adequate exposure to the zygoma and root .
posteriorly , the zygoma was left intact , as a part of the specimen , because the root was heavily involved with tumor .
the temporal muscle was incised below the superior temporal line and reflected posteriorly to gain exposure to the squama for a subtemporal craniectomy .
next , a subtotal petrosectomy was performed to expose the intratemporal seventh nerve distal to the geniculate ganglion , identify and control the sigmoid sinus , and obtain negative bone margins at the zygomatic root .
a subtemporal craniectomy was performed , and the floor of the middle fossa was resected as far medially as the foramen ovale .
the tumor could be seen entering the dura , and a durotomy was made lateral to this point .
intradurally , the tumor was clearly invasive and adherent to the brain , so , microscopically , the lesion was resected down to normal - appearing white matter at all margins .
grossly , the tumor involved the third division of the trigeminal nerve , which was sacrificed at the foramen ovale .
finally , the roof of the glenoid fossa was mobilized and an osteotomy was made in the mandible at mid - ramus , resecting the condyle , the coronoid , and the temporomandibular joint . after dissecting and mobilizing the infratemporal portion of the tumor , the upper mandible , glenoid , a portion of the floor of the middle fossa , and the tumor were removed en bloc .
the dura was repaired with a dural substitute and sealed with a polyethylene glycol based sealant ( fig .
a portion of the temporal lobe and dura were removed and repaired with a dural substitute and sealant .
the temporal floor , zygoma , and portion of the upper mandibular ramus were invested with tumor and resected .
zygomatic arch ( asterisk ) , temporal lobe ( double asterisk ) , and duraplasty ( arrow ; superior temporal line ) .
( b ) sagittal ( c ) coronal , and ( d ) axial views of 19-month follow - up magnetic resonance imaging showing no tumor recurrence and good reconstruction with a rectus free flap .
the patient tolerated the procedure well and was taken sedated to the neurosurgical intensive care unit for monitoring .
she was electively returned to the operating room the following day for reconstruction of the defect with a rectus abdominis free tissue transfer and split thickness skin graft . at 15-month follow - up ,
d ) . she had mild dullness to sensation on the right side of her face , her right seventh nerve palsy had completely resolved , and she had conductive hearing loss in her right ear due to the eam being sewn over .
histologic analysis of the biopsy specimen revealed an infiltrative lesion beneath the epidermis ( fig .
admixed among the mononuclear cells were many multinucleated giant cells and hemosiderin pigment consistent with giant cell tumor . throughout the lesion were many hemosiderin - laden macrophages and hemorrhage .
( a ) infiltrative giant cell tumor ( gct ) beneath the epidermis showing characteristic mononuclear cells , hemosiderin pigment , and multinucleated giant cells . heavy infiltration of gct in ( b , c ) brain and ( d ) bone .
analysis of the resected lesion showed giant cell tumor within the right middle ear , eustachian tube , invading the bone and dura of the middle fossa as well as the adjacent temporal brain tissue ( fig .
six lymph nodes were dissected from the right neck near the infratemporal region of the tumor , and these were negative for malignancy .
features that may suggest a more aggressive lesion , such as vascular invasion , were not identified , and there were no sarcomatous changes .
at the time of surgery , the patient had no focal motor deficits , and her only sensory deficit was decreased hearing in the right ear . audiology reports revealed moderate conductive hearing loss on the right with a speech reception threshold of 35 db in the right ear and 25 db in the left ear .
there was no pathologic adenopathy identified , but there was fullness and tenderness over the right temporomandibular joint .
there was excursion and drift of the mandible to the left and a cross - bite deformity .
initially , a tracheostomy was performed with the patient supine and , after securing a number 6 shiley tracheostomy tube in place , the patient was positioned left lateral with the right side of the head facing upward and the vertex of the head slightly down .
the skin flap , incised to the temporalis facia , was rotated forward . to facilitate this ,
the external auditory canal was transected and oversewn , and the facial nerve was identified and dissected from the parotid gland .
the frontal branch had to be completely dissected and mobilized inferiorly with a cuff of the temporalis facia to gain adequate exposure to the zygoma and root .
posteriorly , the zygoma was left intact , as a part of the specimen , because the root was heavily involved with tumor .
the temporal muscle was incised below the superior temporal line and reflected posteriorly to gain exposure to the squama for a subtemporal craniectomy .
next , a subtotal petrosectomy was performed to expose the intratemporal seventh nerve distal to the geniculate ganglion , identify and control the sigmoid sinus , and obtain negative bone margins at the zygomatic root .
a subtemporal craniectomy was performed , and the floor of the middle fossa was resected as far medially as the foramen ovale .
the tumor could be seen entering the dura , and a durotomy was made lateral to this point .
intradurally , the tumor was clearly invasive and adherent to the brain , so , microscopically , the lesion was resected down to normal - appearing white matter at all margins .
grossly , the tumor involved the third division of the trigeminal nerve , which was sacrificed at the foramen ovale .
finally , the roof of the glenoid fossa was mobilized and an osteotomy was made in the mandible at mid - ramus , resecting the condyle , the coronoid , and the temporomandibular joint . after dissecting and mobilizing the infratemporal portion of the tumor , the upper mandible , glenoid , a portion of the floor of the middle fossa , and the tumor were removed en bloc .
the dura was repaired with a dural substitute and sealed with a polyethylene glycol based sealant ( fig .
a portion of the temporal lobe and dura were removed and repaired with a dural substitute and sealant .
the temporal floor , zygoma , and portion of the upper mandibular ramus were invested with tumor and resected .
zygomatic arch ( asterisk ) , temporal lobe ( double asterisk ) , and duraplasty ( arrow ; superior temporal line ) .
( b ) sagittal ( c ) coronal , and ( d ) axial views of 19-month follow - up magnetic resonance imaging showing no tumor recurrence and good reconstruction with a rectus free flap .
the patient tolerated the procedure well and was taken sedated to the neurosurgical intensive care unit for monitoring .
she was electively returned to the operating room the following day for reconstruction of the defect with a rectus abdominis free tissue transfer and split thickness skin graft . at 15-month follow - up ,
d ) . she had mild dullness to sensation on the right side of her face ,
her right seventh nerve palsy had completely resolved , and she had conductive hearing loss in her right ear due to the eam being sewn over .
histologic analysis of the biopsy specimen revealed an infiltrative lesion beneath the epidermis ( fig .
admixed among the mononuclear cells were many multinucleated giant cells and hemosiderin pigment consistent with giant cell tumor . throughout the lesion were many hemosiderin - laden macrophages and hemorrhage .
( a ) infiltrative giant cell tumor ( gct ) beneath the epidermis showing characteristic mononuclear cells , hemosiderin pigment , and multinucleated giant cells . heavy infiltration of gct in ( b , c ) brain and ( d ) bone .
analysis of the resected lesion showed giant cell tumor within the right middle ear , eustachian tube , invading the bone and dura of the middle fossa as well as the adjacent temporal brain tissue ( fig .
six lymph nodes were dissected from the right neck near the infratemporal region of the tumor , and these were negative for malignancy .
features that may suggest a more aggressive lesion , such as vascular invasion , were not identified , and there were no sarcomatous changes .
bertoni et al reviewed 546 mayo clinic cases of gct and identified 4 cases involving bones of the skull base.1 a further review of 1500 mayo consultation cases revealed just 11 more cases of skull gct .
these lesions preferentially arise from the sphenoid and/or temporal bones,2
3 which may be due to the fact that the skull base and portions of the temporal bone form by endochondral ossification , rather than intramembranous ossification like other bones of the skull .
this is the same process by which bone forms in the epiphyses of long bones where gcts are primarily found.4 the multinucleated giant cell of the gct closely resembles the osteoclast that is prevalent in these ossification centers of long bones.5
6
defining cellular and molecular characteristics of more aggressive gcts have been difficult to elucidate .
studies of gct in long bones has shown that a higher count of mitotic figures per given field has not been consistent in identifying more aggressive lesions .
in fact , sanerkin 's study of 90 gcts revealed lesions possessing as many as 6 to 10 mitoses / mm were not necessarily aggressive ; nor did they recur more often.7 however , all of the tumors possessing 10 mitoses / mm were aggressive , defined by their invasiveness and recurrence .
malignant potential also could not be consistently determined by higher mitotic rate , abnormal mitoses , or vascular invasion .
in general , malignant gcts had higher mitotic counts , but one of the defining properties of lesions with malignant metastases was sarcomatous degeneration.7
8
9 therefore , with the exception of sarcomatous change , the histologic features of a gct often do not seem to correlate with the lesion 's local aggressive nature or metastatic potential .
the tumor discussed in this report was locally destructive and invaded bone , dura , and brain , but its histologic appearance was that of a standard benign gct .
recurrence of giant cell tumors in the skull , and in long bones , seems to be directly related to the extent of resection.3
7
10
it is not clear what molecular properties of the gct permit it to be locally invasive or metastatic .
some lesions barely affect adjacent tissue , whereas others invade and dissolve local tissue and bone .
ueda et al showed that giant cells can express matrix metalloproteinase-9 ( mmp-9 ) , which can dissolve type iv collagen.11 this collagen is a major structural component in the basement membranes of blood vessels .
mmp-9 also has the capacity to degrade type i and v collagen , a major component of bone .
although this may allow the tumor to be locally aggressive , vascular invasion and tissue destruction alone is not sufficient for metastasis of gct .
the large rare gct presented here was invasive to dura , brain , and the fifth cranial nerve and was treated with radical resection .
we are aware of only two existing reports of invasive adult gct.12
13 one of these lesions was invasive to adjacent dura only and the other to dura and brain . because portions of each of these lesions were unresectable , both
radiotherapy is recommended for tumors such as these that can not be completely resected , for patients who can not undergo surgery because of comorbid factors , or for patients with progressing or multiple recurrences after surgical excision .
some advocate treating patients that they feel are at especially high risk of recurrence . nonetheless , treating gcts with radiation is still somewhat controversial .
earlier studies reported sarcomatous malignant degeneration in some tumors that were irradiated.3
7
14 it seems , however , that sarcomatous transformation occurs with similar frequency in tumors that have been irradiated , as well as those treated with surgery alone.15
16 more recent reports with follow - up ranging from 2 to 34 years revealed no cases of malignant transformation when using 35 gy in 15 doses.17 radiotherapy should be used as an adjunct . when used as a single modality
, recurrence rates of 60 to 70% have been reported.18 the patient in this study is > 2 years out from surgery with no sign of recurrence .
we believed we could achieve a gross total resection based on a thorough analysis of the lesion size , location , and invasiveness .
time will tell if resection alone is an adequate and durable treatment for this lesion . | giant cell tumors ( gcts ) are rare , usually affecting the epiphyses in long bones of the extremities .
they seldom occur in the skull , where they preferentially affect the sphenoid and temporal bones .
considered to be benign , locally aggressive lesions , they may cause cranial nerve deficits by compression but infrequently invade the dura and parenchyma of the brain .
several case reports with follow - up describe gross total resection of skull base gct to be curative .
anything short of total resection usually results in recurrence within 4 years .
radiation therapy , although controversial , is reserved for lesions that can not be completely resected .
some argue , however , against the use of radiation because there are reported cases of malignant transformation . here
we describe the case of a large gct that was invasive to the dura , temporal lobe , as well as the third division of the trigeminal nerve , and to date gross total resection has been curative of this lesion . the patient has not undergone radiation therapy . |
patients with end stage renal disease ( esrd ) can be sustained with dialysis therapy . however , patient s survival rate is higher with renal transplantation , which makes it the preferred method of treatment .
in addition , renal transplant recipients have better quality of life than those under dialysis ( 1 ) . however , long - term allograft survival is still limited .
many factors affect patient - graft survival in renal transplantation such as donor or recipient age , gender , race , primary cause of renal failure , cold ischemia time , hla matching , prior transfusion , blood groups and preservation methods ( 2 - 5 ) .
acute rejection ( ar ) is a common complication in renal transplantation and associated with reduced graft survival ( 6 ) . it can occur less than three months ( early acute rejection , ear ) or after three months of transplant ( late acute rejection , lar ) .
while rejection episodes and delayed graft function ( dgf ) increase the risk of renal allograft loss ( 7 , 8) , strong immunosuppressive therapies reduce the frequency of acute rejection ( 9 ) .
the aim of this study was to investigate the timing of ar in a sample population and the relative impact of early and late ar on patients - grafts survival .
a historical cohort study was performed to assess the influence of acute rejection episodes ( ares ) on patient - graft survival rates .
a thorough review of the files of patients with renal transplanted was conducted between 1990 and 2011 in the renal transplant unit of taleghani hospital of shahid beheshti university in tehran , iran .
all patients received cyclosporine ( 7 - 8 mg / kg ) , 500 - 1000 mg of intravenous methyl prednisolone before the operation , as well a 3-day bolus of intravenous methyl prednisolone therapy after the transplantation .
a standard triple therapy comprising cyclosporine a ( csa ) , azathioprine ( aza ) or mycophenolate mofetil ( mmf ) , and prednisone was administrated as primary maintenance immunosuppressive regimen . after the operation , aza ( 1.52 mg / kg / day ) or mmf ( 1.52 g / day )
csa ( 7 - 8 mg / kg / day ) was administered two days after the transplantation when the level of creatinine reduced and the dosage was adjusted according to trough level concentrations .
oral prednisone was subsequently tapered at a daily dosage of 0.6 mg / kg for one month , after which the daily dosage was tapered to 7.5 - 5 mg in three months .
we defined ear as a rejection occurred in less than three months and lar as a rejection happened after the first 3 - 6 months .
recipients were divided into three groups : group-1 ( no rejection ) , group-2 ( ear ) and group-3 ( lar ) .
the degree of allograft function was evaluated by measuring serum creatinine , urea , electrolytes and daily urine output .
any rejection episode was determined through clinical information , color doppler ultrasonography and renal dtpa isotope scan .
, there was no initial response to corticosteroids , polyclonal or monoclonal antibodies were used as a follow - up therapy .
finally , all patients were followed up routinely at the outpatient center of the hospital .
factors considered to have influence on patient - graft survival were donors and recipients age and gender , the type of donor ( cadaver , living related and living unrelated ) , number of transplantations , duration of hemodialysis or peritoneal dialysis and status of panel reactivity antibody .
after gathering information , the transplanted patients - grafts data were divided into four groups of active , graft loss , deaths and inconclusive .
finally , based on the collected information , patient - graft survival rates were calculated .
graft loss was defined as the need for resumption of dialysis or patient dying due to any reason , even though the graft was functioning .
the overall patient - graft survival was calculated for each group using the kaplan - meier method .
means of quantitative variables were compared using student t - test between the two groups and anova test among the three groups , while chi - square test was applied for discontinuous variables .
the overall patient - graft survival was calculated for each group using the kaplan - meier method .
means of quantitative variables were compared using student t - test between the two groups and anova test among the three groups , while chi - square test was applied for discontinuous variables .
we reviewed data of 663 kidney transplant cases , performed between 1990 and 2011 at taleghani hospital .
there were 407 ( 61.4% ) male and 256 ( 38.6% ) female recipients , whose mean age was 34.87 12.96 years .
as for the donors , 526 ( 79.3 % ) of them were males and 137 ( 20.7% ) females , whose mean age was 28.87 7.03 years .
panel reactivity antibody had positive results in five patients ( 0.75% ) and negative in 658 patients ( 99.25% ) .
the type of transplantations was as follows : living related 93 cases ( 14% ) , living unrelated 550 cases ( 82.9% ) , cadaver 15 ( 2.3% ) and inconclusive data 5 ( 2% ) . of 663 patients , 638 patients were first time transplant recipients , 23 of them were second time recipients and two patients were inclusion data transplant recipients .
demographic characteristics of patients and donors along with other pre - transplantation patients data are shown in table 1 .
only four patients were evaluated by kidney biopsy and the rest were diagnosed by renal scanning , measuring creatinine level and observing clinical symptoms . among the variables listed in table 1 , the recipients gender and donors age were more influential .
lar was associated with male recipients ( p = 0.001 ) and donors age with higher than mean age had association with rejection ( p = 0.0002 ) .
the overall patient survival rates of one and five years after renal transplantation were 94.87% and 93.8% , respectively ; while , the overall graft survival rates of one and five years were 92.6% and 81.9% , respectively .
the incidence of rejection episodes were as follows ; 461 ( 69.5% ) had no rejection , 125 ( 18.8% ) developed ear and 77 ( 11.7% ) had lar .
the group-1 patient survival rate of one and five years were 97.4% and 96.8% , for group-2 were 85.6% and 84% , and for group-3 were 92.2% and 89.4% , respectively as shown in table 2 .
graft survival rates at one and five years for group-1 after transplantation were 96.7% and 94.5% , 72% and 61% for group-2 and 84.4% and 36.8% for group-3 , respectively as demonstrated in table 3 .
patient survival rates showed a slight trend to higher values in group-1 at one and five years as presented in figure 1 .
as shown in figure 2 , five - year graft survival rates were higher for group-1 with significance statistical difference compared to the other two groups ( p = < 0.0001 ) .
the five - year graft survival decreased significantly in group-3 ( p = 0.0001 ) . in group-1 ,
cox regression model revealed that the age and gender of donors and recipients and the type of transplant were not effective factors in patients and graft survival rate in renal transplants .
however , creatinine level at the time of discharge from hospital was an effective factor in patients and graft survival rate as shown in table 4 .
patients survival was significantly affected by the combination of dgf and rejection ( 6 , 10 , 11 ) .
long - term survival of renal allograft is significantly associated with ares ( 12 ) .
some studies indicated that dgf appears to be a stronger predictive factor for poor graft survival than ar in the first 90 days of post - transplantation ( 13 ) .
( 8) argued that although dgf is a risk factor of ar and for suboptimal function at one - year duration , it does not appear to increase the rate of graft loss .
ares had no adverse effect on long - term renal graft function if they were successfully treated ( 14 ) .
in the recent years , the incidence of ares has decreased due to introduction of newer immunosuppressive medications ( 15 ) .
the main factors for a more successful long - term allograft outcome are immunologic such as low rate of are and advancement of immunosuppressant agents ( 16 , 17 ) . however , new immunosuppressive agents have little effect on chronic graft dysfunction and long - term renal allograft survival is still a concern ( 18 ) .
recent research has confirmed that with new immunosuppressive therapy , an ear is no longer a risk factor of long - term graft failure and chronic allograft nephropathy ( 19 ) . in preventing severe ar in spk ( simultaneous pancreas
kidne ) transplant patients , tacrolimus - based immunosuppressive therapy has shown significant advantage over cyclosporine ( 20 ) .
today , ar is still a critical risk factor of late transplant failure despite a decrease in occurrence of acute rejections compared to the past ( 21 ) . in this study ,
the incidence of rejection episodes was as follows ; 69.5% had no rejection , 18.8% developed ear and 11.7% had lar .
some previous studies argued that ears are as much of a risk factor as lars in chronic graft loss ( 22 , 23 ) .
in contrast , another research showed that only lars have adverse effect on long - term graft function ( 24 ) .
our study showed that ears were not as significant risk factors of chronic graft loss compared with lars . both the unos and the u.s .
however , ear does not appear to be a risk factor of long - term successful results , it is still a major complication that increases hospital costs .
studies have shown that the number of are has negative impact on graft survival ( 26 , 27 ) .
furthermore , recipients with one are have been found to show better survival rates than those with two or more episodes of rejection ( 27 ) .
the degree of renal damage is estimated by the difference in serum creatinine before and after ares , which affect allograft survival ( 28 ) . as discussed earlier , many factors affect patient - graft survival ( 2 - 5 )
. in our study , among those variables , gender of recipients and age of donor had some influence .
late acute rejection was associated with male recipients ( p = 0.001 ) , while donors with a higher mean age showed association with rejection ( p = 0.0002 ) .
the purposes of the present study were to evaluate the patient - graft survival rates in renal transplantation at our center and to analyze different factors that might have affected patient - graft survival in the sample population with an emphasis on rejection .
this study showed that the timing of an acute rejection episode was associated with the success rate of patient - graft survival .
overall , one and five - year outcomes of graft survival were affected by graft rejection .
the highest amount of graft loss occurred in the late acute rejection group ( p < 0.0001 ) and the best graft survival rates related to patients who did not have acute rejection during the first year after transplantation . in conclusion
, according to this study , late acute rejection episodes are significant predictive factors for the failure of graft survival . | background : patients with end stage renal disease ( esrd ) can be sustained with dialysis therapy.objectives:in this study , we followed up the effect of early acute and late acute rejections on survival rates of patients grafts.patients and methods : we investigated the timing and frequency of acute rejection episodes related to long - term patient - graft survival in taleghani hospital between 1990 and 2011 .
recipients were divided into three groups as group-1 ( no rejection ) , group-2 ( early acute rejection [ ear ] : less than 3-months ) and group-3 ( late acute rejection [ lar ] : after 3 months of transplant).results : one and five - year patient s survival rates were 94.87% and 93.8% , and graft survival ( gs ) rates were 92.6% and 81.9% .
ear and lar occurred in 125 ( 18.8% ) and 77 ( 11.7% ) patients , respectively .
graft and patient survival rates at one and five years were as follows ; group-1 ( graft 96.7% and 94.5% patient : 97.4% and 96.8% ) , group-2 ( graft : 72% and 61% , patient : 85.6% and 84% ) , group-3 ( graft : 84.4% and 36.8% , patient : 92.2% and 89.4% ) .
recipient age , type and length of dialysis , number of transplantations and the status of panel reactivity antibody ( pra ) had no effect on the type of rejection .
lar was more commonly associated with males ( p = 0.001 ) and donors age was associated with rejection ( p = 0.0002 ) .
five - year gs rate among the three groups was lower in the lar group ( p < 0.0001).conclusions : lar had a negative impact on long - term renal allograft survival and the risk of chronic graft dysfunction increased in patients with a history of lar . |
one of the main objectives of root canal preparation is cleaning and shaping of the root canal system , while retaining the original shape of the canal without creating iatrogenic events , such as instrument fracture , apical transportation , ledge or perforation .
deviation from the original canal path and occurrence of apical transportation are some of the procedural errors during root canal preparation .
stainless steel hand instruments are especially more prone to produce these mishaps , due to the stiffness of the file and intense lateral pressure on the walls of the canals . in the recent decades , many new systems and techniques have been introduced for the preparation of root canals with ni -ti rotary instruments .
also , a number of rotary ni - ti instruments with different designs and shapes have been developed for easier root canal preparation and reducing the operating time .
many of these systems have been investigated with regard to their efficacy in cleaning and shaping of root canals , safety and function .
yared used f2 protaper for single file canal preparation in reciprocating motion , despite the fact that this system has not been designed for this motion . in 2008 ,
both waveone ( dentsply maillefer , ballaigues , switzerland ) and reciproc ( vdw , munich , germany ) were internationally launched as single - file shaping techniques .
this study introduces a method of canal preparation with only one rotary file discarded after a single use .
this method needs only one file , reduces the cost and time of canal preparation , increases the accuracy of work and does not need a special expensive engine motor in contrary to the waveone and reciproc systems .
race ( reamer with alternating cutting edges ) rotary files have a noncutting tip and a triangular cross - section .
alternating cutting edges avoid the screwing effect and have the advantage of operating with extremely low torque .
mtwo instruments have a s - shaped cross - sectional design , a noncutting safety tip , positive rake angle with two cutting edges and increasing pitch length from the tip to the shaft .
this design prevents the screwing effect in continuous rotation and reduces the extrusion of debris beyond the apex .
some previous studies reported that mtwo ( vdw , munich , germany ) and race ( fkg dentaire sa , la chaux - de - fonds , switzerland ) rotary file systems maintained the root canal curvature , were associated with few instrument fractures , were more rapid than the manual techniques and were easy to use [ 35 ] .
the aim of this study was to compare the efficacy of mtwo and race rotary file systems in straightening the canal curvature using only one file or the conventional method .
in this experimental study , 60 recently extracted human mandibular first and second molars were collected and kept in normal saline .
mature teeth without visible apical resorption or cracks and with separate mesial and distal roots were selected .
the roots with multiple or abrupt apical curvatures with less than 3 mm radius were excluded .
the teeth were immersed in 5.25% sodium hypochlorite solution for 30 minutes and then stored in 1% chloramine t solution for one week .
after access cavity preparation , apical patency of both mesiobuccal and mesiolingual root canals was examined using a # 10 k - file .
then , based on the degree of mesiodistal canal curvature ( < 25 , > 25 and < 35 , and > 35 ) three groups ( n=20 ) were created .
finally , each group was divided into four subgroups ( n=10 mesial canals each ) .
the access cavity was sealed with cavit ( detrey/ dentsply , konstanz , germany ) after placing a cotton pellet in it and then the root apex was covered with a small piece of wax .
then , the roots were embedded in molds filled with self - polymerizing acrylic resin . to determine the working length , a # 15 k - file was inserted into the canal 1 mm short of the radiographic apex .
occlusal surfaces of the teeth were flattened using a 0.5 mm - thick diamond disk ( d & z , berlin , germany ) to achieve a working length of about 18 mm and a reliable reference point during canal preparation .
the radiographs were taken using a digital radiography sensor ( kodak rvg 5100 digital radiography system , eastman kodak co. , trophy radiology , marne - la - valle , france ) and a platform ( fig .
1 ) , which stabilized the mounted teeth and enabled the superimposition of pre- and post - instrumentation radiographs . next ,
# 15 k - file was inserted into the canal and the initial radiograph was taken using a digital radiography sensor and a radiographic device ( x - genius , de gotzen , roma , italy ) with 70 kvp , 8 ma and 0.3 seconds exposure settings and a cylindrical 12-inch tube .
the initial radiographs were obtained in both buccolingual and mesiodistal directions . the platform : ( a ) superior view ( b ) lateral view root curvatures
were determined using schneider 's method and adobe photoshop cs3 extended ( middle east version 10.0 ) software program .
root canal preparation was done using race ( fkg , lachaux - de - fonds , switzerland ) and mtwo ( vdw , munich , germany ) systems in the conventional method or with the use of only one file .
initially , all the mesial canals were prepared with # 10 and # 15 k - files and well - prep ( vericom co. , anyang , korea ) was used as a lubricant . in the subgroup 1
mtwo files including 10/0.04 , 15/0.05 , 20/0.06 and 25/0.06 were used , respectively in single - length technique according to the manufacturer 's instructions . in the subgroup 2 ,
mesial canals were prepared with only one rotary mtwo file 25/0.06 . in this technique , after the initial preparation with # 10 and # 15 k files , mtwo file 25/0.06 was introduced into the canal .
the coronal two - thirds of the canal was prepared with active working on the walls without excessive pressure on the tip of the file .
after this pre - flaring and determination of the new working length , the file was used for preparation of the entire length of the canal . if the rotary file did not reach the working length , # 20 k - file was used with watch - winding and pulling motion until it reached the working length .
canal irrigation was done with % 5.25 sodium hypochlorite using max - i - probe 30-gauge needle ( dentsply , elgin , illinois , usa ) .
finally , the canals were irrigated with % 17 edta ( ariadent , asia chemi teb co. , tehran , iran ) followed by % 5.25 sodium hypochlorite to remove the smear layer . in the subgroup 3
, the canals were prepared using race rotary files numbers 40/0.10 , 35/0.08 , 25/0.06 , 25/0.04 and 25/0.02 , respectively .
when the file # 25/0.06 reached to the working length , the canal preparation was considered complete . in the subgroup 4 ,
the canals were prepared with only one rotary race file # 25/0.06 using the new method described for the subgroup 2 .
each file was used for preparing two roots or four root canals and in case of any change in the appearance of the file it was discarded . to compare the changes in canal curvatures , a # 25 k - file was introduced into each canal and the radiographs
pre- and post - preparation images were superimposed using adobe photoshop cs3 extended ( middle east version 10.0 ) software .
if the two files were seen separately at the apical third on the superimposed radiograph , it was considered as a deviation from the original path that is apical transportation ( fig . 2a and 2b ) .
superimposition of pre- and post - preparation images in ( a ) buccolingual direction and ( b ) mesiodistal direction .
preparation time for each canal including instrumentation , irrigation and changing the instruments was recorded using a digital chronometer .
the measurements were made blindly and the data were recorded using spss version 16 software ( microsoft , il , usa ) .
data analysis was done with three - way anova and tukey s post hoc test .
the homogeneity of variances was tested with levene 's test and there were no significant differences among the variances of the groups .
statistical indicators regarding deviation of canal curvature ( in degrees ) in buccolingual dimension ( n=10 ) sd : standard deviation regarding the mean deviation of canal curvature in buccolingual direction ( table 1 ) , there was no significant difference between mtwo and race ( p=0.716 ) or between the two canal preparation techniques ( p=0.586 ) .
comparison of deviation of canal curvature in subgroups ( 2535 curvature , greater than 35 and less than 25 ) revealed a significant difference ( p<0.001 ) , and the highest rate of curvature changes was observed in > 35 subgroup .
there were no significant differences in the buccolingual dimension for the following interactions : file type and preparation method ( p=0.302 ) , preparation method and degree of canal curvature ( p=0.743 ) , file type and degree of canal curvature ( p=0.377 ) and finally the interaction of preparation method , file type and degree of canal curvature ( p=0.285 ) .
statistical indicators for deviation of canal curvature ( in degrees ) in the mesiodistal direction ( n=10 ) sd : standard deviation there was no significant difference between mtwo and race ( p=0.550 ) or between the two canal preparation methods regarding the deviation of canal curvature in the mesiodistal direction
. however , comparison of deviation of canal curvature in subgroups ( 2535 curvature , greater than 35 and less than 25 ) revealed significant differences ( p<0.001 ) and the highest rate of curvature changes was observed in the subgroup with curvature > 35. there were no significant differences in the mesiodistal direction for the interactions mentioned earlier .
the mean time of canal preparation with only one rotary file had a significant difference with the conventional method ( p<0.001 ) .
sd : standard deviation no significant differences were observed in terms of canal preparation time with mtwo and race files ( p=0.669 ) , or with regard to the degree of canal curvature ( p=0.426 ) .
assessment of the following interactions with three - way anova revealed no significant effects : preparation method and file type ( p=0.229 ) , preparation method and degree of canal curvature ( p=0.378 ) , file type and degree of canal curvature ( p=0.103 ) , file type and the method of canal preparation and degree of canal curvature ( p=0.622 ) .
regarding the file fracture , none of the race files broke but an mtwo file # 25/0.06 broke in the conventional method in canal curvature of > 35.
recently introduced rotary devices are claimed to be superior to previous instruments in canal preparation .
it is necessary to precisely analyze canal geometry in three dimensions and to compare the pre- and post - instrumentation shapes to confirm or refute such claims . in this study ,
two rotary niti file systems available in the market namely mtwo and race files were evaluated in terms of their ability to maintain the canal curvature , speed of canal preparation and safety of use .
this study focused on the comparison of these systems used in the conventional method of canal preparation versus only one rotary file with the aim of reducing the number of files , cost of treatment and the time of canal preparation . due to the accumulation of debris in file flutes and the problem of cross contamination and the difficult process of cleaning and sterilization , a new technique using only one single - use rotary file may be precious . in some studies , simulated canals in resin blocks were used . the limitations of resin blocks such as toughness and wear properties , which are different from those of dentin , cause differences in the performance of instruments compared to the natural root canals .
the heat generated during instrumentation with rotary files in resin blocks softens the resin ; the softened resin clings to the blades of the files and may lead to breakage . in order to compare the shaping ability of different canal preparation techniques or morphological changes and differences between instruments ,
however , we did not have similar apical canal diameters , which might have affected the results and only the size of final apical preparation file ( # 25/0.06 ) was acceptable due to minimal risk of apical perforation or apical canal deviation . in this study ,
mesial root canals of the first and second mandibular molars were used to reduce the variation range of the three - dimensional shape of the canals . in most cases , these teeth had the greatest root canal curvature . in our study ,
roots with canal curvatures of 15 to 45 were selected and each of the three groups was divided into four subgroups based on their curvature .
assessment of canal straightening in both buccolingual and mesiodistal directions was performed with the purpose of describing the three - dimensional morphological changes during root canal preparation .
acceptable range for transportation of the apical foramen has not been evaluated ; although an earlier study suggests that straightening of canals in the range of 57.7 is acceptable .
another study reported that apical transportation more than 300 micrometers can have an adverse effect on the seal of root canal fillings .
only one file ( mtwo # 25/0.06 ) broke in our study during preparation with the conventional method in a mesiobuccal canal with a curvature of > 35. file fracture is still a serious concern in the use of ni - ti rotary instruments because most fractures occur without any visible deformation .
because of the low incidence of file fracture , no conclusion has been drawn regarding the higher tendency of one specific rotary system for breakage .
the rate of rotary file fracture has reported to be 3 to 5% . similar to our study ,
paque et al , schirrmeister et al , and aguiar and camara reported no fractures in race files in their studies .
however , merrett et al . reported the fracture of one race file # 25/0.06 with no previous observable distortion .
uroz - torres et al . reported the fracture of two mtwo files # 25/0.06 in their study .
possible reasons for these fractures may be related to the usual way of using this system in single - length ; thus , the entire length of the file is under stress .
this is likely to increase the risk of blocking of the file in the depth of the canal .
in fact , torsional fracture occurs when the file shaft rotates and some parts of the instrument are blocked in the canal .
however , caution must be taken during the use of mtwo files because there is a high incidence of permanent deformation in the file in > 35 curved canals .
therefore , the files should be carefully evaluated every time they are going to be used and files with permanent deformation must be discarded . in our study ,
the time of canal preparation was lower for mtwo files in both canal preparation methods ; but there was no statistically significant difference between mtwo and race in this regard . in both systems
when only one rotary file was used , the preparation time was significantly less than that in the conventional method due to the use of fewer files and less time required for changing the files . in a previous study ,
mtwo system maintained the original canal curvature significantly better than k3 or race systems and it was significantly faster than them regarding canal preparation .
faster canal preparation may be related to the design of mtwo file with a s - shaped cross - section and positive rake angle that can effectively cut dentin . in our study , the double radiography technique was used to evaluate the deviation from the main canal curvature . in the buccolingual and mesiodistal directions , there were no significant differences between the two files or preparation techniques .
schafer et al . compared the performance of race and mtwo files and showed lower frequency of canal transportation with mtwo files .
martin - mico et al . reported that mtwo , race and protaper systems had tendency to straighten the canal with no significant difference with one another .
based on our study , the tendency for canal transportation increased as the curvature exceeded 35. both mtwo and race systems showed some degrees of canal transportation , which were consistent with the results of previous studies by weine et al , lim and webber and kosa et al .
schafer et al , and burklein and schafer reported that the degree of canal straightening was between 1 to 2.69 in canals with curvatures of 25 and 35. differences in the results of various studies may be related to the different preparation methods and use of resin blocks instead of the extracted teeth .
our results showed that there was no difference between the only one rotary file and the conventional methods of canal preparation for preservation of initial canal curvature .
but the time of canal preparation was significantly shorter in only one rotary file technique compared to the conventional method and it may not be necessary to buy special or newly designed expensive rotary files with reciprocating motion .
further studies using other niti rotary file systems are required to evaluate other factors related to cleaning and shaping of the root canals .
according to the results of this experimental study , the technique based on using only one rotary mtwo or race file was faster than the conventional method ; but the tendency to deviate the canal curvature was similar in both techniques . | objectives : cleaning and shaping of the root canal system is an important step in endodontic therapy . new instruments
incorporate new preparation techniques that can improve the efficacy of cleaning and shaping .
the aim of this study was to compare the efficacy of mtwo and race rotary file systems in straightening the canal curvature using only one file or the conventional method.materials and methods : sixty mesial roots of extracted human mandibular molars were prepared by race and mtwo nickel - titanium ( niti ) rotary files using the conventional and only one rotary file methods .
the working length was 18 mm and the curvatures of the root canals were between 1545. by superimposing x - ray images before and after the instrumentation , deviation of the canals was assessed using adobe photoshop cs3 software .
preparation time was recorded .
data were analyzed using three - way anova and tukey s post hoc test.results:there were no significant differences between race and mtwo or between the two root canal preparation methods in root canal deviation in buccolingual and mesiodistal radiographs ( p>0.05 ) .
changes of root canal curvature in > 35 subgroups were significantly more than in other subgroups with smaller canal curvatures .
preparation time was shorter in one file only technique.conclusion:according to the results , the two rotary systems and the two root canal preparation methods had equal efficacy in straightening the canals ; but the preparation time was shorter in one file only group . |
heart failure ( hf ) is known to have consequences for physical function affecting daily life of the patient and his / her partner .
patients with hf may report a decrease in sexual performance , a loss of sexual pleasure or satisfaction , a decrease of sexual interest , and a decrease in the frequency of sex.1 , 2 , 3 , 4 for a lot of hf patients , sexual health is important , with 52% of the men and 38% of the women with hf reporting that sex was important and sexual health was impacting their quality of life.5 , 6
although not every patient with hf suffers from sexual problems and the relationship between the patient and the partner is not always affected,7 several patients and partners have questions and worries .
they may have questions about when to resume sexual activity , about possible dangers and what to do in case symptoms occur . in the american heart association ( aha ) scientific statement on sexual activity and cardiovascular disease,8 sexual activity
is described to be reasonable for patients with compensated and/or mild [ new york heart association ( nyha ) class i or ii ] hf ( class iia ; level of evidence b ) .
sexual activity is not advised for patients with decompensated or advanced hf ( nyha class iii or iv ) until their condition is stabilized and optimally managed ( class iii ; level of evidence c ) .
although the majority of health care professionals feel a certain responsibility to discuss patients ' sexual health , in practice , they seldom address sex with their patients , even during cardiac rehabilitation or in general practice.9 , 10 , 11
from the patient side , patients also experience barriers to discuss their worries or questions around sexual function .
some feel embarrassed to address the topic , others do not want to embarrass the health care providers , or others fear that their health care provider is not experienced enough to understand his / her problems.9 , 11
not discussing sexual concerns with patients might lead to unnecessary worries or sadness of patients .
some patients even might skip their medication because they fear for side effects of their cardiac medications .
some patients start using substances that might help increase their potency or sexual desire , without knowing the possible consequences such as interaction with cardiac medications , vasoactive or sympathomimetic effects , and elevating or reducing systemic blood pressure ( bp).8
this short summary article addresses the prevalence of sexual problems in hf patients and factors that are related to sexual problems and provides some basic information on energy , risk , and treatment .
approximately 60% to 87% of patients with hf report sexual problems.8 these problems include a marked decrease in sexual interest and activity , and one quarter of patients with hf report that they have stopped sexual activity altogether.1 , 12 other studies have found that normal sexual activity was observed only in 31% of patients younger than 70.5 when hf patients ( n = 438 ) were compared with healthy elders ( n = 459 ) , the selfreported amount of sexual dysfunction was similar , at 59% and 56%.2 however , hf patients reported significantly more often ed ( 37% vs. 17%).2 sexual problems include a lack of interest in or fear for having sex , orgasmic difficulties , or erectile dysfunction ( ed ) in men .
the prevalence is estimated to be 50% in 60yearold men.13 , 14 but ed is reported in up to 81% of cardiac patients14 across different cultures and ethnic groups.15 although in most studies more male patients report sexual problems , also women with cardiac disease are known to have more frequent sexual problems compared with women in the general population.16 , 17 women may experience other types of sexual dysfunction than men , including decline in sexual interest or desire , decline in sexual arousal , orgasmic disorder , or painful sexual intercourse.17 in a general population , 27% of women ( age 5059 ) reported lack of interest in sexual activity , and 23% of women were not able to have an orgasm.18 in a hf population , 80% of the female hf patients reported reduced lubrication and 76% reported frequent unsuccessful intercourse.16
some sexual problems already are present prior to the onset of hf , but such problems also can develop during different phases in the hf trajectory.6 in a previous study , 27% of the patients without sexual problems at 1 month after discharge developed sexual problems over time .
in 70% of the patients who had difficulties at 1 month after discharge , the sexual problems remained . at the same time , 30% of the patients , who reported sexual problems at 1 month after discharge , did not report difficulties in sexual activity at followup.6
most patients attribute their sexual problems to their hf symptoms ; they perceived that shortness of breath ( 20% ) , fatigue ( 20% ) , medication use ( 10% ) , and limited circulation ( 11% ) are causing their sexual problems.2
however , the mechanism behind sexual problems is complex .
sexual problems can be related to various demographic , clinical , and treatment factors5 ( figure 1 ) .
factors related to sexual problems in heart failure patients . hf specific factors that are related to sexual problems are hf symptoms such as dyspnea , fatigue , and activity intolerance .
in addition , hf patients often suffer from comorbidity ; up to 35% of hf patients suffers from copd,19 and the overall prevalence of diabetes in hf is 2025%.20 these comorbid conditions are known to be closely related to sexual problems.5 there is an over threefold increased risk of ed in diabetic vs. nondiabetic men,21 and diabetic women are more likely to report problems with lubrication ( or[95%ci ] = 2.37[1.354.16 ] ) and orgasm ( or[95%ci ] = 1.80[1.013.20 ] ) than nondiabetic women.22
hf medications are often perceived to cause problems with sexual performance or libido , although newer generations of drugs appear to have fewer sexual sideeffects . on top of hf medication , other medication
is prescribed for comorbid or underlying conditions , which might be the underlying reason for sexual problems .
in particular , thiazide diuretics may impact erectile function.23 thiazide diuretics can cause endothelial dysfunction and increased vascular oxidative stress , as well as hyperlipidemia , insulin resistance , a new onset of diabetes mellitus , and stimulation of the sympathetic system and the renin angiotensin aldosterone system .
digoxin and mineralocorticoid receptor antagonist are also described to have an effect on sexual performance or libido.23 furthermore , the use of beta blockers can reduce sexual function;8 , 23 however , data on third generation beta blockers currently used for hf treatment are inconsistent.23 hf patients have even reported an improvement of sexual performance with beta blockers , which is likely to be a result of both a reduction of hf severity and the ancillary properties of some of the third generation beta blockers.17 in addition , a nocebo effect , in which a patient 's knowledge that a drug has been associated with ed , is often at least as important a contributing factor to a patient 's ed as any physiological effect , particularly with contemporary blockers.8 in patients who develop sexual problems as a result of medication therapy , it can be helpful to switch to another drug from the same class or find a reasonable alternate strategy.8
sexual problems can be related to other hf treatment such as device therapy or heart transplantation.24 , 25 in a small study of 31 patients , 29% left ventricular assist device ( lvad ) patients and 71% heart transplant patients reported being content with sexual activity ; however , satisfaction with sex life was lower in transplant patients compared with htx patients ( 7.6 3.1 for htx on a visual analogue scale vs. 3.9 4.0 for lvad patients , p = 0.017).24
in addition to hf symptoms , comorbidity , and hf treatment , several other factors such as psychological factors ( depression , anxiety ) can contribute to sexual problems.5 more general factors that are related to sexual function might be relevant to consider in the hf population as well , such as age , lifestyle , or problems in a relationship.5 , 26
most studies on the actual energy consumption of sexual activity is performed in healthy young couples or normotensive men.27 , 28 the energy required for sexual activity depends on its intensity and a person 's physical condition . in the aha statement , the physical effort related to sexual activity in cardiac patients is described to be considered as mild to moderate physical activity and the related energy expenditure in the range of three to five metabolic units ( mets).8 the amount of energy needed for sexual activity is often compared with the energy needed to climb three flights of stairs , general housework , or gardening . at the same time , it was found that patients with hf had a vo2 < 10 ml / kg / min ( i.e. 2.8 met ) and had an impaired sexual function.5
bp and heart rate ( hr ) can increase mildly during foreplay , with increases occurring transiently during sexual arousal and the greatest increases occur during the 10 to 15 s of orgasm , and a quick return to baseline bp and hr.8 , 27 in hf patients , it was also found that they had an increased hr , right ventricular pressure , and diastolic pulmonary pressure during sexual activity ( especially during orgasm).29
there is not a lot of data on the risk for exacerbation in patients ' hf as a result of sexual activity . for patients deemed to be at high risk , sexual activity
should be deferred until their condition is optimally managed and stabilized.8 although some patients approach their physical limit during sexual activity , patients might still be able to have sex by their partner actively ensuring that they practice passive sex or sex helped by drugs or implants .
some patients with hf might need specific information about activities they can undertake , as well as clear information and treatment to help cope with sexual problems.30 because sexual problems might occur during the disease trajectory , sexual concerns need to be discussed more than once during treatment and should become an integral part of hf management and patient education .
first , hf to be optimally managed and patient 's condition should be stabilized before they resume sex.8 furthermore , there are several practical advices that can help patients to optimally enjoy their sex life and intimacy ( table 1 ) .
issues to consider when advising heart failure patients on sexual activity this information needs to be adapted to the personal situation of patients , sexual preferences , and culture.30 , 31 one of the approaches that can be used to guide health professionals in determining needs of patients and in providing information related to sexuality is the socalled plissit model32 ( figure 2 ) .
the plissit model to guide education and counselling.32
the basic idea of this approach is that all patients with hf should be given the opportunity to talk about sexual health in a more general way by informing them that this is an issue that patients might worry about and that they are welcome to discuss this with the health care provider .
some patients might need more information ( limited information ) that explains some basic facts and general information .
from those , some patients might wish more detailed and specific information ( specific suggestion ) , and even a smaller group might be in need of referral to specialist ( intensive therapy ) ( figure 2 ) .
phosphodiesterase type 5 ( pde5 ) inhibitors are generally safe and effective for the treatment of ed in patients with systemic arterial hypertension , stable coronary artery disease , and compensated hf.8 however , pde5 inhibitors should be used with caution in cases of intermediate cardiac risk and should be avoided in patients with high cardiac risk or patients who are concurrently being treated with nitrates.8
sexual problems are common in men and women with hf and should be addressed to avoid possible fears and worries , to proactively prevent problems such as medication nonadherence or using medications or herbs that might endanger the health , and to prevent problems in the relationship between patient and partner . | abstractapproximately 60% to 87% of patients with heart failure ( hf ) report sexual problems , and numbers as low as 31% of hf patients younger than 70 have normal sexual function .
when compared with healthy elders , the amount of perceived sexual dysfunction might be similar ( around 56% ) , but patients with hf are reporting more erectile dysfunction ( ed ) and also perceive that their hf symptoms ( 20% ) or hf medication ( 10% ) is the cause for their problems .
the prevalence of ed is highly prevalent in men with cardiac disease and reported in up to 81% of cardiac patients , compared with 50% in the general older population . in total 2576% of women with hf report sexual problems or concerns.the physical effort related to sexual activity in cardiac patients can be compared to mild to moderate physical activity .
the related energy expenditure of sexual activity falls in the range of three to five metabolic units ( mets ) , which can be compared to the energy needed to climb three flights of stairs , general housework , or gardening.information about sexual activity is often overlooked by health care professionals treating hf patients .
advice and counselling about this subject are needed to decrease worries of patients and partners , avoid skipping medication because of fear for side effects , or prevent inappropriate use of potency enhancing drugs or herbs . |
a 43-year - old woman presented with recurrent paroxysmal involuntary movements of her right upper limb and quadriparesis .
she had four previous relapsing and progressive episodes presenting as right upper limb monoparesis , quadriparesis , and paresthesias during three years .
the previous weakness resolved incompletely with steroid treatment . on admission , her wide right forearm - flinging movements , which were occasionally provoked by left knee flexion ,
lying on the bed , the attacks involved flexion of the right elbow , repeated flexion and extension of the ipsilateral wrist .
her only medication was baclofen , which she had been taking for the last three years .
she had a quadriparesis ( grade 34 , medical research council ) , worse on the right limbs and distal muscles .
rough touch , pain , and temperature sensation were slightly decreased on all extremities , but there were no deep sensory abnormalities .
laboratory studies including routine chemistries , various serological markers related with connective tissue diseases , thyroid function tests , and serum and urine copper levels were all normal .
csf examination showed mild elevation of protein ( 80.5 mg / dl ) and igg index ( 0.70 ) , but oligoclonal bands and nmo - igg antibodies were negative .
median and posterior tibial nerve somatosensory evoked potentials showed no n13 , n20 and p37 .
mri revealed diffuse swelling and heterogeneous enhancement of spinal cord from the cervicomedullary junction to c7 ( figure 1 ) .
six months after the disappearance of involuntary movement , she developed decreased visual acuity with prolonged latencies of p100 components in visual evoked potentials .
the occurrence of ballism - chorea and dystonia implies development of lesions involving the basal ganglia and their afferent or efferent connections , such as thalamus , subthalamic nucleus , basal ganglia , and putamen .
however , the unique feature of this patient is choreic movement without responsible brain lesions .
ballism has never been reported in spinal cord lesions whereas chorea has been seen very rarely.4 it was uncertain whether she exhibited a pure ballism or chorea but presumably it is not important .
while ballism and chorea are distinguishable based upon the type and distribution of movements , they may represent two different symptoms or a spectrum of the same basic disease processes.5 why a patient with a spinal cord lesion develops chorea but not ballism is unknown .
ballism , a coarse proximal movement , could evolve into more distal and lower amplitude chorea .
athetosis , which is considered a slow form of chorea , often evolves further into dystonia.5 why movement disorders occur in only a minority of patients with myelopathy is also unknown .
one mechanism involves the posterolateral cervical cord , the projections from supraspinal centers on the ia inhibitory neurons , and other interneurons concerned with reciprocal inhibition.13 disruption of the somatosensory pathways or motor cortex to the striatum also may produce abnormal movement without sensory loss.1 for our purposes , considering paroxysmal chorea and dystonia together as involuntary movements , we can infer the effect of provocative movements in this patient from the results of previous studies .
paroxysmal chorea could be easily provoked by movement , either passive or volitional , and by tactile stimulation.4 it often crosses to the contralateral trunk or limbs .
the so called trigger zone was found at either the same spinal segment or below the clinically estimated segment .
although this patient had relapsing - remitting weakness , it did not fulfill the conditions of definite nmo when the involuntary movements occurred .
however , she must have had nmo , considering the visual symptoms afterwards . despite the lack of lesions located appropriately for the involuntary movements ,
however , we could not evaluate the effectiveness of such therapy because our patient was treated with steroid and clonazepam .
our case suggests that ballism - chorea could present in a spinal cord lesion without involvement of striatum and deep sensory abnormalities .
thus , acute exacerbation of recurrent myelopathy in nmo may be associated with transient movement disorders . | movement disorders secondary to intrinsic spinal cord disease are rare .
paroxysmal chorea has not yet been reported in the neuromyelitis optica ( nmo ) .
we report a 43-year - old woman with relapsing - remitting cervical myelopathy who developed paroxysmal chorea during clinical exacerbation of nmo .
mri scan of the cervical spine revealed a long segmental enhancing lesion , but brain mri did not show any responsible abnormalities .
acute exacerbation of recurrent myelopathy in nmo may be associated with transient movement disorder . |
painful hips with periprosthetic masses may be caused by high wear , but if this can be ruled out , metal hypersensitivity should be considered .
the first generation hip resurfacing arthroplasty ( hra ) developed in the 1970s used a cobalt - chromium or titanium alloy femoral component bearing against a polyethylene metal - backed acetabular component .
these hras generated large volumes of polyethylene wear debris and were highly susceptible to osteolysis and , because of this , they were largely abandoned .
the reintroduction of metal - on - metal ( m - m ) bearings in thas has encouraged their use in hra as well [ 1 , 28 ] .
one design , the birmingham hip resurfacing ( bhr ) , has been in wide use in the united kingdom , europe , and australia for a decade and received fda approval for use in the united states in 2006 . a dozen or more hra designs are available in europe ; some of these are undergoing clinical trials and are pending fda approval for use in the united states
. targeted to young and active patients , hras are expected to account for an increasing number of hip arthroplasties in the future .
thus , it is important to understand the limitations and complications associated with these devices before their widespread use . femoral neck fractures and aseptic loosening account for the majority of hra failures [ 6 , 12 ] whereas , unlike their historical metal - polyethylene predecessors , osteolysis is not a common cause of failure in modern m - m hras [ 2 , 19 , 34 ] .
this is consistent with their ability to operate with very low wear if factors such as surface smoothness and diametric clearance ( the difference between the diameters of the femoral head and acetabular cup ) are optimized [ 17 , 32 ] .
however , it is becoming evident that socket placement outside of a recommended range ( 30 to 50 abduction and 15 to 25 of anteversion ) [ 9 , 10 , 23 ] can lead to a greater amount of metal release , particularly in small - diameter components [ 10 , 22 , 29 ] .
under such conditions , large quantities of particulate cobalt - chromium debris and associated corrosion products can lead to a variety of adverse reactions , including osteolysis [ 6 , 9 ] , periprosthetic soft tissue masses [ 9 , 13 , 14 ] and extensive necrosis [ 4 , 29 ] .
recent reports from one large - volume resurfacing surgery center described pseudotumors forming in the hips of some female patients with m - m hras which led the authors to speculate that a preoperative sensitization to metal may be a factor .
this complication was estimated to occur in 1% of patients undergoing hra within 5 years , but the incidence could be higher with longer followup and in patients with bilateral implants .
subsequent studies by this group reported higher metal wear in patients with pseudotumors compared to patients without pseudotumors .
pseudotumor - like , enlarged , fluid - filled bursae in hras with malpositioned acetabular components had been previously reported by our group [ 6 , 9 ] and others .
however , pseudotumor - like reactions have also been reported in m - m hras without evidence of high wear or metal hypersensitivity as well as in non - m - m bearing hips [ 16 , 24 ] .
the histology of pseudotumors includes features consistent with metal wear reactions ( eg , macrophages with particles [ 9 , 30 ] ) as well as metal hypersensitivity ( eg , lymphocytic aggregates , granulomas [ 26 , 31 ] ) although both may occur together or extensive necrosis may prevent detailed histological characterization .
the aim of this study was to compare the histopathologic features ( synovial lining integrity , inflammatory cell infiltrates including lymphocytes , macrophages , plasma cells , giant cells , as well as tissue organization , necrosis and metal wear particles ) in pseudotumor - like tissues from m - m hips revised for suspected high wear with pseudotumor - like tissues from m - m hips revised for unexplained pain and suspected metal hypersensitivity .
we selected from archived m - m hip retrievals 32 specimens that were submitted with an unusual soft tissue reaction described by the revising surgeons as an aseptic soft tissue mass , enlarged bursa , or a cyst which could be considered as pseudotumor - like .
twenty - seven of the 32 cases were hip resurfacings ( four articular surface replacements ( asr , depuy international , leeds , uk ) , 20 birmingham hip resurfacings ( bhr , smith and nephew , memphis , tn ) , two conserve plus hip resurfacings ( wright medical technology , memphis , tn ) , and one mcminn resurfacing ( corin , cirencester , uk ) .
the remaining five cases were conventional total hip arthroplasties ( one biomet m2 tha , biomet , warsaw , in ) , one big femoral head tha ( wright medical technology ) , and three metasul bearing total hips ( zimmer , warsaw , in ) .
there were 23 females and nine males with average ages of 54 years ( range , 1868 years ) and 62 years ( range , 4882 years ) , respectively .
as documented by the revising surgeons , the reasons for revision were acetabular malposition ( steep abduction angle , excessive or insufficient anteversion , n = 15 ) , unexplained pain ( i.e. , in the absence of infection , radiographic loosening or malposition , and where metal sensitivity was suspected , n = 9 ) , and aseptic loosening ( n = 5 ) .
we calculated the wear depth of 24 of the 32 explanted components including hras that had not been sectioned and were still intact by digitizing 300 to 400 points on the bearing surface with a coordinate measurement machine ( bmt 504 ; mitotoyo , aurora , il ) .
the resolution of this equipment was approximately 4 m , so wear depths at or below this level were considered
acetabular cup abduction angles were measured by the revising surgeons on ap radiographs in 28 of the 32 cases using standard radiographic techniques ( four cases had poor - quality radiographs deemed unsuitable for this analysis ) .
this involved measuring the angle between a line connecting the ischial spines and another line drawn tangent to the opening of the cup , representing the large diameter of the ellipse .
twenty - two of the tissue samples submitted were large , smooth - walled sacs , and some of the tissues were clearly metal - stained ( fig . 1 ) .
they were weighed , measured , photographed , and their gross appearance was noted . from two to five tissue samples from several sites , especially if there were variations in color or texture of the specimen , were embedded in paraffin blocks for routine sectioning and staining with hematoxylin and eosin .
three of us ( pc , sn , kt ) examined at least six tissue specimens per case semiquantitatively for lymphocytes , macrophages , plasma cells , giant cells , necrosis and metal wear particles using the method of doorn et al . ,
i.e. , where a zero to 3 plus score is given as features of interest become more numerous in a high power 40 microscopic field of view .
1an enlarged fluid - filled bursa excised from the hip of a male patient during revision surgery for acetabular malpositioning 13 months after metal - on - metal hip resurfacing arthroplasty is shown .
there is light gray discoloration and the wear measurement of the explanted component showed an annual femoral wear rate of 12.8 m .
an enlarged fluid - filled bursa excised from the hip of a male patient during revision surgery for acetabular malpositioning 13 months after metal - on - metal hip resurfacing arthroplasty is shown .
there is light gray discoloration and the wear measurement of the explanted component showed an annual femoral wear rate of 12.8 m .
each case was also given an alval ( aseptic lymphocytic vasculitis - associated lesion ) score of 1 to 10 ( table 1 ) ; alval has been applied to a unique , lymphocyte - dominated reaction in m - m periprosthetic tissues . to check the reproducibility of scoring
, two of us ( pc , kt ) performed the scoring in a blinded fashion on two separate occasions .
the kappa coefficient for interobserver variability showed a correlation between the two observers of 0.71 and between the two separate measurements of each observer of 0.68 . using only the histologic features and the alval scores , each observer predicted whether each case failed in association with high - wear , suspected metal hypersensitivity , or some other cause .
the kappa coefficient for interobserver variability was used to determine the validity of these predictions against the actual wear measurement from the retrievals .
the kappa coefficient for the agreement between the first observer s prediction and the retrieval findings was between 0.69 and 0.81 and the second observer s between 0.43 and 0.73 .
the kappa coefficient for the prediction regarding the association of a case with high wear was higher for the observer with many years of experience with histologic analysis of retrieval tissues ( pc ) compared with the second , less experienced observer.table 1histologic scoring criteria for alval scorepointssynovial lining0intact synovial lining1focal loss of synovial surface , fibrin attachment may occur2moderate to marked loss of synovial surface , fibrin attachment3complete loss of synovium , abundant attached fibrin and /or necrosis of lining tissuepointsinflammatory infiltrate0minimal inflammatory cell infiltrates1predominantly macrophages , occasional lymphocytes may occur2mix of macrophages and lymphocytes , either diffuse and/or small ( < 50% of hpf ) perivascular aggregates3mix of macrophages and lymphocytes , large ( > 50% hpf ) perivascular aggregates may occur4predominantly lymphocytes , mostly in multiple , large ( > 50% hpf ) perivascular aggregates , follicles may be presentpointstissue organization0normal tissue arrangement1mostly normal tissue arrangement , small areas of synovial hyperplasia , focal necrosis may occur2marked loss of normal arrangement , appearance of distinct cellular and acellular zones , thick fibrous layers may occur3perivascular lymphocytic aggregates mostly located distally , thick acellular areas may occursumlow = 04moderate = 58high = 910alval = aseptic lymphocytic vasculitis - associated lesion ; hpf = high - power field .
histologic scoring criteria for alval score alval = aseptic lymphocytic vasculitis - associated lesion ; hpf = high - power field .
the independent variable considered in this study was whether the patients were revised with suspected high wear or with suspected metal hypersensitivity .
the dependent variables were the histologic features related to the intensity of the inflammatory reaction : alval score , lymphocytes , macrophages , plasma cells , giant cells , necrosis , and metal particles .
univariate analysis was used to determine the mean , median , sd , and distribution for each variable as necessary .
the mann - whitney test was used to compare the femoral wear and wear rate of patients with suspected high wear with those suspected to have metal hypersensitivity .
the mann - whitney test was determined to be appropriate because the dependent variables were not normally distributed .
when comparing the histologic features in the tissues of patients revised for suspected high wear with those revised for pain and suspected metal sensitivity , the higher wear cases had a lower ( p < 0.001 ) alval score , fewer lymphocytes but more macrophages and metal particles ( table 2 ) .
histologically , there was considerable variability in the amount and distribution of metal debris , the number and type and arrangement of inflammatory cells , and the degree of necrosis .
very few tissues demonstrated an intact synovial lining and there was often a layer of adherent fibrin , organized fibrin , or necrosis on the joint cavity side of the tissue .
macrophages and lymphocytes were present in all cases , but those with extensive infiltrates of macrophages tended to have smaller lymphocyte aggregates ( fig . 2 ) .
this was in contrast to the appearance of very large , dense lymphocyte aggregates , often arranged distal to the surface ( fig .
3 ) , which were more often seen in association with small to moderate amounts of macrophages .
most of the tissues had focal to moderate necrosis and one case had necrosis that dominated most of the tissue sections ( fig .
4).table 2results of the semiquantitative evaluation of histologic features for cases revised for suspected high wear and for unexplained pain / suspected metal sensitivityvariable ( p value)suspected wear relatedsuspected metal sensitivityaveragesdaveragesdalval score ( p < 0.001)3.62.58.51.4macrophages ( p < 0.001)2.70.51.70.5lymphocytes ( p = 0.001)1.40.62.50.7metal particles ( p = 0.008)1.50.70.70.5necrosis ( p = 0.29)1.00.71.40.7tidemark ( p = 0.03)0.20.40.70.5layering organization ( p = 0.024)0.280.50.80.4alval = aseptic lymphocytic vasculitis - associated lesion.fig .
2light micrograph showing typical histologic features of high wear cases , including organized fibrin ( f ) , a diffuse , extensive infiltration of slate blue / gray macrophages , and a small aggregate of lymphocytes ( arrows ) ( stain , hematoxylin and eosin , original magnification 40 ) .
this received an alval score of 5 ( 2 for synovial lining , 2 for inflammatory infiltrate , and 1 for tissue organization).fig .
3light micrograph showing typical histologic features of cases revised for suspected metal sensitivity , including a thick , mostly acellular tidemark area lined by fibrin ( f ) and thick , dense aggregates of lymphocytes at the rear of the tissue ( arrows ) ( stain , hematoxylin and eosin , original magnification 40 ) .
this received an alval score of 10 ( 3 for synovial lining , 4 for inflammatory infiltrate , and 3 for tissue organization).fig .
the tissues were from a male patient with a tha that was revised for pain after 2 years .
extensive necrosis was found at revision , but the component wear was within normal range ( stain , hematoxylin and eosin , original magnification 40 ) .
results of the semiquantitative evaluation of histologic features for cases revised for suspected high wear and for unexplained pain / suspected metal sensitivity alval = aseptic lymphocytic vasculitis - associated lesion .
light micrograph showing typical histologic features of high wear cases , including organized fibrin ( f ) , a diffuse , extensive infiltration of slate blue / gray macrophages , and a small aggregate of lymphocytes ( arrows ) ( stain , hematoxylin and eosin , original magnification 40 ) .
this received an alval score of 5 ( 2 for synovial lining , 2 for inflammatory infiltrate , and 1 for tissue organization ) .
light micrograph showing typical histologic features of cases revised for suspected metal sensitivity , including a thick , mostly acellular tidemark area lined by fibrin ( f ) and thick , dense aggregates of lymphocytes at the rear of the tissue ( arrows ) ( stain , hematoxylin and eosin , original magnification 40 ) .
this received an alval score of 10 ( 3 for synovial lining , 4 for inflammatory infiltrate , and 3 for tissue organization ) .
the tissues were from a male patient with a tha that was revised for pain after 2 years .
extensive necrosis was found at revision , but the component wear was within normal range ( stain , hematoxylin and eosin , original magnification 40 ) .
the average femoral wear rate for the components from patients revised for suspected high wear was 19.9 microns per year ( standard deviation 18.1 , range 3.176.2 microns per year ) and was higher ( p = 0.003 ) than that for components from patients revised for pain and suspected metal sensitivity ( average 3.7 , sd 2.2 , range 1.56.7 microns per year ) .
pseudotumors , masses and enlarged bursae have been reported in hips with m - m bearings associated with pain and swelling .
the cause of these reactions is unclear but several authors have suggested it is a reaction to high wear [ 20 , 30 ] or to metal hypersensitivity [ 15 , 26 , 30 ] .
this study was conducted to compare the histology of pseudotumor - like tissues from hips suspected to have high wear with those from patients with pain and suspected metal sensitivity and our results support the formation of pseudotumors from both wear and hypersensitivity reactions .
first , we did not provide any specific morphologic criteria for the tissue specimens we studied because we were confident experienced orthopaedic surgeons would recognize an unusual , adverse reaction .
for this reason , we have used the term pseudotumor - like and we are confident that the submitted specimens , even though labeled as masses , cysts or enlarged bursae , were comparable to the cystic or solid pseudotumors described by pandit et al . .
similar histological features were noted in pseudotumor and pseudocapsule tissues in our analysis ( results not shown ) , a finding also reported by mahendra et al . .
thus , even if some of our samples were actually misclassified thickened capsules , the results of our analysis remain valid .
second , we can not prove a presumptive diagnosis of metal sensitivity . unlike component wear or serum ion levels , which can be measured with a known degree of accuracy , there are currently no definitive blood tests or histopathologic criteria to diagnose metal hypersensitivity .
however , we devised a working postulate to diagnose hypersensitivity : early onset of pain , the absence of other reasons for pain ( such as loosening , impingement , infection , or high wear ) , and the resolution of symptoms after the removal of the cobalt - chromium components .
other clinical reports have noted similar features in patients suspected to have a metal hypersensitivity reaction [ 7 , 37 ] .
we recognize that it is possible for metal hypersensitivity to coexist with any of these other causes of pain and we also recognize that there is variability in the clinical presentation that will confound this working definition .
third , we observed a range of intra- and interobserver statistics for light microscopic tissue features .
this is to be expected given that we were using a semiquantitative scoring system and that the importance of histologic features is subject to individual interpretation .
the histologic rating is meant to be used in conjunction with the case history , radiographic findings , and retrieval findings and when so used , the interpretation of histologic features is more likely to predict their cause .
our semiquantitative analyses demonstrated substantial differences in the histological features of pseudotumor - like tissues from patients with high wear compared with those tissues from patients suspected to have metal hypersensitivity .
the tissues from both groups contained macrophages and lymphocytes in variable amounts and distributions but applying the alval rating allowed clear patterns to emerge .
in particular , there was generally less disruption of the synovial surface , and greater preservation of the normal tissue architecture in the high wear group .
in contrast , the most extensive damage to the tissues and the densest lymphocyte aggregates occurred in patients suspected to have a metal hypersensitivity reaction and typically this occurred in the absence of high wear .
the variability we noted is consistent with other histologic reports ; pandit et al . noted scattered , focally heavy macrophage and lymphocytic infiltrates , including lymphoid aggregates , in formal biopsy samples of pseudotumors from 10 hip resurfacings revised for pain and/or pseudotumor formation .
similar findings were reported in two female patients with masses causing femoral neuropathy around unilateral hip resurfacings .
wear measurements were not provided for the implants associated with these pseudotumors and it is not clear if the patients had risk factors for high wear such as small component size and implant malposition .
one recent study by langton et al . analyzed tissues from 17 patients with m - m hips following revision for an adverse response to metal debris including pseudotumor formation .
they reported substantially higher component wear and blood ion levels in these patients compared with those revised for other reasons .
their histological examination noted alval features such as synovial ulceration and perivascular lymphocytes which ranged from absent to moderate .
the lack of lymphocytes in some of their cases and the absence of high levels of lymphocytic infiltrates in this group of patients with high wear is consistent with our observations .
we suggest that using the alval score will promote more standardized reporting of the histological features of tissues removed from m - m hips . | backgroundpseudotumor - like periprosthetic tissue reactions around metal - on - metal ( m - m ) hip replacements can cause pain and lead to revision surgery .
the cause of these reactions is not well understood but could be due to excessive wear , or metal hypersensitivity or an as - yet unknown cause .
the tissue features may help distinguish reactions to high wear from those with suspected metal hypersensitivity.questions/purposeswe therefore examined the synovial lining integrity , inflammatory cell infiltrates , tissue organization , necrosis and metal wear particles of pseudotumor - like tissues from m - m hips revised for suspected high wear related and suspected metal hypersensitivity causes.methodstissue samples from 32 revised hip replacements with pseudotumor - like reactions were studied .
a 10-point histological score was used to rank the degree of aseptic lymphocytic vasculitis - associated lesions ( alval ) by examination of synovial lining integrity , inflammatory cell infiltrates , and tissue organization .
lymphocytes , macrophages , plasma cells , giant cells , necrosis and metal wear particles were semiquantitatively rated .
implant wear was measured with a coordinate measuring machine .
the cases were divided into those suspected of having high wear and those suspected of having metal hypersensitivity based on clinical , radiographic and retrieval findings .
the mann - whitney test was used to compare the histological features in these two groups.resultsthe tissues from patients revised for suspected high wear had a lower alval score , fewer lymphocytes , but more macrophages and metal particles than those tissues from hips revised for pain and suspected metal hypersensitivity .
the highest alval scores occurred in patients who were revised for pain and suspected metal hypersensitivity .
component wear was lower in that group.conclusionspseudotumor-like reactions can be caused by high wear , but may also occur around implants with low wear , likely because of a metal hypersensitivity reaction .
histologic features including synovial integrity , inflammatory cell infiltrates , tissue organization , and metal particles may help differentiate these causes.clinical relevancepainful hips with periprosthetic masses may be caused by high wear , but if this can be ruled out , metal hypersensitivity should be considered . |
iron overload disturbs many physiological functions and provides iron deposition in different organs including kidney .
kidney iron deposition ( kid ) has been observed in patients with kidney diseases , sickle cell disease , and aplastic anemia .
kid has been also developed in experimental animals for different research purposes.[58 ] under some conditions , the kid may not disturb the renal function , but iron overload potentially could enhance the formation of hydroxyl radical , which promote organ damaging including tubular damage . it has been reported that nitric oxide protected the rats kidney from iron - induced nephrotoxicity , and sesame oil is nephroprotectant against iron - induced injury in mice .
clinically , thalassemic patients usually struggle with iron overload and deferoxamine ( df ) as an iron chelator is the most common drug to prevent increasing of the iron level in plasma or specific organs tissues in patients[1315 ] and experimental animals .
silymarin ( sm ) , as an antioxidant agent , is derived from the herb milk thistle ( silybum marianum ) and potentially has the ability to chelate iron.[1821 ] the antioxidant effect of sm in the renal ischemia / reperfusion injury and renal toxicity has been investigated before .
the patients are usually treated by df , but due to the administration procedure , it is a subject of complaints from the patients and their relatives . to simulate this process , we designed an iron overload model , which consisted of two phases . in phase 1 ,
iron overload was induced by administration of iron dextran and in phase 2 , iron overload was continued and the protective role of sm , df , or combination of both against kid was studied .
thirty - six adult male wistar rats ( animal centre , ahvaz university of medical sciences , ahvaz , iran ) with the mean weight of 201 4 g were used .
the experimental procedures were in advance approved by the isfahan university of medical sciences ethics committee .
the animals were randomly divided into six experimental groups as follows : group 1 ( n = 6 , negative control group ) : the animal received vehicle ( 0.5 ml of saline ) every other day during the 4 weeks of study .
group 2 ( n = 6 , positive control group ) : the animals received iron dextran ( vifor inc . , switzerland )
groups 3 to 6 : the animals in these groups had regimen the same as group 2 , except that they received sm ( 200 mg / kg , orally by feeding tube , group 3 ) , df ( 50 mg / kg , i.p . , group 4 ) , sm ( 400 mg / kg , orally , group 5 ) , and combination of sm and df ( 200 mg / kg , orally and 50 mg / kg , i.p .
blood samples were obtained 2 weeks after overloading ( before treatment ) and 4 weeks after overloading ( end of the experiment ; after treatment ) .
the levels of serum creatinine ( cr ) , blood urea nitrogen ( bun ) , and iron were determined using quantitative diagnostic kits ( pars azmoon , iran ) .
the serum level of nitrite [ stable nitric oxide ( no ) metabolite ] was measured using a colorimetric elisa kit ( promega corporation , usa ) that involves the griess reaction .
the serum level of ferritin was measured using enzyme immunoassay elisa kit for rat ( immunology consultants laboratory inc .
the removed kidneys were fixed in 10% neutral formalin solution and embedded in paraffin for staining to examine iron deposition in the kidney .
the tissue was also subjected to hematoxylin and eosin staining to examine the tissue damage .
the kid was evaluated by two independent pathologists who were totally blind to the study . on the basis of the intensity of kid and tissue damage ,
the kidney tissue damage score ( ktds ) was graded from 1 to 5 , while score 0 was assigned to normal kidney tissue without damage and iron deposition .
the after treatment serum levels of bun , cr , iron , nitrite , and ferritin were compared between the groups using univariate analysis of variance ( anova ) and before treatment parameters as covariate .
thirty - six adult male wistar rats ( animal centre , ahvaz university of medical sciences , ahvaz , iran ) with the mean weight of 201 4 g were used .
the experimental procedures were in advance approved by the isfahan university of medical sciences ethics committee .
the animals were randomly divided into six experimental groups as follows : group 1 ( n = 6 , negative control group ) : the animal received vehicle ( 0.5 ml of saline ) every other day during the 4 weeks of study .
group 2 ( n = 6 , positive control group ) : the animals received iron dextran ( vifor inc . , switzerland )
groups 3 to 6 : the animals in these groups had regimen the same as group 2 , except that they received sm ( 200 mg / kg , orally by feeding tube , group 3 ) , df ( 50 mg / kg , i.p . , group 4 ) , sm ( 400 mg / kg , orally , group 5 ) , and combination of sm and df ( 200 mg / kg , orally and 50 mg / kg , i.p . , group 6 ) instead of vehicle .
blood samples were obtained 2 weeks after overloading ( before treatment ) and 4 weeks after overloading ( end of the experiment ; after treatment ) .
the levels of serum creatinine ( cr ) , blood urea nitrogen ( bun ) , and iron were determined using quantitative diagnostic kits ( pars azmoon , iran ) .
the serum level of nitrite [ stable nitric oxide ( no ) metabolite ] was measured using a colorimetric elisa kit ( promega corporation , usa ) that involves the griess reaction .
the serum level of ferritin was measured using enzyme immunoassay elisa kit for rat ( immunology consultants laboratory inc .
the removed kidneys were fixed in 10% neutral formalin solution and embedded in paraffin for staining to examine iron deposition in the kidney .
the tissue was also subjected to hematoxylin and eosin staining to examine the tissue damage .
the kid was evaluated by two independent pathologists who were totally blind to the study . on the basis of the intensity of kid and tissue damage ,
the kidney tissue damage score ( ktds ) was graded from 1 to 5 , while score 0 was assigned to normal kidney tissue without damage and iron deposition .
the after treatment serum levels of bun , cr , iron , nitrite , and ferritin were compared between the groups using univariate analysis of variance ( anova ) and before treatment parameters as covariate .
the serum levels of iron and ferritin were increased in all iron dextran receiver groups and the levels were significantly different from the values obtained for the negative control group ( p < 0.05 ) . however , no statistically significant differences in serum levels of bun , cr , and nitrite were observed between the groups [ table 1 ] .
these data supported that iron overload have no effect on bun and cr as kidney function biomarkers .
serum levels of bun , cr , iron , ferritin , and nitrite in six groups of experiment 2 weeks after iron overload ( before treatment ) .
iron dextran receiver groups were significantly different from the negative control group in this respect ( p < 0.05 ) the data indicated that sm200 , df + sm200 , and sm400 had no significant effects on serum levels of bun , cr , iron , ferritin , and nitrite [ figure 1 ] .
with regard to the serum levels of bun and cr , it seems that there was no renal dysfunction , but based on histological investigations there was iron deposition in the kidney tissue of groups 2 to 6 [ figure 2 ] .
the serum levels of bun , cr , iron , ferritin , and nitrite after 2 weeks of treatment with sm ( 200 mg / kg ; group 3 ) , df ( 50 mg / kg ; group 4 ) , sm ( 400 mg / kg ; group 5 ) , and combination of sm and df ( 200 and 50 mg / kg , respectively ; group 6 ) compared with the positive control group ( group 2 ) . no significant differences were detected among treatment and control groups .
the group 1 is not shown in the figure because the treated groups ( groups 3 to 6 ) were compared with group 2 ( positive control group ) only the kidney weight per 100 g of body weight and kidney pathology score ( ktds ) , which corresponds to kid after 2 weeks of treatment with sm ( 200 mg / kg ; group 3 ) , df ( 50 mg / kg ; group 4 ) , sm ( 400 mg / kg ; group 5 ) , and combination of sm and df ( 200 and 50 mg / kg , respectively ; group 6 ) compared with the positive control group ( group 2 )
. the star ( ) indicates significant difference from the positive control group ( group 2 ) ( p < 0.05 ) .
the group 1 is not shown in the figure because the treated groups ( groups 3 to 6 ) were compared with group 2 ( positive control group ) only the kidneys from all animals in the negative control group ( group 1 ) were free of iron deposition with 0 score , and significant differences were observed between this negative control group and others groups ( p < 0.05 ) .
the lower intensity of kid and damage in group 3 , which was treated by sm ( 200 mg / kg ) revealed the lower ktds , and group 3 was significantly different from the positive control group in this regard ( p < 0.05 ) .
however , no statistical difference in ktds was detected between the positive control group on the one hand and groups 4 - 6 on the other hand [ figure 3 ] .
the sample images of kidney tissue stained with persian blue to examine iron deposition in the kidney of six experimental groups ; a= vehicle , b= fe , c= sm200+fe : d : df50+fe , e= sm400+fe , f = sm200+df50+fe . a received vehicle during the study , and the groups 2 to 6 received iron dextran for a period of 4 weeks but during weeks 3 and 4 , they were treated by vehicle ( b ; positive control group ) , sm ( 200 mg / kg ; c ) , df ( 50 mg / kg ; d ) , sm ( 400 mg / kg ; e ) , and combination of sm and df ( 200 and 50 mg / kg , respectively ; f ) , respectively .
the serum levels of iron and ferritin were increased in all iron dextran receiver groups and the levels were significantly different from the values obtained for the negative control group ( p < 0.05 ) . however , no statistically significant differences in serum levels of bun , cr , and nitrite were observed between the groups [ table 1 ] .
these data supported that iron overload have no effect on bun and cr as kidney function biomarkers .
serum levels of bun , cr , iron , ferritin , and nitrite in six groups of experiment 2 weeks after iron overload ( before treatment ) .
iron dextran receiver groups were significantly different from the negative control group in this respect ( p < 0.05 )
the serum levels of iron and ferritin were increased in all iron dextran receiver groups and the levels were significantly different from the values obtained for the negative control group ( p < 0.05 ) . however , no statistically significant differences in serum levels of bun , cr , and nitrite were observed between the groups [ table 1 ] .
these data supported that iron overload have no effect on bun and cr as kidney function biomarkers .
serum levels of bun , cr , iron , ferritin , and nitrite in six groups of experiment 2 weeks after iron overload ( before treatment ) .
iron dextran receiver groups were significantly different from the negative control group in this respect ( p < 0.05 )
the data indicated that sm200 , df + sm200 , and sm400 had no significant effects on serum levels of bun , cr , iron , ferritin , and nitrite [ figure 1 ] . with regard to the serum levels of bun and cr
, it seems that there was no renal dysfunction , but based on histological investigations there was iron deposition in the kidney tissue of groups 2 to 6 [ figure 2 ] .
the serum levels of bun , cr , iron , ferritin , and nitrite after 2 weeks of treatment with sm ( 200 mg / kg ; group 3 ) , df ( 50 mg / kg ; group 4 ) , sm ( 400 mg / kg ; group 5 ) , and combination of sm and df ( 200 and 50 mg / kg , respectively ; group 6 ) compared with the positive control group ( group 2 ) .
the group 1 is not shown in the figure because the treated groups ( groups 3 to 6 ) were compared with group 2 ( positive control group ) only the kidney weight per 100 g of body weight and kidney pathology score ( ktds ) , which corresponds to kid after 2 weeks of treatment with sm ( 200 mg / kg ; group 3 ) , df ( 50 mg / kg ; group 4 ) , sm ( 400 mg / kg ; group 5 ) , and combination of sm and df ( 200 and 50 mg / kg , respectively ; group 6 ) compared with the positive control group ( group 2 ) .
the star ( ) indicates significant difference from the positive control group ( group 2 ) ( p < 0.05 ) .
the group 1 is not shown in the figure because the treated groups ( groups 3 to 6 ) were compared with group 2 ( positive control group ) only the kidneys from all animals in the negative control group ( group 1 ) were free of iron deposition with 0 score , and significant differences were observed between this negative control group and others groups ( p < 0.05 ) .
the lower intensity of kid and damage in group 3 , which was treated by sm ( 200 mg / kg ) revealed the lower ktds , and group 3 was significantly different from the positive control group in this regard ( p < 0.05 ) .
however , no statistical difference in ktds was detected between the positive control group on the one hand and groups 4 - 6 on the other hand [ figure 3 ] .
the sample images of kidney tissue stained with persian blue to examine iron deposition in the kidney of six experimental groups ; a= vehicle , b= fe , c= sm200+fe : d : df50+fe , e= sm400+fe , f = sm200+df50+fe .
a received vehicle during the study , and the groups 2 to 6 received iron dextran for a period of 4 weeks but during weeks 3 and 4 , they were treated by vehicle ( b ; positive control group ) , sm ( 200 mg / kg ; c ) , df ( 50 mg / kg ; d ) , sm ( 400 mg / kg ; e ) , and combination of sm and df ( 200 and 50 mg / kg , respectively ; f ) , respectively .
the data indicated that sm200 , df + sm200 , and sm400 had no significant effects on serum levels of bun , cr , iron , ferritin , and nitrite [ figure 1 ] . with regard to the serum levels of bun and cr
, it seems that there was no renal dysfunction , but based on histological investigations there was iron deposition in the kidney tissue of groups 2 to 6 [ figure 2 ] .
the serum levels of bun , cr , iron , ferritin , and nitrite after 2 weeks of treatment with sm ( 200 mg / kg ; group 3 ) , df ( 50 mg / kg ; group 4 ) , sm ( 400 mg / kg ; group 5 ) , and combination of sm and df ( 200 and 50 mg / kg , respectively ; group 6 ) compared with the positive control group ( group 2 ) .
the group 1 is not shown in the figure because the treated groups ( groups 3 to 6 ) were compared with group 2 ( positive control group ) only the kidney weight per 100 g of body weight and kidney pathology score ( ktds ) , which corresponds to kid after 2 weeks of treatment with sm ( 200 mg / kg ; group 3 ) , df ( 50 mg / kg ; group 4 ) , sm ( 400 mg / kg ; group 5 ) , and combination of sm and df ( 200 and 50 mg / kg , respectively ; group 6 ) compared with the positive control group ( group 2 ) .
the star ( ) indicates significant difference from the positive control group ( group 2 ) ( p < 0.05 ) .
the group 1 is not shown in the figure because the treated groups ( groups 3 to 6 ) were compared with group 2 ( positive control group ) only the kidneys from all animals in the negative control group ( group 1 ) were free of iron deposition with 0 score , and significant differences were observed between this negative control group and others groups ( p < 0.05 ) .
the lower intensity of kid and damage in group 3 , which was treated by sm ( 200 mg / kg ) revealed the lower ktds , and group 3 was significantly different from the positive control group in this regard ( p < 0.05 ) .
however , no statistical difference in ktds was detected between the positive control group on the one hand and groups 4 - 6 on the other hand [ figure 3 ] .
the sample images of kidney tissue stained with persian blue to examine iron deposition in the kidney of six experimental groups ; a= vehicle , b= fe , c= sm200+fe : d : df50+fe , e= sm400+fe , f = sm200+df50+fe .
a received vehicle during the study , and the groups 2 to 6 received iron dextran for a period of 4 weeks but during weeks 3 and 4 , they were treated by vehicle ( b ; positive control group ) , sm ( 200 mg / kg ; c ) , df ( 50 mg / kg ; d ) , sm ( 400 mg / kg ; e ) , and combination of sm and df ( 200 and 50 mg / kg , respectively ; f ) , respectively .
the main objective of this study was to investigate the protective role of sm and df against deposition of iron in an iron overload rat model .
our finding indicated that sm at the dose of 200 mg / kg reduced the kid .
iron is required for metabolic processes in the kidney , but iron overload enhances hydroxyl radical formation , resulting tubular damage and may disturb physiological function of the cellular environment of the kidney . clinically , thalassemic patients are the best example of iron overload , and usually they use df as an iron chelator . however , renal dysfunction is one of the side effects of iron overload in these patients .
sm as an antioxidant agent is also an iron chelator.[1820 ] in the study , iron overload was induced by iron dextran , which is a safe agent , but due to formation of oxidative stress , it may damage the organs .
serum levels of both iron and ferritin increased significantly after 2 weeks of iron overload [ table 1 ] .
however , based on the serum levels of bun and cr , no renal dysfunction occurred .
this may be attributed to the lower potency of iron dextran in enhancing the oxidative stress and location of iron deposition in the kidney that does not disturb the regular renal function .
there is also little evidence available that accumulated iron via polynuclear iron complexes participate in free radical reactions to disturb the functions of various organ .
however , a dose of 600 mg / kg iron dextran for 24 h increased the serum level of urea and cr .
high iron stores may disturb endothelial function in patients , and iron chelators may improve the endothelial function . in this study , we measured no metabolite ( nitrite ) as a marker of endothelial function ; however , no change in nitrite level was observed .
it is reported that chronic iron overload enhances inducible no synthase expression in some organ ; however ,
our model was an acute iron overloading model , and it seems for such acute model iron - induced endothelial dysfunction may not achieve . the data obtained in our study indicated that sm , df
, or combination of both did not demonstrate an iron chelation effect in reducing the iron serum level .
the reduction of the serum level of iron by sm in a different iron overload model was reported before . in this model ,
sm ( 200 mg / kg ) was administrated accompanied with iron dextran for 2 weeks , but the usage dose of iron dextran was half of our usage dose .
sm also used as iron chelator , protective agent against gentamicin - induced nephrotoxicity , or treatment of liver diseases .
it seems that our model and the high usage dose of iron dextran ( 200 mg / kg ) was suitable to obtain a kid animal model , but possibly the dose of iron dextran was high enough to saturate the iron and ferritin capacities , so sm , df , or combination of both were not enough potentiate to reduce the saturated level of iron and ferritin in animal serum . on the contrary , the histopathological data revealed that sm ( 200 mg / kg ) alone reduced the kid significantly .
this finding supports the antioxidant effects of sm similar to other experimental models.[2225 ] df also has antioxidant effect in rat kidneys , and it reduced the kid in our study nonsignificantly .
future studies are needed to compare the antioxidant potency of sm and df . in our iron overload model ,
coadministration of sm and df did not protect the kidney tissue from iron deposition , possibly due to pharmacokinetic agents interactions , and finally our findings demonstrated unexpected results that kid obtained from the treated animals with 200 mg / kg of sm was less than kid obtained from treated animals with 400 mg / kg of sm .
the effect of some flavonoids antioxidant such as galangin in fructose - fed - induced renal damage was dose - dependent , and the lower dose provide a better protection effect for renal damage , so possibly the sm as a flavonoids at the dose of 200 mg / kg was more effective than the higher dose .
our finding demonstrated that sm with the dose of 200 mg / kg potentially was more protective than df against kid .
this observation need to be verified in serial experimental studies with different intensity of iron overload . | background : kidney iron deposition ( kid ) is caused by iron overload that is observed in kidney diseases and anemia .
the protective effects of deferoxamine ( df ) and silymarin ( sm ) were studied against iron overload - induced kid in rat model.methods:rats received iron dextran ( 200 mg / kg ) for a period of 4 weeks every other day , but at the beginning of week 3 , they also were subjected to a 2-week ( every other day ) treatment with vehicle ( group 2 , positive control ) , sm ( 200 mg / kg ; group 3 ) , df ( 50 mg / kg ; group 4 ) , sm ( 400 mg / kg ; group 5 ) , and combination of sm and df ( 200 and 50 mg / kg , respectively ; group 6 ) .
group 1 , as the negative control , received saline alone during the study .
the levels of serum creatinine ( cr ) , blood urea nitrogen ( bun ) , iron , ferritin , and nitrite were determined , and the kidney was removed for histopathological investigations.results:before treatment , the serum levels of iron and ferritin in all iron dextran receiver groups were significantly higher than those of the negative control group ( p < 0.05 )
. however , the serum levels of bun , cr , and nitrite were not different between the groups .
no statistical differences were detected in kidney weight and the serum levels of bun , cr , iron , ferritin , and nitrite after 2 weeks of treatment with sm , df , or combination of both .
the sm and df treatments reduced the intensity of the kid , but only in the sm ( 200 mg / kg ) group , a significant reduction in kid was observed ( p < 0.05).conclusion : it seems that sm is a nephroprotectant agent against kid in acute iron overload animal models . |
h and 13c nmr spectra were recorded at ambient temperature in cdcl3 and dmso on a bruker amc instrument operating at 300.12 and 75.47 mhz , respectively or a bruker drx instrument operating at 500 , respectively 125 mhz .
si gel for chromatography was silica gel 60 ( 0.20.5 mm merck ) for analytical work and for preparative work tlc silica gel 60 gf 254 merck .
haliclona cymaeformis ( esper , 1794 ) order haplosclerida , family chalinidae , was collected by scuba diving in the gulf of thailand near koh chang ( 2 meter depth ) , trad province in march 2004 .
haliclona baeri ( wilson , 1952 ) was also collected by scuba diving in the in the coastal area of chonburi province ( 3 meter depth ) .
sumaitt putchakarn and the vouchers of the specimens , registered as bims - i 953 and bims - i 1766 , respectively were deposited at bangsaen institute of marine science ( bims ) , burapha university , bangsaen , chonburi , thailand .
the materials were frozen immediately at 20c prior to extraction . the sample ( 1.3 kg net weight )
was thawed , homogenized with etoh ( 3 l ) , allowed to stand for 24 h in a dark chamber and filtered .
the residue on the filter paper was again extracted with etoh ( 2 3l ) , the aqueous alcoholic extracts were combined , evaporated at reduced pressure to ca .
the etoac extracts were combined and concentrated at reduced pressure to give the crude etoac extract .
this ( 10 g ) was chromatographed over a si gel column ( 120 g ) and eluted with petrol - chcl3 and chcl3-me2o , 350 ml fractions being eluted as follows .
frs 162 ( petrol - chcl3 , 1:1 ) , 6378 ( petrol - chcl3 , 3:7 ) , 7995 ( petrol - chcl3 , 1:9 ) , 96182 ( chcl3-me2o , 4:1 ) , 183206 ( chcl3- me2o , 3:2 ) .
frs 101114 were combined ( 107.8 mg ) and purified by tlc ( si gel , chcl3-me2o
frs 125198 were combined ( 185.6 mg ) and purified by tlc ( si gel , chcl3-meoh - hco2h , 93:7:1 ) to give 13.1 mg 3,4-dihydroxybenzoic acid ( 2 ) and 8.0 mg of tetillapyrone ( 5and 6 ) .
the sample of haliclona cymaeformis ( 7.6 kg net weight ) was thawed , homogenized with etoh ( 10l ) , allowed to stand for 24 h in the dark chamber and filtered .
the residue on the filter paper was again extracted with etoh ( 2 10l ) , the aqueous alcoholic extracts were combined , evaporated at reduced pressure to give ca .
the etoac extracts were combined and concentrated at reduced pressure to give a crude etoac extract ( 21 g ) .
this extract was applied to flash chromatography column of si gel ( 150 g ) and eluted with chcl3 ( frs .
15 ; 5 200 ml ) , chcl3-meoh , , 49:1 ( frs 615 ; 10 200 ml ) , chcl3-meoh , , 19:1 ( frs1620 ; 5 200 ml ) , chcl3-meoh , , 9:1 ( frs 2122 ; 2 500 ml ) , etoac ( frs 2324 ; 2 500ml ) and meoh ( frs 2526 ; 2 500ml ) .
frs 1824 were combined ( 3.1 g ) , applied on the flash chromatography column of ods - a gel ( 35 g ) and eluted with mixture of meoh - h2o ; 7:3 ( sfr 1 ; 200ml ) , meoh - h2o ; 4:1 ( sfr 2 ; 200ml ) , meoh - h2o ; 9:1 ( sfr 3 ; 2 100ml ) , meoh ( sfr 4 ; 400ml ) , chcl3-meoh - h2o ; 14:6:1 ( sfr 5 , 400ml ) .
sfr 1 ( 156 mg ) was purified by tlc si gel , chcl3-meoh - hco2h , 19:1:0.1 ) to give 3.5 mg of p - hydroxybenzaldehyde ( 3 ) and 15 mg of tetillapyrone ( 5 and 6 ) .
sfr 5 ( 103 mg ) was purified by tlc ( si gel , chcl3-meoh - hco2h , 19:1:0.1 ) to give 7 mg of phenylacetic acid ( 4 ) and 17 mg of nortetillapyrone ( 7 and 8) .
maleimide-5-oxime ( 1 ) : yellow viscous mass ; ei hrms 122.02723 ( m),cald for c4h4n2o2 , 122.02728.hnmr ( 300 mhz , dmso ) : 11.03 brs ( nh ) , 10.86 brs ( = n - oh ) , 7.40d ( j=7.6 hz , h-4 ) , 5.45d ( j=7.6 hz , h-3 ) , cnmr ( 75.47 mhz , dmso ) : 164.37 ( co-2 ) , 151.54 ( c-5 ) , 142.22 ( ch-4 ) , 100.24 ( ch-3 ) .
the effect of compounds on the growth of human tumor cell lines were evaluated according to the procedure adopted by national cancer institute ( nci , usa ) for the in vitro anticancer drug discovery screen which uses the protein - binding dye sulforhodamine b ( srb ) to assess cell growth inhibition [ 13 , 14 ] and according to the method described by kijjoa et al . .
antifungal activity of maleimide-5-oxime ( 1 ) , p - hydroxybenzaldehyde ( 3 ) , phenylacetic acid ( 4 ) , tetillapyrone ( 5 and 6 ) and nortetillapyrone ( 7 and 8) was evaluated against candida , aspergillus and dermatophyte strains : three yeasts clinical strains , one of cryptococcus neoformans ph1 , one of candida dubliniensis ph10 and one of c. glabrata d10r , as well as , four american type culture collection ( atcc ) type strains ( c. albicans attc 10231 , c. tropicalis atcc 13803 , c. krusei atcc 6258 and c. parapsilosis atcc 90018 ) ; one aspergillus clinical strain ( a. flavus f44 ) , as well as , two atcc type strains ( a. niger atcc 16404 , and a. fumigatus atcc 46645 ) ; five dermatophyte clinical strains ( microsporum canis ff1 , m. gypseum ff3 , trichophyton rubrum ff5 , t. mentagrophytes ff7 and epidermophyton floccosum ff9 ) .
the fungal isolates were identified by standard microbiology methods and stored on sabouraud dextrose broth with glycerol at 70c and subcultured in sabouraud - chloramphenicol agar . minimal inhibitory concentration ( mic )
was determined by a microdilution method according to the clsi reference method m27-a2 for yeasts , and m38-a for filamentous fungi .
the test was performed in sterile 96 flat - bottom well microtitration plates and the medium used was mops - buffered rpmi 1640 .
briefly , mics were determined for all yeasts strains with the adjusted final inoculum of 14 10 cfu / ml , prepared from a 2448 hours culture . with filamentous fungi ( except for epidermophyton floccosum ) , the final inoculum used was 48 10cfu / ml , prepared from a sporulated culture .
for epidermophyton floccosum , the final inoculum was 8 10 to 1.6 10 cfu / ml .
two fold serial dilutions of compounds in dmso , ranging from 250 to 15.125 g / ml , were tested .
in addition , reference antifungal compounds , fluconazole ( pfizer , united kingdom ) for yeasts and dermatophytes or amphotericin b ( sigma ) for aspergillus , were used as the standard antifungal drugs .
twofold serial dilutions ranging from 128 to 0.25 g / ml for fluconazole and 16 to 0.063 g / ml for amphotericin b were used .
plates were incubated at 30c for 48 hours for candida species , 72 hours for cryptococcus neoformans , 45 days for aspergillus species and 8 days for dermatophytes .
quality control determinations of the mic of fluconazole were ensured by testing c. parapsilosis atcc 90018 .
h and 13c nmr spectra were recorded at ambient temperature in cdcl3 and dmso on a bruker amc instrument operating at 300.12 and 75.47 mhz , respectively or a bruker drx instrument operating at 500 , respectively 125 mhz .
si gel for chromatography was silica gel 60 ( 0.20.5 mm merck ) for analytical work and for preparative work tlc silica gel 60 gf 254 merck .
haliclona cymaeformis ( esper , 1794 ) order haplosclerida , family chalinidae , was collected by scuba diving in the gulf of thailand near koh chang ( 2 meter depth ) , trad province in march 2004 .
haliclona baeri ( wilson , 1952 ) was also collected by scuba diving in the in the coastal area of chonburi province ( 3 meter depth ) .
sumaitt putchakarn and the vouchers of the specimens , registered as bims - i 953 and bims - i 1766 , respectively were deposited at bangsaen institute of marine science ( bims ) , burapha university , bangsaen , chonburi , thailand .
the sample ( 1.3 kg net weight ) was thawed , homogenized with etoh ( 3 l ) , allowed to stand for 24 h in a dark chamber and filtered .
the residue on the filter paper was again extracted with etoh ( 2 3l ) , the aqueous alcoholic extracts were combined , evaporated at reduced pressure to ca .
the etoac extracts were combined and concentrated at reduced pressure to give the crude etoac extract .
this ( 10 g ) was chromatographed over a si gel column ( 120 g ) and eluted with petrol - chcl3 and chcl3-me2o , 350 ml fractions being eluted as follows .
frs 162 ( petrol - chcl3 , 1:1 ) , 6378 ( petrol - chcl3 , 3:7 ) , 7995 ( petrol - chcl3 , 1:9 ) , 96182 ( chcl3-me2o , 4:1 ) , 183206 ( chcl3- me2o , 3:2 ) .
frs 101114 were combined ( 107.8 mg ) and purified by tlc ( si gel , chcl3-me2o
frs 125198 were combined ( 185.6 mg ) and purified by tlc ( si gel , chcl3-meoh - hco2h , 93:7:1 ) to give 13.1 mg 3,4-dihydroxybenzoic acid ( 2 ) and 8.0 mg of tetillapyrone ( 5and 6 ) .
the sample of haliclona cymaeformis ( 7.6 kg net weight ) was thawed , homogenized with etoh ( 10l ) , allowed to stand for 24 h in the dark chamber and filtered .
the residue on the filter paper was again extracted with etoh ( 2 10l ) , the aqueous alcoholic extracts were combined , evaporated at reduced pressure to give ca .
the etoac extracts were combined and concentrated at reduced pressure to give a crude etoac extract ( 21 g ) .
this extract was applied to flash chromatography column of si gel ( 150 g ) and eluted with chcl3 ( frs .
15 ; 5 200 ml ) , chcl3-meoh , , 49:1 ( frs 615 ; 10 200 ml ) , chcl3-meoh , , 19:1 ( frs1620 ; 5 200 ml ) , chcl3-meoh , , 9:1 ( frs 2122 ; 2 500 ml ) , etoac ( frs 2324 ; 2 500ml ) and meoh ( frs 2526 ; 2 500ml ) .
frs 1824 were combined ( 3.1 g ) , applied on the flash chromatography column of ods - a gel ( 35 g ) and eluted with mixture of meoh - h2o ; 7:3 ( sfr 1 ; 200ml ) , meoh - h2o ; 4:1 ( sfr 2 ; 200ml ) , meoh - h2o ; 9:1 ( sfr 3 ; 2 100ml ) , meoh ( sfr 4 ; 400ml ) , chcl3-meoh - h2o ; 14:6:1 ( sfr 5 , 400ml ) .
sfr 1 ( 156 mg ) was purified by tlc si gel , chcl3-meoh - hco2h , 19:1:0.1 ) to give 3.5 mg of p - hydroxybenzaldehyde ( 3 ) and 15 mg of tetillapyrone ( 5 and 6 ) .
sfr 5 ( 103 mg ) was purified by tlc ( si gel , chcl3-meoh - hco2h , 19:1:0.1 ) to give 7 mg of phenylacetic acid ( 4 ) and 17 mg of nortetillapyrone ( 7 and 8) .
maleimide-5-oxime ( 1 ) : yellow viscous mass ; ei hrms 122.02723 ( m),cald for c4h4n2o2 , 122.02728.hnmr ( 300 mhz , dmso ) : 11.03 brs ( nh ) , 10.86 brs ( = n - oh ) , 7.40d ( j=7.6 hz , h-4 ) , 5.45d ( j=7.6 hz , h-3 ) , cnmr ( 75.47 mhz , dmso ) : 164.37 ( co-2 ) ,
the effect of compounds on the growth of human tumor cell lines were evaluated according to the procedure adopted by national cancer institute ( nci , usa ) for the in vitro anticancer drug discovery screen which uses the protein - binding dye sulforhodamine b ( srb ) to assess cell growth inhibition [ 13 , 14 ] and according to the method described by kijjoa et al . .
antifungal activity of maleimide-5-oxime ( 1 ) , p - hydroxybenzaldehyde ( 3 ) , phenylacetic acid ( 4 ) , tetillapyrone ( 5 and 6 ) and nortetillapyrone ( 7 and 8) was evaluated against candida , aspergillus and dermatophyte strains : three yeasts clinical strains , one of cryptococcus neoformans ph1 , one of candida dubliniensis ph10 and one of c. glabrata d10r , as well as , four american type culture collection ( atcc ) type strains ( c. albicans attc 10231 , c. tropicalis atcc 13803 , c. krusei atcc 6258 and c. parapsilosis atcc 90018 ) ; one aspergillus clinical strain ( a. flavus f44 ) , as well as , two atcc type strains ( a. niger atcc 16404 , and a. fumigatus atcc 46645 ) ; five dermatophyte clinical strains ( microsporum canis ff1 , m. gypseum ff3 , trichophyton rubrum ff5 , t. mentagrophytes ff7 and epidermophyton floccosum ff9 ) .
the fungal isolates were identified by standard microbiology methods and stored on sabouraud dextrose broth with glycerol at 70c and subcultured in sabouraud - chloramphenicol agar .
minimal inhibitory concentration ( mic ) was determined by a microdilution method according to the clsi reference method m27-a2 for yeasts , and m38-a for filamentous fungi .
the test was performed in sterile 96 flat - bottom well microtitration plates and the medium used was mops - buffered rpmi 1640 .
briefly , mics were determined for all yeasts strains with the adjusted final inoculum of 14 10 cfu / ml , prepared from a 2448 hours culture . with filamentous fungi ( except for epidermophyton floccosum ) , the final inoculum used was 48 10cfu / ml , prepared from a sporulated culture . for epidermophyton floccosum ,
two fold serial dilutions of compounds in dmso , ranging from 250 to 15.125 g / ml , were tested .
in addition , reference antifungal compounds , fluconazole ( pfizer , united kingdom ) for yeasts and dermatophytes or amphotericin b ( sigma ) for aspergillus , were used as the standard antifungal drugs .
twofold serial dilutions ranging from 128 to 0.25 g / ml for fluconazole and 16 to 0.063 g / ml for amphotericin b were used .
plates were incubated at 30c for 48 hours for candida species , 72 hours for cryptococcus neoformans , 45 days for aspergillus species and 8 days for dermatophytes .
quality control determinations of the mic of fluconazole were ensured by testing c. parapsilosis atcc 90018 .
the effect of compounds on the growth of human tumor cell lines were evaluated according to the procedure adopted by national cancer institute ( nci , usa ) for the in vitro anticancer drug discovery screen which uses the protein - binding dye sulforhodamine b ( srb ) to assess cell growth inhibition [ 13 , 14 ] and according to the method described by kijjoa et al . .
antifungal activity of maleimide-5-oxime ( 1 ) , p - hydroxybenzaldehyde ( 3 ) , phenylacetic acid ( 4 ) , tetillapyrone ( 5 and 6 ) and nortetillapyrone ( 7 and 8) was evaluated against candida , aspergillus and dermatophyte strains : three yeasts clinical strains , one of cryptococcus neoformans ph1 , one of candida dubliniensis ph10 and one of c. glabrata d10r , as well as , four american type culture collection ( atcc ) type strains ( c. albicans attc 10231 , c. tropicalis atcc 13803 , c. krusei atcc 6258 and c. parapsilosis atcc 90018 ) ; one aspergillus clinical strain ( a. flavus f44 ) , as well as , two atcc type strains ( a. niger atcc 16404 , and a. fumigatus atcc 46645 ) ; five dermatophyte clinical strains ( microsporum canis ff1 , m. gypseum ff3 , trichophyton rubrum ff5 , t. mentagrophytes ff7 and epidermophyton floccosum ff9 ) . the fungal isolates were identified by standard microbiology methods and stored on sabouraud dextrose broth with glycerol at 70c and subcultured in sabouraud - chloramphenicol agar
. minimal inhibitory concentration ( mic ) was determined by a microdilution method according to the clsi reference method m27-a2 for yeasts , and m38-a for filamentous fungi .
the test was performed in sterile 96 flat - bottom well microtitration plates and the medium used was mops - buffered rpmi 1640 .
briefly , mics were determined for all yeasts strains with the adjusted final inoculum of 14 10 cfu / ml , prepared from a 2448 hours culture . with filamentous fungi ( except for epidermophyton floccosum ) , the final inoculum used was 48 10cfu / ml , prepared from a sporulated culture . for epidermophyton floccosum ,
two fold serial dilutions of compounds in dmso , ranging from 250 to 15.125 g / ml , were tested .
in addition , reference antifungal compounds , fluconazole ( pfizer , united kingdom ) for yeasts and dermatophytes or amphotericin b ( sigma ) for aspergillus , were used as the standard antifungal drugs .
twofold serial dilutions ranging from 128 to 0.25 g / ml for fluconazole and 16 to 0.063 g / ml for amphotericin b were used .
plates were incubated at 30c for 48 hours for candida species , 72 hours for cryptococcus neoformans , 45 days for aspergillus species and 8 days for dermatophytes .
quality control determinations of the mic of fluconazole were ensured by testing c. parapsilosis atcc 90018 . | a new compound maleimide-5-oxime was isolated , together with 3,4-dihydroxybenzoic acid , tetillapyrone , from the ethyl acetate extract of the marine sponge haliclona baeri while tetillapyrone , nortetillapyrone , p - hydroxybenzaldehyde and phenylacetic acid were isolated from the ethyl acetate extract of haliclona cymaeformis , collected from the gulf of thailand .
the structures of tetillapyrone and nortetillapyrone were re - examined using hmbc correlations .
maleimide-5-oxime , tetillapyrone and nortetillapyrone were found to be inactive against three human tumor cell lines ( the estrogen - dependent er(+ ) mcf-7 , the estrogen - independent er( ) mda - mb-231 and nci - h460 .
maleimide-5-oxime , p - hydroxybenzaldehyde , phenylacetic acid , tetillapyrone and nortetillapyrone were evaluated for their growth inhibitory effect against seven yeasts and eight filamentous fungi .
only nortetillapyrone showed antifungal activity , with a preponderance on the dermatophytic filamentous fungi . |
bipolar dislocation of clavicle , was first reported by poral in 1831.1 scapinallium ( 2004)2 though collected 26 published cases yet it is considered a rare entity . although relatively minor trauma was also reported as the cause of bipolar dislocation , floating clavicle most principally results from major trauma which produces deforming force on the lateral aspect of shoulder or a severe pressure on the shoulder together with the torsion of the trunk.2 nonsurgical treatment , adopted in the past , was highly recommended out of its easiness and low cost . however , redislocation , deformity , and dysfunction after conservative treatment are more and more emphasized .
open reduction and internal fixation ( orif ) gradually became the first choice of treatment for floating clavicle because it ensured better stability and earlier recovery .
we report a case with bipolar dislocation of clavicle , which was successfully treated by open reduction and internal fixation by oblique t - plate and kirschner wires with 2 years followup demonstrating good outcome .
the patient was informed that the case would be presented for publication and she agreed .
a 41-year - old lady met with a car accident in june 2009 and was admitted to the hospital with the chief complaint of chest and back pain and restrictions of movements of right shoulder . during the accident ,
the patient was knocked down by a car when she was riding a motorcycle and trying to cross a street .
there was no history of coma , nausea , vomiting , respiratory distress , and shortness of breath .
active abduction and flexion were limited to 60 , while there was limitation of extension and adduction .
preoperative photograph of the patient , showing deformity at the right sternoclavicular joint with prominent medial end of the clavicle radiographic examination showed dislocation of right acromioclavicular joint and sternoclavicular joint , multiple rib fractures , and minor right pleural effusion [ figure 2a ] .
3d ct reconstruction [ figure 2b ] of the injured clavicle demonstrated posterior dislocation of the lateral end of the clavicle and anterosuperior dislocation of the sternoclavicular joint .
preoperative ( a ) x - ray and ( b ) 3d ct reconstruction showing anterosuperior dislocation of the sternoclavicular joint and superioposterior dislocation of acromioclavicular joint open reduction and internal fixation was performed under general anesthesia through a 10-cm curved incision from acromian and a 5-cm horizontal incision on the right sternoclavicular joint . after exposure , superioposterior dislocation was at the acromio claviculer joint , while the medial end of the clavicle was found subluxed anteriorly and upward .
the sternoclavicular joint was fixed first . to relocate the clavicle , we firstly removed the adherent dense fibrous connective tissue in the joint space . next , with a satisfied reduction of the clavicle , in the sternoclavicular joint ,
an oblique t - type steel plate was transfixed by four screws , while at the same time another lag screw was inserted from the superior surface of clavicle toward sternum [ figure 3a ] .
the wires were inserted from the lateral end of acromion , parallel to the articulation [ figure 3b ] .
intraoperative photograph showing the fixation of ( a ) sternoclavicular and ( b ) acromioclavicular joints with oblique t - plate and kirschner wires , respectively after wound closure , the patient was asked to wear an abduction device for a month .
passive motion exercises began after 1 week while the active ones began 2 weeks later .
removal of both kirschner wires and plate was performed under general anesthesia 6 months after surgery .
x - ray conducted 1 week [ figure 4a ] , 6 months [ figure 4b ] , 1 year [ figure 4c ] and 2 years [ figure 4d ] after the operation showed stable fixation and good relationship of both acromioclavicular and sternoclavicular joints . at 2 year
followup , the patient had an abduction of approximately 150 , forward flexion of 160 , backward extension of 60 , and normal rotation [ figure 5a c ] .
besides , the power of the shoulder girdle muscles recovered and no instability of the dislocated joints was noted .
x - ray followup ( a ) 1 week , ( b ) 6 months , ( c ) 1 year , and ( d ) 2 years after surgery shows normal alignment and good relationship of both joints rehabilitation of joint abduction ( a ) flexion , ( b ) extension , and ( c ) rotation is satisfactory 2 years after surgery
in contrast to low - energy trauma like falling,34 bike riding,5 undoubtedly , a high - impact collision is considered the most common reason for bipolar clavicle dislocation.269 two hypotheses have been suggested as the mechanism of the synchronous dislocation of both the joints .
one theory suggests that elastic potential energy , originating from trauma related initial clavicle transformation , is released after the elimination of outer force , causing the synchronous dislocation of both acromioclavicular and sternoclavicular joints as well as the injury of ligaments.7 the other theory indicates that a backward violence firstly tears the articular capsule together with the acromioclavicular and coracoclavicular ligaments , causing a superioposterior dislocation of the lateral end of clavicle , and then elevates and pivots the medial end of clavicle from the first rib , causing the former to be dislocated anteriorly and upward.2410 the conservative treatments including sling immobilization and plaster application appeared to be the preference by the most satisfactory outcome was reported in some of the cases , the residual pain , dysfunction and deformity resulting from various means of external fixation.25711 beckman6 tried for the first time to consolidate the articular capsule by suturing between periosteum and the surrounding soft tissue , but without an effective outcome .
however , with the promising result brought about by the use of the kirschner wires , reported by echo6 and benabdallah,7 more and more surgeons became interested in open surgical treatment which was characterized by simultaneous treatment at the two related joints.211 the following studies also proved that strong fixation not only ensures earlier rehabilitative training but also prevents deformity secondary to redislocation.89 nevertheless , it could also cause unpredictable intra and postoperative complications .
so , thorough considerations of surgery indications become critical . in general , surgery should be adopted in comparatively younger patients or patients with high requirement of their sport ability , as it guarantees better stability .
the use of kirschner wires in sternoclavicular joint remains controversial , concerning their relatively inadequate stabilization which may contribute to higher risks of redislocation or subluxation as well as deformity .
more seriously , the migration and rupture of kirschner wires , which are not rare , could lead to severe complications such as hemorrhage threatening patient 's life when part of it migrates into mediastinum , heart , or lung.1214 therefore , more reliable internal fixation is required in the case of sternoclavicular joint dislocation .
schemitsch et al.,9 had used balser plate instead of kirschner wires to fix the dislocation of sternoclavicular joint and achieved success , proving that the selection of implant material is the key to the therapy of bipolar dislocation .
but in our opinion , t - plate is superior to other inter - fixation materials due to the following three reasons : firstly , though is in a smaller size , it is stronger ; secondly , it has high plasticity ; and finally , it resembles skeleton owing to its elasticity modulus .
our patient was young with high requirement of sport ability , we synchronously conducted bilateral internal fixation . on one hand , we used steel plate along with screws to deal with sternoclavicular joint first . on the other end , we fixed acromioclavicular joint with kirschner wires joined and a figureof - eight tension band .
two years followup showed that the patient had satisfactory outcome with no signs of redislocation , good mobility , and no pain . | bipolar dislocation of the clavicle at acromioclavicular and sternoclavicular joint is an uncommon traumatic injury .
the conservative treatments adopted in the past is associated with redislocation dysfunction and deformity .
a 41 years old lady with bipolar dislocation of right shoulder is treated surgically by open reduction and internal fixation by oblique t - plate at sternoclavicular joint and kirschner wire stabilization at acromioclavicular joint .
the patient showed satisfactory recovery with full range of motion of the right shoulder and normal muscular strength .
the case reported in view of rarity and at 2 years followup . |
epilepsy is a common disease with a cumulative lifetime risk of at least 3%.1 it is associated with significant morbidity and mortality , as well as decreased quality of life in a variety of social , educational , and vocational measures.24 specifically , mortality has been demonstrated to be up to threefold higher in developed countries.5 further , adults with seizures are at risk for lower levels of education , higher rates of unemployment , and psychological and physical ailments.6 a number of new antiseizure medications have been developed and marketed in the last two decades . despite these new therapies , medically intractable epilepsy , defined as persistent seizures after an adequate trial of two appropriate antiseizure medications , continues to occur in approximately one - third of cases.7,8 this makes the development of antiseizure medications with novel mechanisms of action an important goal to further broaden the possible success in intractable epilepsy of various etiologies .
however , with novel mechanisms of action there is a risk of unique side effects .
one of these new antiseizure medications with a novel mechanism of action is ezogabine ( ezg ) .
ezg is an ethyl n-(2-amino-4-{[(4-fluorophenyl)methyl]amino } phenyl ) ( figure 1 ) and is known by the international nonproprietary name of retigabine in europe and most of the world .
this drug was recently approved by the european medicines agency and the us food and drug administration ( fda ) for adjunctive treatment of partial - onset seizures in adults .
it appears to work by a unique mechanism of action compared with other currently available antiepileptic drugs .
a number of other recent reviews have also highlighted different aspects of ezg,913 but this review focuses especially on the adverse effects of ezg .
first developed through the national institutes of health antiepileptic drug development program after the identification of a structural analog s ( flupirtine ) efficacy against seizures , ezg s mechanism of action is novel among currently available antiseizure medications.14 ezg exerts its effects on the kv7 family of potassium channels15,16 by binding to the cytoplasmic aspects of the s5 and s6 parts of the activation gate of these potassium channels involved in the m - current.17,18 in doing so , ezg increases rates of channel opening , maintains potassium channels in the open state for longer periods of time , and alters the voltage characteristics of the cell , making channel opening more likely.16 the channels bound by ezg consist of the kv7 homoand heteromeric subunits kcnq25.15,16,19 ezg s primary site of action is the kcnq2/3 heteromer,15,16 which is expressed in the nervous system and accounts for its antiseizure efficacy .
there is no enhancement of the potassium currents mediated by the kcnq1 channel subunit , which is found primarily in cardiac tissue18 and also in the gastrointestinal system and brain.20 however , kcnq4 and kcnq5 potassium channels are activated by ezg and may be found in cochlear hair cells21 or smooth muscle.22 ezg causes relaxation of smooth muscle in the bladder , which probably accounts for the adverse effects of ezg on urinary function.23
ezg is absorbed rapidly after oral administration , with a peak plasma concentration at 1.5 hours after ingestion.24 ezg is metabolized primarily by acetylation and glucuronidation in the liver and is subsequently eliminated by renal excretion.25,26 due to this dual metabolism , it is recommended that individuals with low creatinine clearance ( < 50 ml / min ) , undergoing hemodialysis , or with moderate to severe hepatic impairment start at a lower starting dose ( 50 mg / d ) and titrate to a lower maximum dose ( 600 mg / d for renal failure or severe hepatic impairment ; 750 mg / d for moderate hepatic impairment ) . in patients
6681 years of age , it has been noted that ezg clearance was diminished compared with younger study participants ( 1840 years of age ) and the authors attributed this to the normal decrease in creatinine clearance that occurs with age .
no side effects from this difference were noted.27 as in vitro studies have demonstrated little or no potential for ezg to inhibit or induce the major cytochrome,28 p450 isoenzymes , minimal drug interactions of clinical significance have been reported .
ezg does not interact with oral contraceptive agents.29 moreover , it seems to be well tolerated in individuals taking other antiseizure medications as well , with minimal direct interactions .
while ezg and lamotrigine had reciprocal effects on renal clearance , both drugs were tolerated equally well alone and in combination , thus these interactions were not thought clinically significant.30 no clinically significant interactions have been found between ezg and propofol , valproate , or imipramine26 and an in vivo study has demonstrated lack of interaction between phenobarbitone and ezg.31 in clinical studies , no significant pharmacokinetic effect has been seen on phenytoin , carbamazepine , valproic acid or topiramate.32 overall , ezg has few clinically significant interactions with other pharmaceutical agents .
three double - blind placebo - controlled multicenter trials have taken place investigating the efficacy of ezb in adults with medically intractable partial - onset epilepsy as add - on therapy in combination with other antiepileptic medications.3335 a recent paper has reported an integrated analysis of these three controlled trials.36 after pooling data from the three studies , 1240 patients were included , with 813 patients randomized to ezg and 427 to placebo . in all trials , after an initial 8-week baseline phase , patients were randomized to placebo or ezb ( titrated to 600 , 900 , or 1200 mg / day , depending on the trial ) , followed by an 812-week maintenance phase .
two primary efficacy measures were used : ( 1 ) responder rate , defined as the percentage of patients experiencing a 50% reduction in seizure frequency compared with the baseline phase ; and ( 2 ) the percent change in partial seizure frequency .
a number of secondary efficacy endpoints were also assessed , including more specific categorization of seizure frequency reduction rates and the proportion of seizure - free days and seizure - free patients .
analysis of the integrated data found statistically significant effects of ezb on both primary efficacy measures.36 responder rates ( 50% reduction in seizure frequency ) were 35% and 45% for ezg at 600 and 900 mg / day , respectively , compared with 21% for placebo ( p
< 0.001 ) and 50% for ezg at 1200 mg / day , compared with 24% for placebo ( p < 0.001 ) .
average reduction in seizure frequency was 26% , 37% , and 39% for 600 , 900 , and 1200 mg / d ezg , respectively , all of which were statistically significantly greater than placebo ( 15% ) .
for example , the proportion of patients experiencing a 50%75% or 75% reduction in seizures showed a dose - dependent increase with ezg compared with placebo . at all doses
, there was a significant increase in seizure - free days with ezg compared with placebo , although the effect was modest ( 81%84% for ezg vs 78% for placebo ) .
in addition , while in the individual trials there had been no statistically significant effect of ezg on the percentage of seizure - free patients , in the pooled analysis of ezg at 1200 mg / d , but not at 600 or 900 mg / d , there was a statistically significant increase in the proportion of seizure - free patients during the maintenance phase ( 15% with 1200 mg / d ezg vs 5% with placebo ) .
subgroup analyses did not demonstrate any differences in the magnitude of the effect of ezg based on age ( 1844 years vs > 44 years ) , sex , race ( white vs nonwhite ) , region ( us vs non - us ) , number of baseline antiseizure medications , and seizure subtypes ( simple partial , complex partial , secondary generalized ) .
overall , these randomized placebo - controlled trials demonstrated that ezg is effective as add - on therapy for adults with intractable partial - onset epilepsy .
the magnitude of the effects of ezg on seizures is similar to most other new antiseizure medications used as adjunctive therapy for intractable epilepsy and only a small percentage of patients become seizure - free on ezg .
over the course of the three major placebo - controlled trials,3335 ezg was generally quite well tolerated .
based on the pooled analysis , adverse events that were considered severe were similar in the placebo ( 10% ) , ezg 600 mg / d ( 10% ) , and ezg 900 mg / d ( 12% ) groups but were higher in the ezg 1200 mg / d group ( 20%).36 all other adverse events were noted by investigators to be mild to moderate .
three deaths ( 0.7% ) related to sudden death in epilepsy , injury , and respiratory failure occurred in the 427 patients on placebo , whereas only two deaths ( 0.2% ) in the cumulative ezg groups due to sudden death in epilepsy and diabetic ketoacidosis occurred in the 813 patients .
discontinuation of the study due to adverse events occurred in 10% , 15% , 24% , and 28% of patients in the placebo , 600 mg / d , 900 mg / d , and 1200 mg / d groups , respectively.36 reasons for discontinuation of the study and the overall most common adverse events were generally central nervous system ( cns ) related ( table 1 ) .
the most common side effects included dizziness and somnolence and , to a lesser extent , confusion , fatigue , and asthenia.3335 porter et al33 noted speech difficulties in no patients in the placebo arms and five ( 5% ) , twelve ( 12.6% ) , and 17 ( 16% ) patients in the 600 mg / d , 900 mg / d , and 1200 mg / d arms , respectively .
ezg has been noted to decrease gastrointestinal smooth muscle contractility.37,38 constipation was not mentioned in two studies33,35 but occurred in three ( 2% ) patients in the placebo arm and nine ( 5.9% ) patients in the 1200 mg / d group french et al s study.34 alterations in liver enzyme levels were noted rarely but were transient and did not require discontinuation from the studies.3335 no changes in electrocardiography have been reported .
adverse events related to cardiac abnormalities were similar in the placebo ( 4% ) and ezg ( 5% ) groups .
while most of the reported side effects of ezg are similar to many other antiseizure medications in that they most commonly affect the cns , ezg has one relatively unique and specific adverse effect , which is related to urinary function.37 ezg had initially been noted to relax rat bladders39 as well as decrease bladder tension and contraction amplitude.40 as a result of this basic science data , urinary adverse events were recorded during the placebo - controlled trials . in brodie
et al s trial,35 three patients were discharged from these trials for urinary symptoms , although the groups to which these patients were assigned were not delineated : one for nephritis and two for urinary hesitation .
french et al34 reported nine ( 5.9% ) patients in the 1200 mg / d arm and one ( 0.7% ) in the placebo arm with urinary hesitation and eight ( 5.2% ) patients in the 1200 mg / d arm and two ( 1.3% ) in the placebo arm with dysuria . in the pooled analysis , urinary or renal symptoms were reported in the ezg 1200 mg / d group ( 25% ) almost twice as frequently as in the placebo ( 13% ) group , although similar rates as in the placebo group were reported with 600 mg / d ( 14% ) and 900 mg / d ( 13%).35,36 other urinary symptoms and signs reported included urinary tract infections , polyuria , hematuria , proteinuria , and chromaturia .
these adverse events were noted to diminish with time on the medication and were noted to be mild to moderate .
very few cases led to discontinuation ( 1% for both ezg and placebo ) . due to the urinary symptoms ,
some data from the open - label extension trial have now been published in a recent review.37 there continues to be no evidence of direct renal adverse events , although one participant did have reversible renal failure related to obstructive uropathy.37 of note , four participants have now been reported with nephrolithiasis in the 1200 mg / d group.37 however , based on the overall incidence of nephrolithiasis in the usa and ezg s mechanism of action , it is not felt that ezogabine is an independent risk factor for nephrolithiasis at this time .
urinary crystals with an appearance similar to bilirubin have been reported in some participants.37 this could result in an abnormal urinalysis result but does not appear to be related to a risk for nephrolithiasis .
overall , it would seem prudent to monitor any patients on ezg for urologic symptoms and be cautious with those with risk factors for urinary obstruction .
in addition , there is a lack of established safety data on infants of breastfeeding mothers on ezg .
these include in neuromyotonia,41 neuropathic pain,42,43 mania , and addiction to psychostimulants.44 other rodent models have demonstrated a possible anti - dystonic effect.45 an in vitro study has demonstrated a possible anxiolytic effect of ezg.46 however , these other applications are still under investigation .
epilepsy , despite several new medications that have been developed in the last few decades , continues to be refractory to medications in one - third of patients and can be a significant cause of morbidity and mortality .
it is a potassium - channel activator primarily active in the cns , with alternate sites of action in the urinary bladder , gastrointestinal smooth muscle , and vascular smooth muscle.47,48 ezg has proven efficacy and is approved both in the usa and europe for treatment of adults with intractable partial - onset epilepsy . given its novel mechanism of action , there is hope that ezg may be more effective for some patients with intractable epilepsy than other available antiseizure medications , although , overall , the efficacy data are fairly comparable to studies of other drugs in intractable patients .
early data demonstrate minimal interaction with medications other than lamotrigine and those interactions were deemed to be clinically insignificant.30 ezg is generally well tolerated and is rarely discontinued due to adverse events .
the number of reported adverse events is also likely to be influenced by the forced fast titration schedule deemed necessary by the authors of the placebo - controlled trials .
specifically , on the urinary bladder both in animal models and in human trials .
urinary or secondary renal adverse effects are collectively relatively common , occurring in about 25% of patients in the highest - dose ezg group.36,37 symptoms from urinary adverse events were usually noted to be minor or moderate , sometimes only involving laboratory abnormalities on urinalysis and rarely leading to discontinuation from the trial . however , urinary hesitancy and retention can be clinically significant problems .
thus , due to this novel side effect and the lack of a significant quantity of long - term data as to the severity and prevalence of urinary adverse events , the fda has recommended a risk evaluation and mitigation strategy to further track the use of ezg in this regard .
overall , no long - term data currently exist as to the safety of ezg . | one - third of patients with epilepsy continue to have seizures despite current treatments , indicating the need for better antiseizure medications with novel mechanisms of action .
ezogabine ( retigabine ) has recently been approved for adjunctive treatment of partial - onset seizures in adult patients with epilepsy .
ezogabine utilizes a novel mechanism of action , involving activation of specific potassium channels .
the most common side effects of ezogabine are shared by most antiseizure medications and primarily consist of central nervous system ( cns ) symptoms , such as somnolence , dizziness , confusion , and fatigue .
in addition , a small percentage of patients on ezogabine experience a unique adverse effect affecting the bladder , which results in urinary hesitancy ; thus , patients on ezogabine should be monitored carefully for potential urological symptoms .
overall , ezogabine appears to be well tolerated and represents a reasonable new option for treating patients with intractable epilepsy . |
enamel is the outermost covering of vertebrate teeth and the hardest tissue in the vertebrate body . during tooth development , ectoderm - derived ameloblast cells create enamel by synthesizing a complex protein mixture into the extracellular space where the proteins self - assemble to form a matrix that patterns the hydroxyapatite woven to form a tough , wear - resistant composite material .
the mature enamel composite contains almost no protein and is a hard , crack - tolerant , and abrasion - resistant tissue . during enamel biomineralization ,
the dominant protein of mammalian enamel is amelogenin , a hydrophobic protein that self - assembles to form nanospheres that in turn influence the crystal habit and packing of the crystallites .
in contrast to the mesenchyme - controlled biomineralization of bone , which uses collagen and remodels both the organic and inorganic phases over a lifetime , enamel contains no collagen and does not remodel .
mineralized dentin is synthesized by odontoblasts that line the centrally located dental pulp chamber and is deposited beneath the enamel and cementum .
dentin , otherwise to the enamel , is soft flexible and able to absorb energy , and resists fracture .
it is less mineralized than enamel , and it is a sort of sponge crossed by channels of one micron wide radically departing from the odontoblasts .
these channels called dentinal tubules , are occupied by a part of the odontoblasts whose cytoplasm body underlies the dentin - dental pulp interface .
dentin is formed by mineralization of the dentin matrix mainly composed of collagen type i and some specific noncollagenous matrix proteins .
the deposition of the dentin occurs over the life of the teeth . sometimes in the immature
odontoblasts can be formed from dental pulp stem cells following a differentiation process induced by required signals .
it is also known that , in response to stimulation with recombinant bmps , dental pulp cells differentiate into dentin - forming odontoblasts .
however , it is still unknown what is the required ideal combination of signals and the minimum set of cells , to engineer all the cellular components of a fully functional dental pulp , while the allegation that dental pulp stem cells may have the potential to differentiate into most cells of the dental pulp has not yet been strongly demonstrated in vivo .
the use of calcium hydroxide to stimulate reparative dentin is an example of therapeutic strategy .
tissue engineering enhances dentistry to move forward in the application of regeneration as important principle for the treatment of dental disease .
it is based on fundamental approaches that involve the identification of appropriate cells , the development of conductive biomaterials , and an understanding of the morphogenic signals required to induce cells to regenerate the lost tissue .
extended research has started to emerge in the field of enamel and other dental tissue regeneration applying material - cell - based strategies .
it is expected that strategies involving the use of tissue engineering , nanotechnology , and stem cells to have an increasing participation in clinical dentistry over the next 520 years .
there are major issues to overcome before such strategies be introduced into the clinic and used regularly to treat dental diseases .
however , there is evidence that suggest tissue engineering as the main approach in the future of operative dentistry , for the development of new dental structures .
odontoblasts are found in the dental pulp of erupted teeth . in their absence , undifferentiated dental pulp cells or dental pulp stem cells
can be differentiated into odontoblasts and restore the capability of the dental pulp to synthesize reparative dentin .
however , ameloblasts which specialize in making enamel are not present in teeth with complete crown development .
consequently , an endogenous regeneration of enamel is not feasible , while the development of synthetic enamel and/or in situ cell - based approaches are being achieved by using the principles of tissue regeneration and nanotechnology .
surfactants were used as reverse micelles or microemulsions to synthesize enamel , as they can mimic the biological action of enamel proteins .
the synthesized nanoscale structures may self - assemble into one dimensional building blocks leading to the development of hydroxyapatite nanorods similar to natural enamel crystals .
the fabricated nanorods can potentially be applied as flowable restorative material for the restoration of lost enamel .
chen et al . based on the biological processes involved in amelogenesis , combined with new approaches in nanotechnology , fabricated enamel prism - like structures consisted of fluorapatite nanorods ( figure 1(a ) ) precipitated directly from solution under controlled chemical conditions without the use of surfactants , proteins , or cells .
the fabricated nanorods present similar characteristics to those of the natural enamel crystals isolated from rat incisor enamel , as it is confirmed from the scanning electron microscope ( sem ) images in figure 1(b ) .
another enamel - based biomaterial having the added benefit of fluorapatite incorporated intrinsically into the composition was also observed .
particularly , amelogenin - driven apatite crystal growth , incorporating fluoride into the process , allowed the synthesis of elongated rod - like apatite crystals with dimensions similar to those observed in natural enamel . although the extended research for engineering advanced biomaterials , it is evidenced that none of the available material today can mimic all the physical , mechanical , and esthetic properties of enamel .
this conclusion was an important parameter toward the establishment of cell - based strategies that could stimulate enamel regeneration .
it has been suggested that extracellular matrix proteins such as fibronectin , laminin , and ameloblastin not only function as a mechanical scaffold for cell attachment and survival but also provide a microenvironment for guiding cell growth and differentiating on .
used an in vitro cell and organ culture system , to study the effect of artificial bioactive nanostructures on ameloblasts with the long - term goal of developing cell - based strategies for tooth regeneration .
particularly , a branched peptide amphiphile molecules containing the peptide motif arg - gly - asp or
( abbreviated brgd - pa ) , known to self - assemble in physiologic environments into nanofibers network , was used in order to mimic the extracellular matrix that surrounds the ameloblasts .
ameloblast - like cells ( line ls8 ) and primary enamel organ epithelial ( eoe ) cells were cultured within brgd - pa hydrogels and formed focal multilayered structures that accumulated minerals .
brgd - pa was also injected into the enamel organ epithelia of mouse embryonic incisors .
at the site of injection , it was observed eoe cell proliferation with differentiation into ameloblasts as evidenced by the expression of enamel - specific proteins .
moreover , it was shown the nanofibers within the forming extracellular matrix , in contact with the eoe cells engaged in enamel formation and regeneration .
finally it was concluded that brgd - pa nanofibers present with enamel proteins participate in integrin - mediated cell binding to the matrix with delivery of instructive signals for enamel formation .
a crosstalk that involves signals of diffusible molecules from the epithelium induces odontoblasts to synthesize extracellular matrix proteins required for dentin formation .
there is a big research in the field of the different inducers of dentin mineralization .
the demineralized dentin powder , likewise the demineralized bone powder , observed to have also the capability to induce mineralization when applied directly to areas of pulp exposure [ 19 , 20 ] . specific functions of dentin seem to contain bone morphogenetic protein ( bmp ) activity , which induces reparative dentin formation , leading to the potentially use of bmps in dentin regeneration [ 16 , 20 , 21 ] .
moreover the use of recombinant human proteins combined with collagen - based matrixes was applied to induce dentin regeneration .
it was observed the induction of reparative dentin at the sites of pulp exposure within a period of 2 to 4 months [ 22 , 23 ] .
the general mechanism of this process is based on the fact that reparative dentin is formed where the stimulating agents were placed in direct contact with the dental pulp .
this consideration was strengthened as it was observed a proportional dependence of the area of the induced reparative dentin with the amount of the applied bmp-7 , which could eventually allow the predetermination of dentin 's amount .
however the induction of reparative dentin was not successful in the case of inflamed dental pulps , which was assigned to insufficient amount of active recombinant protein due to its relative short half - life and to the faster degradation rates of the protein in the presence of the inflamed pulp .
the capability to induce reparative dentin was also found to growth / differentiation factor 11 ( gdf11 ) with a direct delivery to pulp cells applying a gene transfer strategy .
additionally , bone sialoprotein ( bsp ) was observed to stimulate the differentiation of dental pulp cells into cells that can secrete extracellular matrix which is further mineralized into reparative dentin , presenting different morphological characteristics compared to the respective induced by bmp proteins .
this observation enhances the consideration that one day based on the patient 's needs it will be possible to have the capability to select the ideal type of biological inducer for the desired reparative dentin .
in addition , the side population fraction of human dental pulp cells and the periodontal tissue stem cells derived from human - extracted teeth observed to partially regenerate dentin and periodontal tissue by cell transplantation into defects , suggesting that the transplantation of stem cells for partial tissue repair using autologous dental tissue stem / progenitor cells is possible when appropriate signals coexist , as it is schematically presented in figure 2 .
these cells are thought to be already committed to dental cell lineages as they are able to form dental tissues without epithelial - mesenchymal interactions .
in addition to specific cells and signaling molecules , the importance of scaffolds in guiding dentin regeneration has also been evaluated .
tissue engineering using scaffold and cell aggregate methods has been also suggested to produce bioengineered complex dentin - enamel regeneration from dissociated cells .
investigated the capability of epithelial cell rests of malassez ( erm ) to regenerate dental tissues by transplanting subcultured erm seeded onto scaffolds into the omentum of athymic rats .
particularly , in combination with dental pulp cells at the crown formation stage , erm was coseeded into collagen sponge scaffolds .
after 8 weeks transplantation , enamel - dentin complex - like structures were recognized in the implants , as enamel - like tissue and the stellate reticulum - like structures were observed to some degree , while the tall columnar ameloblast - like cells were aligned with the surface of the enamel - like tissues .
similar results were observed in our lab with dental epithelial stem populations isolated by fluorescence activation cell sorting ( facs ) using previously discovered epithelial stem cell markers and subcultured under serum - free and xenon - free conditions . as it is illustrated in figure 3 , the collected human dental epithelial stem cells ( hdescs )
can generate mineralized tissue in vivo when coseeded on plla scaffolds with human dental pulp stem cells ( hdpscs ) and implanted subsequently in the nude mouse .
after 10 weeks postimplantation mineralization is seen in the implants . furthermore , complex dental tissues regeneration was investigated with different types of reassociations between epithelial and mesenchymal tissues and/or cells from mouse embryos which were cultured in vitro before in vivo implantation . in vitro the reassociated tissues developed and resulted in jointed dental structures that exhibited normal epithelial histogenesis and allowed the functional differentiation of odontoblasts and ameloblasts .
after implantation , the reassociations formed roots and periodontal ligament , the latter connected to developing bone .
a potential solution could be the application of appropriate advanced biological systems with therapeutic agents able to control the inflammatory response while inducing mineralization .
an additional important challenge is the development of suitable carriers which can house all the necessary factors for the treatment and regeneration of lost / diseased tooth parts , while they should present biocompatibility , physicochemical , and mechanical properties compatible to their application in restorative dentistry .
these new fabricated carriers should be able to create well - sealed restorations , preventing microleakages and subsequent contamination of the exposure pulp before the mineralization .
the use of composites of synthetic or natural 3d scaffolds with bioactive antibacterial materials seeded with specific dental tissue stem cells could be a potential innovated system fulfilling all these significant requirements .
consequently , extended interdisciplinary research and effective collaboration between basic scientists and clinicians could potentially lead this field to the final goal of regeneration tooth parts or eventually the entire tooth . | the process of tooth mineralization and the role of molecular control of cellular behavior during embryonic tooth development have attracted much attention the last few years . the knowledge gained from the research in these fields
has improved the general understanding about the formation of dental tissues and the entire tooth and set the basis for teeth regeneration .
tissue engineering using scaffold and cell aggregate methods has been considered to produce bioengineered dental tissues , while dental stem / progenitor cells , which can differentiate into dental cell lineages , have been also introduced into the field of tooth mineralization and regeneration .
some of the main strategies for making enamel , dentin , and complex tooth - like structures are presented in this paper .
however , there are still significant barriers that obstruct such strategies to move into the regular clinic practice , and these should be overcome in order to have the regenerative dentistry as the important mean that can treat the consequences of tooth - related diseases . |
thirty - seven subjects were enrolled in this study ; there were 19 men and 18 women with a mean age of 70 years ( age range : 47 - 85 years ) .
the study protocol was approved by the institutional review board of our university and also by the ethics committee of the institute for medical science .
the procedure was explained in detail to all the prospective patients , and a written consent was obtained from all the subjects before the procedure .
the study inclusion criteria were ( i ) the presence of malignant obstruction of the common bile duct below the hilar confluence , and this was caused by unresectable malignant tumor or reocclusion of a previously inserted self - expandable uncovered biliary stent , and ( ii ) obtaining an informed consent .
the exclusion criteria included surgical resectability of the tumor , hilar obstruction or postoperative anastomotic site obstruction due to malignant recurrence , or the presence of a generally fatal patient condition such as sepsis or disseminated intracoagulopathy .
thirty - seven patients had percutaneous biliary drainage performed , of which 34 were treated with a covered stent as the first stent , and three were treated for failure of a standard uncovered stent .
six patients underwent sphincterotomy via endoscopic guidance before stent insertion . in our study , 14 patients had biopsy - confirmed adenocarcinomas and 23 patients lacked a confirming biopsy , but these 23 patients had been diagnosed by the findings on abdominal spiral computed tomography ( ct ) , magnetic resonance ( mr ) imaging , percutaneous cholangiography and endoscopic retrograde cholangiopancreatography ( ercp ) .
thirteen patients had common bile duct carcinoma , eight patients had pancreatic head carcinoma , four patients had gallbladder carcinoma , six patients had periampullary carcinoma and six patients had gastric carcinoma .
the site of obstruction was the proximal common bile duct ( cbd ) in three patients , the middle cbd in six patients and at the distal cbd in 28 patients .
the stents used in this study were self - expanding biliary ptfe covered niti - s stents ( taewoong medical corporation , seoul , korea ) , as are described in figure 1 .
the niti - s stent we used was made of 0.007-inch single nitinol wire , which has excellent biocompatibility and expansile force , and the stent was also self - expanding without any sharp spurs at either end ( low trauma ends ) . to prevent stent migration , a ptfe membrane covered the stent on the inside of the metal wire .
the stents were 5 - 8 cm long when fully expanded to a maximum diameter of 10 mm , and they were positioned using a 9-fr introducer system .
the stent was held , compressed and elongated on the delivery catheter by a cylindrical rolling membrane ( the outer sheath ) .
the delivery catheter had three markers : one at a first - proximal portion ( the proximal marker ) , the second at the actual stent length position ( the stent length marker ) , and the third at the distal position of the loaded stent ( the stent marker ) .
the devised niti - s stent became shortened by 51% after deployment , which is similar to that of wallstents ( 50% ) ; the nitis - s stent had tungsten markers on its proximal and distal ends that helped with manipulation during deployment ( 13 ) .
stent placement was accomplished within two weeks after performing percutaneous transhepatic biliary drainage ( ptbd ) .
stent placement was accomplished in three patients during the follow - up tubogram after protracted ptbd or stent placement .
a right intercostal and/or left epigastric approach was chosen depending on the extent of tumor .
stent insertion was performed over an extra stiff amplatz guidewire ( 180 cm , 0.035 inch , curved tip ; cook inc . ,
care was taken to precisely locate the covered mid - portion of the stent exactly at the stricture .
balloon dilation after stent insertion was not performed in any of our patients ; instead , an 8.5-fr or 10.2-fr biliary drainage catheter was left in place for seven days .
this catheter was used only for flushing and maintaining access to the biliary tree , and not for drainage .
thereafter , imaging was repeated via the drainage catheter to check the stent 's patency , function and position .
the catheter was removed if free bile flowing into the duodenum was documented or if the bilirubin level was normalized or markedly reduced .
all the patients received 200 mg of prophylactic antibiotics that were administered intravenously over a period of 12 hours ( flomoxef sodium ; flumarin , ildong , ansung , korea ; or fosfomycin sodium ; fonofos , chong keun dang , chunan , korea ) before and usually 48 - 72 hours after the procedure .
coagulation tests were performed before the procedure , and if a patient had disseminated intravascular coagulopathy , then stent placement was not performed .
sonography of the upper abdomen was performed , and the bilirubin levels were checked at each examination .
the patients were also followed by conducting telephone interviews with the referring physicians or with the patients when the patients failed to appear at the follow - up examinations .
if stent occlusion was suspected , then the stent was revised percutaneously using a 10-fr drainage catheter .
finally , the cause of death was ascertained for all the patients who died within the observation period .
successful decompression of the biliary system was defined by a decrease of the serum bilirubin level of more than 30% versus the baseline value within a week of stent insertion ( 10 ) .
we measured the distance from the bifurcated line between the right and left intrahepatic ducts to the end of the biliary stent .
proximal stent migration was defined as a stent that migrated into the common hepatic duct , and distal stent migration was defined as a stent that migrated into the duodenum or out of the patient 's body .
the extents of these migrations were established by comparing the stent ends with the stent positions seen on the initial images .
early migration was defined as migration that occurred within one week of deployment , and delayed migration was defined as migration that occurred from one week to one month after stent placement . for the cases of recurrent jaundice , any stent occlusion was confirmed by sonography or ct , which showed dilation of the intrahepatic duct and the presence of soft tissue density inside the stent .
recurrent episodes of inexplicable fever were defined as cholangitis . at the time of death , a stent was assumed to be patent if the patient had normal or only mildly elevated serum bilirubin levels ( < 3 mg / dl ) . if the patients had obvious jaundice or if they had a higher bilirubin level than that before stent placement , then the stent was assumed to be obstructed .
echogenic materials in the stents , as were determined by us , were considered to represent sludge , and floating material in the stent , as determined by percutaneous transhepatic cholangiography , was also considered to represent sludge formation .
the bilirubin and amylase / lipase levels before and during follow - up were statistically analyzed by using the wilcoxon signed - rank test .
the stent patency rate and the patient survival rate were calculated by performing kaplan - meier survival ( life - table ) analysis .
the duration of primary patency was defined as the interval between stent placement and the recurrence of obstructive jaundice .
if obstruction was not evident during a patient 's life , then the patency period was considered to be equal to the survival duration , but this data was censored .
thirty - seven subjects were enrolled in this study ; there were 19 men and 18 women with a mean age of 70 years ( age range : 47 - 85 years ) .
the study protocol was approved by the institutional review board of our university and also by the ethics committee of the institute for medical science .
the procedure was explained in detail to all the prospective patients , and a written consent was obtained from all the subjects before the procedure .
the study inclusion criteria were ( i ) the presence of malignant obstruction of the common bile duct below the hilar confluence , and this was caused by unresectable malignant tumor or reocclusion of a previously inserted self - expandable uncovered biliary stent , and ( ii ) obtaining an informed consent .
the exclusion criteria included surgical resectability of the tumor , hilar obstruction or postoperative anastomotic site obstruction due to malignant recurrence , or the presence of a generally fatal patient condition such as sepsis or disseminated intracoagulopathy .
thirty - seven patients had percutaneous biliary drainage performed , of which 34 were treated with a covered stent as the first stent , and three were treated for failure of a standard uncovered stent .
six patients underwent sphincterotomy via endoscopic guidance before stent insertion . in our study , 14 patients had biopsy - confirmed adenocarcinomas and 23 patients lacked a confirming biopsy , but these 23 patients had been diagnosed by the findings on abdominal spiral computed tomography ( ct ) , magnetic resonance ( mr ) imaging , percutaneous cholangiography and endoscopic retrograde cholangiopancreatography ( ercp ) .
thirteen patients had common bile duct carcinoma , eight patients had pancreatic head carcinoma , four patients had gallbladder carcinoma , six patients had periampullary carcinoma and six patients had gastric carcinoma .
the site of obstruction was the proximal common bile duct ( cbd ) in three patients , the middle cbd in six patients and at the distal cbd in 28 patients .
the stents used in this study were self - expanding biliary ptfe covered niti - s stents ( taewoong medical corporation , seoul , korea ) , as are described in figure 1 .
the niti - s stent we used was made of 0.007-inch single nitinol wire , which has excellent biocompatibility and expansile force , and the stent was also self - expanding without any sharp spurs at either end ( low trauma ends ) . to prevent stent migration , a ptfe membrane covered the stent on the inside of the metal wire .
the stents were 5 - 8 cm long when fully expanded to a maximum diameter of 10 mm , and they were positioned using a 9-fr introducer system .
the stent was held , compressed and elongated on the delivery catheter by a cylindrical rolling membrane ( the outer sheath ) .
the delivery catheter had three markers : one at a first - proximal portion ( the proximal marker ) , the second at the actual stent length position ( the stent length marker ) , and the third at the distal position of the loaded stent ( the stent marker ) .
the devised niti - s stent became shortened by 51% after deployment , which is similar to that of wallstents ( 50% ) ; the nitis - s stent had tungsten markers on its proximal and distal ends that helped with manipulation during deployment ( 13 ) .
stent placement was accomplished within two weeks after performing percutaneous transhepatic biliary drainage ( ptbd ) .
stent placement was accomplished in three patients during the follow - up tubogram after protracted ptbd or stent placement .
a right intercostal and/or left epigastric approach was chosen depending on the extent of tumor .
stent insertion was performed over an extra stiff amplatz guidewire ( 180 cm , 0.035 inch , curved tip ; cook inc . ,
care was taken to precisely locate the covered mid - portion of the stent exactly at the stricture .
balloon dilation after stent insertion was not performed in any of our patients ; instead , an 8.5-fr or 10.2-fr biliary drainage catheter was left in place for seven days .
this catheter was used only for flushing and maintaining access to the biliary tree , and not for drainage .
thereafter , imaging was repeated via the drainage catheter to check the stent 's patency , function and position .
the catheter was removed if free bile flowing into the duodenum was documented or if the bilirubin level was normalized or markedly reduced .
all the patients received 200 mg of prophylactic antibiotics that were administered intravenously over a period of 12 hours ( flomoxef sodium ; flumarin , ildong , ansung , korea ; or fosfomycin sodium ; fonofos , chong keun dang , chunan , korea ) before and usually 48 - 72 hours after the procedure .
coagulation tests were performed before the procedure , and if a patient had disseminated intravascular coagulopathy , then stent placement was not performed .
sonography of the upper abdomen was performed , and the bilirubin levels were checked at each examination .
the patients were also followed by conducting telephone interviews with the referring physicians or with the patients when the patients failed to appear at the follow - up examinations . if stent occlusion was suspected , then the stent was revised percutaneously using a 10-fr drainage catheter .
finally , the cause of death was ascertained for all the patients who died within the observation period .
successful decompression of the biliary system was defined by a decrease of the serum bilirubin level of more than 30% versus the baseline value within a week of stent insertion ( 10 ) .
we measured the distance from the bifurcated line between the right and left intrahepatic ducts to the end of the biliary stent .
proximal stent migration was defined as a stent that migrated into the common hepatic duct , and distal stent migration was defined as a stent that migrated into the duodenum or out of the patient 's body .
the extents of these migrations were established by comparing the stent ends with the stent positions seen on the initial images .
early migration was defined as migration that occurred within one week of deployment , and delayed migration was defined as migration that occurred from one week to one month after stent placement . for the cases of recurrent jaundice , any stent occlusion was confirmed by sonography or ct , which showed dilation of the intrahepatic duct and the presence of soft tissue density inside the stent .
recurrent episodes of inexplicable fever were defined as cholangitis . at the time of death , a stent was assumed to be patent if the patient had normal or only mildly elevated serum bilirubin levels ( < 3 mg / dl ) . if the patients had obvious jaundice or if they had a higher bilirubin level than that before stent placement , then the stent was assumed to be obstructed .
echogenic materials in the stents , as were determined by us , were considered to represent sludge , and floating material in the stent , as determined by percutaneous transhepatic cholangiography , was also considered to represent sludge formation .
the bilirubin and amylase / lipase levels before and during follow - up were statistically analyzed by using the wilcoxon signed - rank test .
the stent patency rate and the patient survival rate were calculated by performing kaplan - meier survival ( life - table ) analysis .
the duration of primary patency was defined as the interval between stent placement and the recurrence of obstructive jaundice .
if obstruction was not evident during a patient 's life , then the patency period was considered to be equal to the survival duration , but this data was censored .
stent deployment was successful in all 37 patients , and this was performed via transpapillary placement of the covered stent ( 37/37 , 100% ) ( fig .
two patients showed that the stent had migrated completely out of the biliary duct during the 7-week and 27-week follow - up period , respectively .
the distance between the bifurcated line and the end of the stent immediately after stent placement was 6 - 35 mm ( mean : 15 mm ) .
one week later , follow - up tubography showed no change of this distance ( range : 6 - 35 mm : mean : 15 mm ) .
the follow - up plain films showed no changes in any of the stents in the right upper quadrant areas except for two patients who experienced delayed stent migration .
the mean bilirubin level decreased from 11.1 mg / dl 6.86 standard deviation ( sd ) before the procedure to 5.12 mg / dl 4.6 sd at the first follow - up , and this difference was statistically significant ( wilcoxon test : p < 0.01 ) .
significant changes in the bilirubin levels were observed between the baseline and the most recent follow - up after stent placement ( p < 0.05 ) .
the amylase and lipase levels were 110.5 iu / l 150.6 and 133.4 iu / l 187.3 , respectively , before the procedure ; they were 94.0 iu / l 90.6 and 135.2 iu / l 185.0 , respectively , one week after the procedure , and they were 48.6 iu / l 38.1 and 50.3 iu / l 54.2 , respectively , at the last follow - up ( fig .
3 ) . the amylase and lipase levels did not increase at one week and at the last follow - up day after the insertion ( p > 0.01 ) .
the amylase and lipase levels were found to be severely elevated one day after stent insertion in 11/37 patients ( 29% ) ; that is , the amylase / lipase level was 505 iu / l ( range : 107 - 1,455)/520
iu / l ( range : 77 - 1,019 ) . despite endoscopic sphincterotomy that was done in six patients ,
four patients revealed severe elevations of their amylase and lipase levels one day after stent insertion .
these 11 patients had pancreatitis symptoms such as pain ; however , their amylase and lipase levels stabilized within seven days .
the 30-day mortality rate was 8% ( 3/37 ) , which reflected fatal septic conditions in two patients and death from an unknown cause in one patient .
three patients showed good patency of their biliary stent , according to one - week follow - up ultrasound sonography .
however , the bilirubin level of two patients before stent insertion was from 11.0 mg / dl to 11.5 mg / dl and 13.5 mg / dl to 14.4 mg / dl , respectively , on the one - week follow - up , and one other patient showed a bilirubin level from 24.7 mg / dl to 22.0 mg / dl .
the discrepancies between the image findings and lab findings were related to each patient 's conditions .
the lesion was completely covered by the stent in four patients who received a 5-cm - long stent , in 12 patients who received a 6-cm - long stent , in 11 patients who received a 7-cm - long stent , in nine patients who received an 8-cm - long stent and in one patient who received a 9-cm - long stent .
no case of biliary side - branch obstruction occurred , except for at the cystic duct .
seventy - six percent ( 28/37 ) of the study patients experienced adequate palliative drainage for the remainder of their lives .
three patients displayed stent sludge occlusion , and ptbd showed some filling defect in these stents ( fig .
however , good stent patency was achieved via irrigation for two patients who both underwent ptbd two times during the follow - up .
two patients had ptbd performed : one patient due to tumor ingrowth ( 34 weeks ) and another due to stent migration ( 7 weeks ) .
the tubogram finding after ptbd showed stone formation , but we did not find the previously inserted ptfe stent in the abdomen ( fig .
one patient underwent percutaneous transhepatic gallbladder drainage at 19 weeks due to acute cholecystitis , and that patient also underwent ptbd at 24 weeks due to overgrowth that was noted at 24 weeks .
the duration of the follow - up study for all the patients was 2 - 81 weeks ( mean : 27.9 weeks ) .
twenty - eight patients died during 2 - 81 weeks and nine patients lived until 43 - 80 weeks . according to the kaplan - meier survival analysis , the primary stent patency rates were 85% and 78% at 20 and 50 weeks , respectively ; the survival rates were 49% and 27% at 20 and 50 weeks , respectively ; and the mean survival and patency durations were 33 weeks and 64 weeks , respectively .
no procedure - related patient deaths were identified , and all the deaths were attributed to the patients ' natural course of disease .
the covered stents showed patent lumens in 28 patients ; this was confirmed by such laboratory values as the bilirubin levels at the final follow - up .
the reported follow - up durations and patency rates of covered stents in the medical literature have varied ( 10 - 19 ) hausegger et al .
( 10 ) have reported that the patency rates after 1 , 3 , 6 and 12 months were 96% , 69% , 47% and 31% , respectively .
rossi et al . ( 16 ) have reported primary patency rates after 3 , 6 and 9 months of 72% , 46% and 46% , respectively .
both of these investigations ( 10 , 16 ) did n't report good patency rates because their stent occlusion rates were somewhat high ( 33 - 37% ) .
( 12 ) reported that the mean patency period of their stents was 37.5 weeks .
( 19 ) , in a large series of covered stents , reported the primary patency rates at 3 , 6 and 12 months to be 90% , 76% and 76% , respectively , and we previously reported stent patency rates of 71% and 48% at 20 and 50 weeks , respectively ( 20 ) . in this present study ,
the primary and stent patency rates were 85% , and 78% at 20 and 50 weeks , respectively .
( 14 ) have reported good patency rates that were the result of lower stent occlusion rates . however
, making comparison with those studies is difficult because each of their patients was treated at a different stage of disease , and the tumors differed for their histologic type , cellular growth and biological behavior ( 12 ) .
it is resistant to bacterial growth , and this reduces the risk of occlusion caused by bile incrustation ( 18 , 22 ) .
moreover , ptfe resists chemicals ( acids and alkalies ) and heat , and ptfe is self - lubricating and not prone to demolding .
moreover , although ptfe has a low coefficient of friction and therefore good patency , it has poor adhesion to other materials ( 23 ) . in the present study ,
first , a 9-fr introducer system was used for implanting the ptfe - covered stents , which is a much smaller system than that required for the schoder et al .
second , in order to prevent stent migration , the end portion of the stent and the outside of the stent are not coated with ptfe .
third , the stent is designed to be covered with ptfe on the inner aspect of the stent .
the stent wire surrounds the ptfe , and so the stent 's outer surface is similar to that of an uncovered stent . in our study ,
the disadvantage of the eptfe / fep - covered stent is that the fabric covering of the expanded stent may cause blockage of the cystic or pancreatic duct , and this can result in possible cholecystitis or pancreatitis ( 18 , 19 ) . in our study , three patients ( 3/37 , 8% ) underwent percutaneous transhepatic gallbladder drainage due to acute cholecystitis .
the most obvious disadvantage of the ptfe stent was that stent insertion was followed by marked elevation in the amylase and lipase levels one day after insertion ; these levels subsequently decreased and then fell to normalized levels within seven days .
the other disadvantage of the stent is the occurrence of stent obstruction due to sludge formation . in our opinion , this elevation of the amylase and lipase levels was caused by pancreatic sphincter hypertension because there was no space between the stent and the sphincter of oddi , as seen on post - endoscopic retrograde cholangiopancreatography ( ercp ) .
the level of the lesion was the proximal cbd in one patient , the mid cbd in two patients and the distal cbd in eight patients .
the cause of disease was cbd carcinoma in three patients , gastric carcinoma in three patients , ampulla of vater carcinoma in two patients and pancreatic head carcinoma in three patients .
the second disadvantage of stenting is the possibility of stent occlusion due to food reflux from the duodenum into the proximal common bile duct .
there are many studies that have made comparisons between covered and uncovered stents ( 24 - 26 ) .
reported have that covered stents successfully prevented tumor ingrowth and they were significantly superior to uncovered stents for treating those patients suffering with distal malignant biliary obstruction ( 24 ) .
stent occlusion occurred in covered stents for eight patients ( 14% ) after a mean of 304 days ( 24 ) .
yoon et al . reported that the stent patency rates were 83% , 78% , 67% and 54% at 100 , 200 , 300 and 400 days , respectively , for covered stents and 83% , 66% , 54% and 36% , respectively , for uncovered stents , which was not significantly different ( 25 ) .
park et al . have reported that the mean stent patency was 148.9 days for covered stents and 143.5 days for uncovered stents .
migration of stent occurred in 0.6% of the patients and sludge formation in stents occurred in 20% of the patients in the above study . in our study , the covered stent showed similar patency rates , a high incidence of mild pancreatitis and a low incidence of sludge formation and stent migration .
one patient had tumor ingrowth in the mid portion of their stent ; however , the image finding was not confirmed by choledochoscopy .
tumor ingrowth may not occur in ptfe materials because the ptfe itself is a strong barrier .
if it does occurs , it would be due to damage or natural degradation of ptfe . in conclusion ,
the ptfe - covered self - expandable nitinol stent is safe with acceptable complication rates .
this study is similar to previous studies in regard to comparing between the patency rates and the survival rates . using the ptfe - covered self - expandable nitinol stent for treatingmalignant biliary obstruction | objectivewe wanted to determine the technical and clinical efficacy of using a ptfe - covered self - expandable nitinol stent for the palliative treatment of malignant biliary obstruction.materials and methodsthirty - seven patients with common bile duct strictures caused by malignant disease were treated by placing a total of 37 nitinol ptfe stents .
these stents were covered with ptfe with the exception of the last 5 mm at each end ; the stent had an unconstrained diameter of 10 mm and a total length of 50 - 80 mm .
the patient survival rate and stent patency rate were calculated by performing kaplan - meier survival analysis .
the bilirubin , serum amylase and lipase levels before and after stent placement were measured and then compared using a wilcoxon signed - rank test .
the average follow - up duration was 27.9 weeks ( range : 2 - 81 weeks).resultsplacement was successful in all cases .
seventy - six percent of the patients ( 28/37 ) experienced adequate palliative drainage for the remainder of their lives .
there were no immediate complications .
three patients demonstrated stent sludge occlusion that required ptbd ( percutaneous transhepatic biliary drainage ) irrigation .
two patients experienced delayed stent migration with stone formation at 7 and 27 weeks of follow - up , respectively .
stent insertion resulted in acute elevations of the amylase and lipase levels one day after stent insertion in 11 patients in spite of performing endoscopic sphincterotomy ( 4/6 ) .
the bilirubin levels were significantly reduced one week after stent insertion ( p < 0.01 ) .
the 30-day mortality rate was 8% ( 3/37 ) , and the survival rates were 49% and 27% at 20 and 50 weeks , respectively .
the primary stent patency rates were 85% , and 78% at 20 and 50 weeks , respectively.conclusionthe ptfe - covered self - expandable nitinol stent is safe to use with acceptable complication rates .
this study is similar to the previous studies with regard to comparing the patency rates and survival rates . |
randomized , controlled clinical studies are considered the gold standard for the evaluation of efficacy , safety , and tolerability of pharmaceutical drugs.1,2 such studies allow for rigorous assessment of these parameters in controlled settings that prevent potential confounds when observing a drug 's effect(s).3 these studies , however , have important characteristics that may limit the generalizability of results , including evaluation of efficacy , safety , and/or tolerability in select patient populations with well - defined and often limited medical comorbidities . these include underrepresentation of the elderly and women , patient selection bias , restrictions regarding concomitant medications , and intensive medical follow - up that is generally not possible in routine clinical care.1,2,4
such limitations have prompted the use of alternative complementary approaches to assess drug treatment outcomes in a clinical practice setting,4 including the assessment of medical claims data , establishment of patient registries , patient surveys , and retrospective review of patient charts from a clinical practice setting .
each of these methods has been employed to evaluate the benefits and risks associated with specific treatments for patients with chronic hepatitis c virus ( hcv ) infection,511 thereby providing valuable information on factors influencing treatment adherence , underrepresentation of patients in clinical studies , economic burden of treatment , management of patients with hcv infection , and treatment outcomes .
the efficacy , safety , and tolerability of telaprevir , an ns3/4a protease inhibitor , in combination with peginterferon alfa and ribavirin ( pr ) have been evaluated in three randomized , well - controlled , phase 3 clinical studies in patients with genotype 1 chronic hcv infection .
the advance12 and illuminate13 studies enrolled treatment - nave patients , and the realize study14 enrolled patients who had not achieved sustained virologic response ( svr ) with prior combination therapy with pr .
telaprevir administered in combination with pr for 12 weeks , followed by 12 or 36 weeks of pr alone , was associated with significantly improved svr rates versus pr alone in treatment - nave ( 74%79% versus 46% ) and previously treated ( 32%86% versus 5%22% ) patients.15 adverse events ( aes ) that occurred more often ( 5% higher frequency ) with telaprevir and pr compared with pr alone included rash ( 56% vs 34% , respectively ) , fatigue ( 56% vs 50% ) , pruritus ( 47% vs 28% ) , nausea ( 39% vs 28% ) , anemia ( 36% vs 17% ) , diarrhea ( 26% vs 17% ) , vomiting ( 13% vs 8% ) , hemorrhoids ( 12% vs 3% ) , anorectal discomfort ( 11% vs 3% ) , dysgeusia ( 10% vs 3% ) , and anal pruritus ( 6% vs 1%).12,14,15 serious skin reactions , including drug reaction with eosinophilia and systemic symptoms and stevens - johnson syndrome , were reported in < 1% of patients who received telaprevir and pr.15 all three phase 3 studies of telaprevir were controlled , with randomized , double - blind study designs , well - defined inclusion and exclusion criteria , and careful patient follow - up , and each study provided important data on treatment response with telaprevir and pr for patients with hcv infection . however , as with all randomized , controlled clinical studies , the study designs inherently limit the generalizability of the results to real - world clinical practice settings . in particular , the rigorous entry criteria defined for these studies excluded adolescents and patients older than 70 years , those with human immunodeficiency virus or hepatitis b virus co - infection , and those with blood chemistry and hematology values that were outside normal limits . to date , there is little information available regarding the similarities and differences between patients enrolled in the phase 3 studies of telaprevir and those receiving telaprevir in a clinical practice setting .
this post - marketing , retrospective study reviewed patient charts obtained from academic and community treatment centers and was conducted to evaluate prespecified baseline characteristics and medical follow - up data during the telaprevir treatment phase for patients in the us treated with telaprevir and pr . while not designed to make direct statistical comparisons , this study aimed to assess general similarities and/or differences between the patient populations in the phase 3 studies of telaprevir and those patients treated with telaprevir in real - world clinical practice settings since marketing approval was granted .
this retrospective study was conducted at four academic and three community treatment centers in the us that were selected based on physicians ' clinical experience and involvement with research activities in chronic hcv infection , geographic diversity , and the number of patient charts available for review .
patient charts from academic centers were provided by multiple physicians per site , whereas charts from community centers were provided by a sole physician .
originally , the study protocol planned for 50 consecutive patient charts to be reviewed at each site . however , some sites provided fewer charts than planned and others were permitted to provide more charts to compensate .
the study was conducted in accordance with the current good clinical practice guidelines of the international conference on harmonization , the principles of the declaration of helsinki , and local applicable laws and regulations .
the institutional review board ( irb ) or independent ethics committee of each participating center ( baylor research institute irb , northwestern university biomedical irb , scripps office for the protection of research subjects irb , temple university office for human subjects protections irb , and western irb ) reviewed and approved the protocol and its revisions . because no procedures were performed on patients and their personal information was de - identified , the institutional review boards or independent ethics committees of all participating centers waived the need for written informed consent from the patients .
charts for the first 50 patients with genotype 1 chronic hcv infection who received telaprevir and pr at one of seven us treatment centers shortly after marketing approval of telaprevir was granted were used ( all patients began therapy between may and september 2011 ) .
some sites were permitted to provide more charts ( no more than 70 consecutive charts ) to compensate for sites that provided fewer than 50 charts .
patient charts were required to include records for at least one dose of telaprevir with pr followed by at least 12 weeks of follow - up .
charts from patients who received treatment with telaprevir during participation in a randomized clinical study were not included .
case report forms were provided to participating sites , and all appropriate patient data were recorded on these forms . to protect patient confidentiality , data collection
was conducted by study site personnel , as designated by the study sponsor , who were trained by the developers of the case report form on the collection of data .
if any data required in the case report form were not available for a given patient , this was indicated on the case report form , and those charts were not excluded because of missing data .
baseline information collected from the patient charts , when available , included sex , age , body mass index , race , ethnicity , medical history , hcv infection history ( time since diagnosis , hcv genotype and viral load , interleukin 28b [ il28b ] genotype , stage of liver fibrosis , and response to prior hcv treatment ) , as well as hematology and clinical chemistry values and aes at baseline and/or on treatment . in some instances ,
baseline blood work was obtained as many as three months prior to initiation of therapy .
key on - treatment data extracted from the patient charts , when available , included telaprevir dosing , including any modifications for anemia , rash , pruritus , or anorectal adverse events ; pr dosing , including modifications based on the above - listed events ; transfusions or administration of erythropoietin - stimulating agents ; concomitant medications ; and aes and serious aes ( saes ) . modifications to telaprevir and/or pr dosing were made at the discretion of the treatment center .
data regarding hospitalizations , clinical chemistry , hematology , and hcv rna levels were also collected , when available .
available information for aes , as interpreted by study site personnel , included classification as serious or not serious , dates of onset and resolution , severity , causal relation to study drug(s ) , action taken , concomitant medications or other treatments given , and outcomes as typically reported during the conduct of clinical studies .
results from 1,797 treatment - nave and -experienced patients who were assigned to 8 weeks ( n=364 ; not an approved treatment regimen ) or 12 weeks ( n=1433 ; approved treatment regimen ) of treatment with telaprevir and pr followed by 12 or 36 weeks of pr alone ( total treatment duration of 24 or 48 weeks ) were pooled , when possible , from the three phase 3 studies of telaprevir . in these phase 3 studies ,
telaprevir was administered at 750 mg every 8 hours ; the peginterferon alfa-2a dose was 180 micrograms per week , and the ribavirin dose was 1,000 mg per day ( patients weighing < 75 kg ) or 1,200 mg per day ( patients weighing 75 kg ) .
the post hoc review of data for these patients aimed to match information collected in the chart review and included baseline demographic characteristics ( sex , age , body mass index , race , and ethnicity ) , medical history , hcv infection history ( time since diagnosis , hcv genotype and baseline viral load , il28b genotype , stage of liver fibrosis at baseline , and response to prior hcv therapy ) , as well as hematology and clinical chemistry values and aes at baseline and/or on treatment . whenever possible , general comparisons were made with pooled data from the phase 3 studies of telaprevir , but no statistical analyses were performed .
this retrospective study was conducted at four academic and three community treatment centers in the us that were selected based on physicians ' clinical experience and involvement with research activities in chronic hcv infection , geographic diversity , and the number of patient charts available for review .
patient charts from academic centers were provided by multiple physicians per site , whereas charts from community centers were provided by a sole physician .
originally , the study protocol planned for 50 consecutive patient charts to be reviewed at each site . however , some sites provided fewer charts than planned and others were permitted to provide more charts to compensate .
the study was conducted in accordance with the current good clinical practice guidelines of the international conference on harmonization , the principles of the declaration of helsinki , and local applicable laws and regulations .
the institutional review board ( irb ) or independent ethics committee of each participating center ( baylor research institute irb , northwestern university biomedical irb , scripps office for the protection of research subjects irb , temple university office for human subjects protections irb , and western irb ) reviewed and approved the protocol and its revisions . because no procedures were performed on patients and their personal information was de - identified , the institutional review boards or independent ethics committees of all participating centers waived the need for written informed consent from the patients .
charts for the first 50 patients with genotype 1 chronic hcv infection who received telaprevir and pr at one of seven us treatment centers shortly after marketing approval of telaprevir was granted were used ( all patients began therapy between may and september 2011 ) .
some sites were permitted to provide more charts ( no more than 70 consecutive charts ) to compensate for sites that provided fewer than 50 charts .
patient charts were required to include records for at least one dose of telaprevir with pr followed by at least 12 weeks of follow - up .
charts from patients who received treatment with telaprevir during participation in a randomized clinical study were not included .
case report forms were provided to participating sites , and all appropriate patient data were recorded on these forms . to protect patient confidentiality , data collection
was conducted by study site personnel , as designated by the study sponsor , who were trained by the developers of the case report form on the collection of data .
if any data required in the case report form were not available for a given patient , this was indicated on the case report form , and those charts were not excluded because of missing data .
baseline information collected from the patient charts , when available , included sex , age , body mass index , race , ethnicity , medical history , hcv infection history ( time since diagnosis , hcv genotype and viral load , interleukin 28b [ il28b ] genotype , stage of liver fibrosis , and response to prior hcv treatment ) , as well as hematology and clinical chemistry values and aes at baseline and/or on treatment . in some instances , baseline blood work was obtained as many as three months prior to initiation of therapy .
key on - treatment data extracted from the patient charts , when available , included telaprevir dosing , including any modifications for anemia , rash , pruritus , or anorectal adverse events ; pr dosing , including modifications based on the above - listed events ; transfusions or administration of erythropoietin - stimulating agents ; concomitant medications ; and aes and serious aes ( saes ) . modifications to telaprevir and/or pr dosing were made at the discretion of the treatment center .
data regarding hospitalizations , clinical chemistry , hematology , and hcv rna levels were also collected , when available .
available information for aes , as interpreted by study site personnel , included classification as serious or not serious , dates of onset and resolution , severity , causal relation to study drug(s ) , action taken , concomitant medications or other treatments given , and outcomes as typically reported during the conduct of clinical studies .
results from 1,797 treatment - nave and -experienced patients who were assigned to 8 weeks ( n=364 ; not an approved treatment regimen ) or 12 weeks ( n=1433 ; approved treatment regimen ) of treatment with telaprevir and pr followed by 12 or 36 weeks of pr alone ( total treatment duration of 24 or 48 weeks ) were pooled , when possible , from the three phase 3 studies of telaprevir . in these phase 3 studies ,
telaprevir was administered at 750 mg every 8 hours ; the peginterferon alfa-2a dose was 180 micrograms per week , and the ribavirin dose was 1,000 mg per day ( patients weighing < 75 kg ) or 1,200 mg per day ( patients weighing 75 kg ) .
the post hoc review of data for these patients aimed to match information collected in the chart review and included baseline demographic characteristics ( sex , age , body mass index , race , and ethnicity ) , medical history , hcv infection history ( time since diagnosis , hcv genotype and baseline viral load , il28b genotype , stage of liver fibrosis at baseline , and response to prior hcv therapy ) , as well as hematology and clinical chemistry values and aes at baseline and/or on treatment .
whenever possible , general comparisons were made with pooled data from the phase 3 studies of telaprevir , but no statistical analyses were performed .
in total , 338 patient charts containing records for patients receiving telaprevir and pr with at least 12 weeks of follow - up were included in the study . of these ,
210 ( 62% ) charts were from patients treated at academic treatment centers , and 128 ( 38% ) were from patients treated at community treatment centers .
of these 338 patients , 269 ( 79.6% ) had completed the recommended 12 weeks of treatment with telaprevir and pr .
the most common reason for treatment discontinuation , which occurred in 69 ( 20.4% ) patients , was aes ( table 1 ) .
almost half of the discontinuations in the clinical practice group ( n=32 ) were attributable to aes .
baseline and demographic characteristics of the patients from clinical practice and patients from the phase 3 studies of telaprevir are shown in table 2 .
patients included in the clinical practice group had a mean age of 55 years , were predominantly male ( 58.9% ) and white ( 79.3% ) , and had been diagnosed with hcv infection for a mean of nine years ; 35.5% of patients had cirrhosis at baseline .
only 30.2% of patients in the clinical practice group had data available for il28b genotype ( 8.0% cc , 14.5% ct , and 7.7% tt ) .
medical comorbidities were common , with hypertension ( 36.4% ) and depression ( 30.2% ) being the two most frequently observed comorbid conditions in this patient population ( table 3 ) . includes patients who were assigned to 8 or 12 weeks of treatment with telaprevir in combination with peginterferon alfa and ribavirin . from the illuminate study.13
bmi , body mass index ;
hcv , hepatitis c virus ; il28b , interleukin 28b ; n / a , not applicable ; pr , peginterferon alfa and ribavirin ; rna , ribonucleic acid ; sd , standard deviation . includes patients who were assigned to 8 or 12 weeks of treatment with telaprevir and peginterferon alfa and ribavirin .
includes data for treatment - nave patients enrolled in the advance and illuminate studies only .
data for treatment - experienced patients enrolled in the realize study were excluded because the source data collection and dictionary encoding methods for comorbid medical conditions were different for the realize study , resulting in different classifications for the medical conditions and preventing pooling of the data .
of the 338 patients in the clinical practice group , 187 ( 55.3% ) had been previously treated with pr therapy ; of those , 80 ( 23.7% ) had a null response to prior treatment .
most patient charts included information regarding the frequency of telaprevir dosing ; 257 ( 76.0% ) and 73 ( 21.6% ) patients received telaprevir three times daily and twice daily , respectively . selected baseline hematology and clinical chemistry variables , including mean hemoglobin , albumin , total bilirubin levels , platelet , and absolute neutrophil counts , are shown in table 4 for patients in the clinical practice group and in the phase 3 studies of telaprevir . in the clinical practice group , 31 ( 9.2% ) patients had baseline hemoglobin levels below the lower limits required per protocol ( 130
g / l for females ) for enrollment in the phase 3 studies of telaprevir .
the mean reductions in hemoglobin levels for patients in the clinical practice group and in the phase 3 studies of telaprevir over the first 12 weeks of treatment are shown in fig .
overall , mean reductions during the first four weeks and the entire 12 weeks of treatment were similar . in the clinical practice group ,
patients who began treatment with telaprevir and pr with other baseline hematology and clinical chemistry values below the per - protocol minimum criteria set for the phase 3 studies of telaprevir were as follows : 18 ( 5.3% ) patients with albumin levels <33 g / l , 9 ( 2.7% ) patients with total bilirubin levels > 38 mol / l , 39 ( 11.5% ) patients with platelet counts < 90 10/l , 19 ( 5.6% ) patients with absolute neutrophil counts < 1.5 10/l , and three ( 0.9% ) patients with thyroid - stimulating hormone levels > 5.6 miu / l ( table 4 ) . among patients who had platelet counts
< 90 10/l , 11 ( 28.2% ) had platelet counts < 60 10/l .
includes patients who were assigned to 8 or 12 weeks of treatment with telaprevir in combination with peginterferon alfa and ribavirin .
bl , baseline . includes patients who were assigned to 8 or 12 weeks of treatment with telaprevir in combination with peginterferon alfa and ribavirin . per - protocol requirement for phase 3 study entry : 130 g / l ( males ) ; 120 g / l ( females ) per - protocol requirement for phase 3 study entry : 33
mol / l per - protocol requirement for phase 3 study entry : 9010/l per - protocol requirement for phase 3 study entry : 1.510/l per - protocol requirement for phase 3 study entry : 5.6 miu / l
overall , aes were reported by 329 of the 338 ( 97.3% ) patients in the clinical practice group .
four patients in the clinical practice group experienced life - threatening aes ( defined as a patient being at immediate risk of death from the event as it occurred ) that included hepatorenal syndrome , rectal cancer , and pneumonia in three patients ( n=1 each ) and mental status change , respiratory distress , and septic shock in one patient .
three ( 0.9% ) of these four patients died , with their deaths attributed separately to pneumonia , septic shock , and hepatorenal syndrome .
thirty - nine patients ( 11.5% ) reported aes rated as severe in intensity , and the most common ( occurring in 2 patients ) were anemia ( n=15 , 4.4% ) , rash ( n=6 , 1.8% ) , thrombocytopenia ( n=4 , 1.2% ) , neutropenia ( n=2 , 0.6% ) , pruritus ( n=2 , 0.6% ) , anxiety ( n=2 , 0.6% ) , and syncope ( n=2 , 0.6% ) .
aes experienced by 20% of patients that occurred at a rate of at least 5% higher in the clinical practice group than the phase 3 pooled data were anemia and dyspepsia .
those that occurred at least 5% higher in the phase 3 pooled data than the clinical practice group were pruritus , headache , pyrexia , and influenza - like illness .
a total of 70 saes occurred in 40 ( 11.8% ) patients in the clinical practice group ; 48 of these saes were considered to be related to treatment in 31 ( 9.2% ) patients .
the most commonly reported saes were anemia and rash , occurring in seven ( 2.1% ) and six ( 1.8% ) patients , respectively ( table 6 ) .
there was also one ( 0.3% ) patient with an sae of drug reaction with eosinophilia and systemic symptoms ; no saes of stevens - johnson syndrome or toxic epidermal necrolysis were reported .
fifty - five percent ( n=22 ) of the patients who experienced saes had documented baseline cirrhosis .
hospitalizations during the treatment period occurred in 38 ( 11.2% ) patients ; of these hospitalizations , 26 were determined to be related to telaprevir and pr treatment , with 11 due to the need for blood transfusions .
includes patients who were assigned to 8 or 12 weeks of treatment with telaprevir in combination with peginterferon alfa and ribavirin .
includes patients who were assigned to 8 or 12 weeks of treatment with telaprevir in combination with peginterferon alfa and ribavirin .
telaprevir treatment was interrupted in 17 ( 5% ) patients in the clinical practice group due to aes .
telaprevir doses were modified because of anemia in five ( 1.5% ) patients , rash in 10 ( 3.0% ) patients , pruritus in five ( 1.5% ) patients , anorectal disorder in zero patients , and other reasons in 20 ( 5.9% ) patients .
peginterferon doses were modified because of anemia in 21 ( 6.2% ) patients , rash in seven ( 2.1% ) patients , pruritus in 2 ( 0.6% ) patients , anorectal disorder in one ( 0.3% ) patient , and other reasons in 57 ( 16.9% ) patients .
ribavirin doses were modified due to anemia in 153 ( 45.3% ) patients , rash in 21 ( 6.2% ) patients , pruritus in seven ( 2.1% ) patients , anorectal disorder in three ( 0.9% ) patients , and other reasons in 39 ( 11.5% ) patients . during treatment with telaprevir and pr , 114 ( 33.7% ) and 23 ( 6.8% ) patients received erythropoietin and blood transfusions , respectively .
this retrospective study of real - life clinical patients who received telaprevir and pr for chronic hcv infection provides valuable insight into the baseline characteristics and safety and tolerability outcomes of the population of treated patients that may not be represented within the controlled setting of a randomized study .
the composite baseline patient profiles in this current study may be considered more difficult to treat than those in the phase 3 studies , as many patients were older ( mean age , 55 vs 49 years ) , a greater proportion had cirrhosis ( 36% vs 14% ) , and more patients were treatment experienced ( 55% vs 29% ) .
in addition , a substantial percentage of patients treated with telaprevir and pr in the clinical practice group would not have met the per - protocol minimum baseline clinical chemistry and hematology criteria for inclusion in the phase 3 studies .
although this would suggest that such patients may have been excluded from the phase 3 studies , it is important to note that there were occasional protocol deviations in the phase 3 studies regarding clinical chemistry and hematology inclusion criteria . in the clinical practice group , the treatment of patients with relatively low levels of hemoglobin , platelets , and albumin suggests that physicians in clinical practice may feel an urgency to treat these patients .
additionally , patients included in the current study had comorbid conditions , including depression , hypothyroidism , asthma , thrombocytopenia , and esophageal varices , which potentially could have excluded them from participation in the phase 3 studies of telaprevir .
many of these observed differences in patient characteristics likely contributed to some of the differences observed in rates of aes , particularly anemia . since the approval of telaprevir in 2011
, telaprevir - based therapy has been purported to be less well tolerated than was expected given the data from the phase 3 studies . in the current study , almost 80% of patients completed the recommended 12-week period of telaprevir and pr treatment , with 10% of patients discontinuing treatment because of an ae .
this suggests that aes experienced by patients in a clinical practice setting were generally manageable , and most of the reported aes were characterized as mild or moderate .
anemia , rash , and pruritus were among the most common aes associated with the use of telaprevir , which is consistent with data from the phase 3 studies.1214 the incidence of pruritus , headache , pyrexia , and influenza - like illness was higher in patients in the phase 3 studies , while the incidence of anemia and dyspnea was higher in patients in the clinical practice group . while 60% of clinical practice patients experienced anemia as an ae , 33% of patients did so in the phase 3 studies .
however , the proportion of patients who reported anemia as an sae was similar between both groups ( 2.1% in the clinical practice group vs 1.8% in the phase 3 studies ) .
in addition , while the pattern of reduction in hemoglobin levels during telaprevir treatment was similar between both groups , the mean levels were lower in patients in the clinical practice group .
it appears that incident anemia was managed successfully in most cases with ribavirin dose reductions and erythropoietin supplementation ; blood transfusions were thought to be necessary occasionally .
relatively few saes ( n=15 ) were associated with anemia , pruritus , or rash . in the clinical practice group ,
. the increased rate of saes observed in patients with cirrhosis is consistent with preliminary findings from the french compassionate use of protease inhibitors in cirrhosis ( cupic ) study , an independent patient registry established in europe to observe patients undergoing hcv treatment in the clinical setting.10,16 in the hcv - target study , a similar registry established in the us , patients with cirrhosis were at increased risk for discontinuing treatment early because of aes.6,17 these data highlight the need for careful and routine monitoring of patients with cirrhosis in the clinical setting . although an attempt was made to collect available data with respect to on - treatment efficacy outcomes , an insufficient number of patients had been assessed at the relevant time points or had an incomplete history for treatment outcomes , which prevented analysis and interpretation of the data .
however , the cupic study in europe10 and the hcv - target study in the us6,17,18 have been designed to collect data regarding on - treatment efficacy outcomes in the clinical setting .
retrospective reviews of patient charts provide information from readily accessible , existing data , are relatively inexpensive to conduct , may be important in hypothesis generation for prospective studies , and may allow the study of rare occurrences or conditions where there is a long latency period between exposure and disease .
however , limitations inherent to retrospective studies include incomplete documentation , problems in interpreting and verifying information ( e.g. , differences in terminology used by clinicians ) , variance in the quality of information as recorded by clinicians , and difficulty in determining cause and effect between treatment exposure and outcomes.19,20 the current study was performed at only seven sites that , despite the diversity in physicians ' clinical experience and involvement with research activities in chronic hcv infection , the geography , and the number of patient charts available for review , may not fully represent all treatment centers in which patients with chronic hcv infection are treated .
data were collected by site personnel who were trained by the developers of the case record form to record all appropriate patient data .
however , uniform approaches of data entry may not have occurred at all sites , and charts did not always contain complete patient information , resulting in some missing data . for example , while obesity was reported as a comorbidity in 6.8% of patients in the clinical practice group , the mean body mass index was 29 kg / m , highlighting the lack of uniformity in approaches to data entry . in some instances ,
baseline blood work was obtained as many as three months prior to the initiation of therapy .
furthermore , while most sites were associated with academic hospitals , which are more likely to test for il28b genotype and hcv subtype , only a few charts included test results for il28b genotype ( 30.2% ) , and the hcv 1 subtype was unknown in approximately 18% of patients . the limitations of obtaining these data highlight the variance in the types and quality of information recorded by clinicians , as well as the lack of a standardized approach in collecting such information .
lastly , while general comparisons were made between chart review data from the clinical practice group and the pooled data from the phase 3 studies of telaprevir , no statistical analyses were performed , as direct comparisons can not be made between studies that were conducted at different times , with different levels of rigor , and not designed in advance with the intent to demonstrate differences .
this retrospective study of real - life patients with chronic hcv infection treated with telaprevir provides insight into baseline patient demographics and clinical characteristics , as well as dosing and safety and tolerability profiles for telaprevir - based treatment in clinical practice settings .
compared to the phase 3 studies , in clinical practice , the composite baseline patient profiles were considered more difficult to treat and anemia was reported twice as often .
aes led to discontinuation of treatment in 10% of patients , suggesting that aes experienced by patients in a clinical setting are generally manageable .
findings from this study complement those reported from rigorous , randomized controlled studies with telaprevir - based treatment and provide a general assessment of the similarities and/or differences between patients from the phase 3 studies of telaprevir and those treated with telaprevir in real - world clinical practice . treatment with telaprevir - based regimens should be individualized based on both clinical practice observations and data from randomized controlled studies .
medical writing and coordination support were provided by erika d. reynoso , phd , who was an employee of vertex pharmaceuticals incorporated and may own stock or stock options in that company . medical writing , editorial , and graphic design support
was provided by bina j. patel , pharmd , of peloton advantage and was funded by vertex pharmaceuticals incorporated . | background and aimsthere is a paucity of information regarding similarities and differences between patients from the phase 3 studies of telaprevir and those receiving telaprevir in clinical practice.methodsthis retrospective chart review evaluated baseline characteristics and follow - up safety and tolerability data for patients with hepatitis c virus ( hcv ) infection treated with telaprevir and peginterferon alfa and ribavirin ( pr ) in clinical practice.resultsin total , 338 charts from patients at four academic and three community us treatment centers who received telaprevir and pr and had at least 12 weeks of follow - up data were included ; 62% were from academic centers and 38% were from community centers . of the 338 patients ,
269 completed 12 weeks of telaprevir and pr ; 32 discontinued due to adverse events .
mean age was 55 years ; patients were predominantly white ( 79.3% ) males ( 58.9% ) with genotype 1a hcv infection ( 61.8% ) ; 35.5% were reported to have cirrhosis at baseline ; and 55.3% previously received pr .
hypertension and depression were the most common comorbidities .
patient characteristics outside the per - protocol minimum criteria used in the phase 3 studies of telaprevir were , e.g. , hemoglobin , 9.2% ; albumin , 5.3% ; platelets , 11.5% ; and neutrophil count , 5.6% .
adverse events occurred in 329/338 ( 97.3% ) patients , with anemia , fatigue , nausea , and rash being the most common .
of 38 hospitalizations , 26 were deemed related to telaprevir and pr .
three patients died due to pneumonia , septic shock , and hepatorenal syndrome ( n=1 each).conclusionsthese findings complement those reported from rigorous , randomized controlled studies with telaprevir - based treatment and provide a general assessment of similarities and/or differences between patients from the phase 3 studies of telaprevir and those treated with telaprevir in clinical practice . |
health technology assessment ( hta ) is an effective policy tool in the evidence - based evaluation of health care technologies .
one of the most important components of any hta is its focus on ethical considerations of technologies .
this matter is both dissscussed as decision criteria and also as an ethical or value basis for all decision makings ( 1 - 4 ) .
previous studies , however , have shown that despite being completely stipulated , ethical issues are less considered in hta reports .
the difficulty and subjectivity inherent in ethical analyzes might be a reason for such a negligence ( 5 ) .
nevertheless , there have been great efforts to develop numerous tools to consider ethical aspects in htas ( 6 - 11 ) .
whether those tools are used or not is a worthy question to ask .
this study intends to review hta reports to describe methods of considering ethics in htas .
such a consideration may range from propounding of ethical issues to effects they might have on final decisions ; i.e. approaches and methods adopted , principles referred to and goals and objectives sought by ethical analysis and also the extent to which these have been exposed during decision - making process .
first , to reach to hta reports we searched and found hta institutions around the world .
so , our study focused on hta reports accessible by the internet and was limited to electronic sources in english .
to this end , we used google search engine by applying the following general terms :
health technology assessment or hta or technology appraisal .
to be more sensitive and to assume that in addition to aforementioned terms , there might be other relevant concepts and words too , we used other queries in refining and revising search strategies and efforts . after finding relevant hta websites ,
thus , hta full reports in english published in 2011 and with available and accessible were selected out .
in the next stage ,
lehoux and williams - jones model and the map were used to conduct a content analysis of hta reports .
the model and map apply for social and ethical issues that might emerge out of conditions inherent to technology use ; issues like doctor - patient relationship , direct or indirect effect on other stakeholders , broader societal issues ( such as conflict with social values and arrangements , human integrity and dignity ) , or issues concerning assessment methods ( such as choice of end points , moral consequences of the hta ) and , finally , issues of resource implications of technology dissemination ( such as distribution of and access to health care services and equity ) ( 6 - 11 ) .
authors assumed that based on a report contents , influence and implication of ethical considerations would be traceable if the issues raised could change the final decisions made , or affect the decision - making process ( e.g. observing
accountability for reasonableness as a moral approach to decision making ) , or add an ethical point to executive summary .
the general approach of the hta institutions to ethical aspects in hta based decision making was also analyzed .
most reports belonged to the united states and the uk and a few ones to sweden and ireland .
no ethical issue was brought up in 55 reports ( 61.8% ) . amongst the rest
34 reports , ethical issues about the nature of the technology , issues of doctor - patient relationship , issues relating to other stakeholders , social values , assessment methods , and equity were discussed in 3 , 3 , 3 , 4 , 0 and all 34 reports respectively . according to the text report , those ethical considerations influenced documented dialectics of decision making or final decision in 1 ( 33% ) , 1 ( 33% ) , 2 ( 66% ) , 1 ( 100% ) , 0 , 15 ( 44% ) reports respectively .
also , influencing final recommendation in terms of ethical points added to executive summary was boldly detectable in 16 reports ( table 1 ) .
however , it was not investigated whether other cases were relevant for a due consideration and change in decision making .
the most common ethical assessment considered was equity - related issues , though inadequately and incompletely .
nevertheless , equity concerns were not even brought forth in 55 reports ( 61.8% ) . amongst countries , these ethical aspects
were fully assessed and considered in all reports issued in ireland and sweden , with obvious effects on final decision statement ( table 2 ) .
nice ( national institute for health and clinical excellence ) of england had the highest number of hta reports accessible ( 26 reports ) .
equity - related issues , as a distinct component , were considered in all of nice technology appraisals ; equality impact assessment influenced final decision statement in 10 reports .
although issues of assessment methods were not considered in any report , nice process for hta - based decision making ( technology appraisal ) conformed with principles of accountability for reasonableness , which have 4 conditions for the legitimacy of a decision - making process : publicity , relevance , appeals , and enforcement ( 12 ) .
among hta reports in which ethical issues were somehow raised ( i.e. 38% of all reports in 2011 ) ,
equity issues were considered in 100% of the reports ; issues concerning societal values in 11.8% ; and issues of technology itself , of doctor - patient relationship , and of other stakeholders in 8.8% of the reports . from extent - of - influence viewpoint ,
equity issues were stated in final document of 15 cases ( 44.1% ) ; issues of other stakeholders in 2 final documents ( 5.9% ) ; and issues of societal values , of technology itself , and of doctor - patient relationship in only one final document ( 2.9% ) .
despite existence of various tools for consideration of ethical aspects in htas ( 6,7,21,22 )
, no reference was made to them in all hta reports retrieved in present study .
although there are numerous studies that point to little attention to ethics compared to other aspects in hta reports ( 23 - 26 ) , more detail about local approaches to and influence of ethical considerations on final decisions in hta reports is missing .
our study tried to answer the question that to what extent each type of ethical issue was considered and implicated in hta documents ?
findings showed that most attention was paid to equity issues while allocating the resources . amongst the countries ,
the highest moral considerations concerning evaluation methods ( ethical hta process ) was related to
accountability for reasonableness for legitimate decision - making in the uk . according to literature
, this might show that structural measures for moral decision - making process seem to be more effective .
some of this success might be attributable to the legal status of hta system and related recommendations in public services as quasi - law statements ( 27 ) .
therefore , it seems that institutionalization of ethical hta would not be possible only by adding a chapter on ethics to the hta reports .
again , it was observed that there are few hta reports with a complete approach to all aspects of ethical consideration .
although there are some accessible tools of addressing ethical and / or equity issues in health technology assessment , we could not judge on whether or not the tools was helpful and had any effect because no reports referred to them .
while few number of ethical issues included in hta reports has real influence on final hta decisions , those ethical considerations defined in the form of planned and written policy and institutionalized in a systematic way , are of the great need to truly influence hta decision makings .
it can be said in countries that are considering the ethical issues in their hta reports ( such as the uk , sweden and ireland ) , the approach is more focused on implications of equality and fairness in the allocation of resources .
the authors only managed to assess the content of hta reports in one year , those with accessible electronic english documents ; therefore , this sample may not represent all relevant hta reports .
moreover , in a review of hta based decision - making systems around the world , there might be some other mechanisms for ethical considerations that are not documented ( as technical evidence ) in the assessment phase of hta reports .
those mechanisms could have a potential influence on decision - making about technologies under question .
our study only focused on assessment consequences and not on other ethical issues that might be relevant to htas .
indeed , the authors could not evaluate subjectivity of ethical concerns about the technologies assessed in the reports . for future studies
, one can recommend that all the stages of assessment , especially scoping phase of the hta project , should be documented and analyzed .
a repeat of the present review for different years in order to assess the trend of attention to ethics in htas can be another recommendation . | background : dealing with ethical considerations is a major component of health technology assessment
( hta ) definitions .
objectives : present study aimed to explore and describe the manner of ethical analyses in hta reports and the
effects it had on hta - related decision making around the world .
methods : by considering the contextual milieu of reports and searching for ethical themes and subjects in
hta full reports , a descriptive analysis of hta reports ' contents and related processes was conducted .
the review
focused on all english hta reports issued in a year .
all ethical aspects , criteria , approaches , and also decision-
making related ethical issues were described and summarized in retrieved reports .
the inclusion of ethical
aspects in final decision - making criteria of hta reports was also considered .
results : eighty - nine hta reports issued in one year were included in this review and analyzed for ethical considerations .
there was no trace of any ethical issues in 60.7% of retrieved hta reports .
dimensions of equity in
resource distribution , stakeholder engagement , social values , essence and nature of technology , and ethical issues
about the method of assessment for decision making , and physician - patient relationship were raised and
discussed in 38.2% , 3.4% , 3.4% , 3.4% , 34.8% and 3.4% of reports respectively .
those issues were also included
in 44.1 , 5.9 , 2.9 , 2.9 , 0 and 2.9% of final reports , respectively . in overall , only in 16 cases ( 17.9% ) of all 89 reports ,
ethical issues were included in hta decision - making orientations .
conclusion : this review shows that ethical issues are occasionally raised and discussed in hta reports .
more
importantly , the inclusion of ethical concerns as a decision criterion in htas is few and insufficient . |
allergic diseases in western countries have steadily increased over the last 20 years , affecting the respiratory system , conjunctiva , and skin.1,2 moreover , an emergent aspect is the association of gastrointestinal involvement with respiratory and/or cutaneous symptoms , arising from allergies and/or intolerance .
gastroenteric expressions of these diseases include acute symptoms ( diarrhea or vomiting ) and chronic symptoms ( bloating , constipation which may alternate with diarrhea ) .
allergy tests , performed with commercial extracts or fresh foods , can be negative or slightly positive in most patients in both situations.3 allergy tests may also be negative in respiratory and cutaneous allergy , and this enables a distinction between , eg , allergic and nonallergic asthma , allergic and nonallergic rhinitis , and allergic and nonallergic dermatitis.4 different explanations for this increase in prevalence have been hypothesized , including genetic predisposition,5,6 environmental factors,7,8 or the hygiene hypothesis,9 but none of these possibilities seems to be completely satisfactory . in this context , since the role of immunoglobulin e ( ige ) antibodies is central to the development of allergies , factors related to the onset of symptoms must also include the possibility of helmintic infections , which induce high ige levels .
the ige increase is proportional to both the severity of infection and invasion of parasites in the tissues , and is the sum of the total ige level plus parasite - specific ige release.10,11 the latter derives from helmintic production of factors that stimulate interleukin-4 production.12 in particular , one study found an increase in circulating ige levels during parasitic infections caused by ascarids,13 while other studies demonstrated a total ige increase in parasitic diseases and their reduction after antiparasitic therapy,14 as well as high ige levels in patients affected by visceral larva migrans with antibodies against toxocara.15 the infestation caused by toxocara canis appears to be very frequent in tropical countries , but also in some areas in europe , especially among children in the first decade of life.16,17 the symptoms caused by toxocara infection in the different target organs in the respiratory , cutaneous , and gastrointestinal systems are often similar to the symptoms of allergic diseases .
the aim of the present study was to evaluate the prevalence of seropositivity to t. canis in a large population of patients referred for symptoms of suspected allergy .
between 2003 and 2008 , we evaluated 9985 new patients presenting with symptoms suggesting an allergic disease .
the clinical symptoms involved seasonal or persistent rhinoconjunctivitis , asthma , urticaria , angioedema , and dermatitis .
all patients underwent clinical examination , careful evaluation of symptoms , and allergy tests , including skin prick tests with inhaled or food allergen extracts and , in patients with dermatitis , patch tests with a standard panel of haptens ( merck , milan , italy ) .
skin prick tests were performed with the panel of allergen extracts from stallergenes , milan , italy .
routine and immunological blood tests and parasitological tests on feces were performed , as well as symptomatic therapy , environmental prophylaxis as preventive treatment , an exclusion diet when food allergy was suspected , and replacement of medication(s ) when a drug allergy was suspected .
a subgroup comprising 753 patients was selected who suffered from chronic recurrent respiratory , eye , skin or gastrointestinal symptoms caused by nonallergic mechanisms , as assessed by results to allergy tests negative or unrelated to clinical history , and these patients underwent further hematochemical and immunological blood tests , ie , enzyme - linked immunosorbent assay ( elisa ) , measured in optical density ( od ) with a range from 0.9 to 1.1 , and western blotting ( wb ) tests for igg antibodies to t. canis , using material from ltbio diagnostics , lyon , france .
these patients mainly suffered from asthma , urticaria , dermatitis , and conjunctivitis ; less frequently they suffered from gastroenteric symptoms , asthenia , headache , dizziness , or drug reactions .
the prevalence of the different clinical presentations was compared using the chi - squared test .
anthelmintic therapy was prescribed for t. canis seropositive patients , using mebendazole ( one 100 mg tablet twice daily for 3 days , 5 ml syrup twice daily in children ) , repeated after 20 days up to three times in an effort to achieve significant symptomatic improvement . in the event of lack of improvement ,
albendazole ( one 400 mg tablet twice daily for 5 days in adults , 5 mg / kg divided in two half doses in children ) was used , and repeated after two months .
these were mainly antihistamines or topical steroids for rhinitis symptoms , inhaled beta - agonists and corticosteroids for asthma symptoms , antihistamines , and topical or systemic corticosteroids for skin symptoms , including urticaria / angioedema , dermatitis and itching , and probiotics ( lactobacillus reuteri ) for intestinal symptoms .
regular control visits were scheduled , the first after 2 months and subsequently every 4 months .
patients who did not come back for the checkup visit were contacted by telephone about their symptoms .
clinical evaluations of symptoms and serological tests with the wb and elisa for antibodies to t. canis were performed in patients who came to the visits .
the study was registered ( eudract number 2010 - 024221 - 20 ) and approved by local authorities .
in the selected group of 753 patients suffering from chronic recurrent symptoms caused by nonallergic mechanisms , 240 ( 31.8% ) tested positive for t. canis by elisa , wb , or both tests ; 64 of them ( 26.7% ) were positive to elisa , 110 ( 45.8% ) to wb , and 66 ( 27.5% ) to both tests .
these patients included 222 adults ( 150 females , 72 males , age range 1872 years ) and 18 children ( seven females , 11 males , age range 217 years ) .
sixty - two of the 753 patients ( 25.8% ) had animals at home ( 28 had a cat , 23 had a dog , and 11 had both animals ) .
fifty - two patients ( 23.3% ) were atopic , with positive results to skin prick testing that were unrelated to clinical symptoms ; in these patients , the mean level of total ige was 280 ku / l . in the 188 nonatopic patients ,
asthma was significantly more frequent compared with the other presentations ( p = 0.029 versus urticaria , p = 0.002 versus dermatitis , p < 0.0001 versus each other presentation ) . among patients with asthma , five had level i , 30 had level ii , 16 had level iii , and eight patients had level iv disease according to the global initiative for asthma ( gina ) classification .
of the 240 t. canis seropositive patients , 162 ( 67.5% ) agreed to undergo anthelmintic therapy , which resulted in considerable improvement in symptoms .
eighty - eight patients did not come for the checkup visit , but they were contacted by phone and confirmed that they did not suffer from symptoms any longer and did not use any more symptomatic drugs . in 14 of the 74 patients who came back to the scheduled control visits , we observed both clinical and serological improvement in wb and elisa tests . in six of these patients , a decrease in the size of bands using wb was found ; in five patients , the size of bands using wb and the od using elisa decreased , and elisa od values decreased from > 1.1 to negative values in the range of 0.91.0 in three patients .
there was a complete remission of symptoms with complete absence of antibodies to t. canis in 26 of the 74 patients .
because we have previously observed cases of t. canis infection that simulated allergic manifestations and were mistakenly considered allergies for a long time,18 we systematically tested allergic patients referred for suspected allergy ( who had negative allergy tests or test results clearly unrelated to history ) for seropositivity to t. canis . of 753 patients with such characteristics , 240 ( 32% ) were positive using in vitro tests ( elisa or wb ) for igg antibodies to t. canis .
the role of t. canis infection in sustaining the symptoms was further demonstrated by the efficacy of anthelmintic treatment , which was evident in more than two thirds of treated patients and was associated with negativization or a decrease in toxocara igg antibodies in 43% of patients who underwent repeat elisa or wb .
human toxocariasis is a zoonosis sustained by parasitic infestation with the nematode t. canis belonging to the order of ascaris , which infects dogs , cats , and many other mammals .
this happens mainly among children when they ingest soil while playing or eating . in human hosts
, the larvae do not develop into adults , but they can migrate to different organs and tissues and survive for long periods of time , varying from a few months to decades , depending on the host immune response.19 the real incidence / prevalence of this infestation in humans has not yet been assessed , because the disease induces nonspecific symptoms
. however , sometimes it mimics a viral infection , and in the mildest cases can be totally asymptomatic .
the first one is called capsulated toxocariasis , which is characterized by very mild symptoms , with no clear clinical manifestations ( for example , nonspecific illness with fever , recurrent hacking cough , insomnia , abdominal pain , headache , and behavioral disorders ) . the second one is called ocular larva migrans , limited to the eye , and may cause a reduction in sight , conjunctival hyperemia , recurrent conjunctivitis , and even granulomas , uveitis , and retinal detachment .
the last one is visceral larva migrans , and its symptoms include fever , asthenia , behavioral depression ( suggesting central nervous system involvement ) , skin afflictions like nodules , chronic hives , recurrent labial and eyelid angioedema and eczematous dermatitis , enteric symptoms such as meteorism and abdominal pain , respiratory symptoms such as dyspnea , persistent cough , recurrent asthmatic and bronchitic manifestations , pleuritis , and bronchopneumonia.17,19 epidemiological studies conducted in europe have shown variable prevalence of seropositivity for t. canis . in spain , 14% of both pediatric and adult patients were found to suffer from eosinophilia , and 6% of these patients showed eye disorders .
20 in sweden , the prevalence of seropositivity was about 7% , indicating a relevant presence of subclinical toxocariasis . in this group ,
eosinophilia was recognized in 32% of the sample , and the most common clinical manifestation was eye problems ( 46% ) while neurological , respiratory and hepatic symptoms were described in 40% of these patients.21 in an italian study that included 2112 healthy adult subjects , the prevalence of t. canis antibody seropositivity was 3.9% .
the percentage increased in the control group of 471 nonhealthy subjects ( 257 affected by epilepsy , 142 by oligophrenia , 76 by strongyloides stercoralis infestation ) , and was 4.35% in epilepsy patients , 9.2% in patients with strongyloidiasis , and 10.6%14.5% in subjects with oligophrenia .
22 thirty - one of 100 toxocara - positive pediatric patients suffered from asthma or bronchitis , while the percentage decreased to only 12% among toxocara - seronegative patients in a study performed in the netherlands.23 toxocara infestation mainly occurs via accidental ingestion of soil , that is why the illness usually affects children , but adults can also be infected . as stated above , the larvae do not develop in the human host but only migrate to different organs and tissues , where they survive for a certain time period depending on the level of the host immune response .
. they may even cause symptoms similar to viral infections or allergy - like symptoms , ie , may present with the clinical expression of allergy .
symptoms can be mild , including fever , recurrent hacking cough , dyspnea , recurrent bronchitis , pleuritis , bronchopneumonia , asthma , skin afflictions like nodules and chronic hives , recurrent labial and eyelid angioedema and eczematous dermatitis , enteric symptoms , or even symptoms limited to the eye causing conjunctiva hyperemia and recurrent conjunctivitis.18,19 most of these symptoms were present in the population we studied , in particular asthma , which was significantly more frequent than any other clinical presentation in patients with positive in vitro tests for toxocara .
this reinforces the suggestion made by cooper that t. canis is an important and neglected environmental risk factor for asthma.24 the role of t. canis infection may be assessed by the detection of specific igg antibodies , it being known that the igg4 subclass is predominantly involved in the response to parasitic infections.25
the findings from this study indicate that t. canis infection , as assessed by seropositivity of igg antibodies by elisa or wb , is not rare in patients presenting with symptoms of apparent allergy but with negative or inconclusive allergy tests .
this suggests that serological tests should be performed to detect toxocara - specific igg antibodies in such patients and to assess the effect of anthelmintic treatment , in an effort to diagnose parasitic infection correctly in patients who are otherwise given a simple ( but incorrect ) diagnosis of nonallergic disease . | backgroundtoxocara canis is an intestinal nematode affecting dogs and cats , which causes human infection when embryonated eggs excreted in dog feces are ingested .
humans are paratenic hosts .
although the larvae do not develop into adult worms in the human body , they may migrate to various tissues and organs where they can survive for several years , giving rise to several clinical symptoms , which can present in allergy - like form.methodsover 5 years , we examined 9985 patients referred for suspected allergies , based on symptoms such as dermatitis , urticaria , rhinitis , asthma , and conjunctivitis ; 753 patients who had allergy tests negative or unrelated to clinical history were tested for seropositivity to t. canis by enzyme - linked immunosorbent assay ( elisa ) or western blotting ( wb).resultsin 240 patients ( 31.8% ) , elisa or wb or both tests were positive for t. canis immunoglobulin g ( igg ) antibodies : in particular , 64 of them ( 26.7% ) were positive to elisa , 110 ( 45.8% ) to wb , and 66 ( 27.5% ) to both tests .
asthma was the most common clinical presentation .
two thirds of patients underwent subsequent anthelmintic therapy and showed a complete remission of symptoms and , in 43% of patients retested by elisa and wb , became negative to toxocara.conclusionthese findings strongly suggest that t. canis plays a significant role in inducing chronic symptoms presenting as suspected allergies . |
herpes zoster typically affects the thoracic and lumbar vertebra ( t3-l3 ) , and sacral herpes zoster is found in only 4% of cases .
it usually occurs on one side of the sacrum , and in less than 1% of cases , it occurs on both sides .
this dysfunction is experienced by only 4% of herpes zoster patients , but it can happen if the virus affects the sacral nerve . voiding dysfunctions caused by herpes zoster have also been reported in korea .
however , the cases identified so far are limited to elderly people or patients with a deteriorated immune system , and no such occurrence has been found among young adults . against this background ,
we present the case of a 25-year - old man with a normal immune system who was hospitalized for scrub typhus and experienced herpes zoster on both sides of the sacral nerve .
a 25-year - old man was hospitalized after seven continuous days of fever , chills , and a general feeling of weakness .
his blood pressure was 140/70 mmhg , his pulse rate was 90 beats a minute , his respiratory rate was 20 breaths per minute , and his body temperature was 39.2. the patient reported feeling fatigued , pain upon urination , and pain in the anal area during urination and bowel movements . a skin rash was observed on the right side of the body , which was probably a eschar ( fig .
1 ) . the patient was a college student with no previous illness , and nothing of note was found in terms of family history , personal history , or travel history .
the patient had climbed a mountain on september 20th , about two weeks before his hospitalization .
the fever and chills started one week before hospitalization , and a dark red eschar of dead skin was found on the right side of the abdomen . because the diagnosis was scrub typhus , the patient was treated with doxycycline ( 100 mg orally 2 times a day for seven days ) .
the fever began to subside three days after hospitalization . on the second day , the patient reported bloody stools and severe pain around the anus .
the patient was examined by a surgeon to see if there was an anal laceration .
a hip bath was advised , and anti - inflammatory medicine was administered . on the sixth day of hospitalization , the pain during urination lessened , but the patient still complained about voiding dysfunction .
he had to suck in his stomach to urinate , and the amount of urine produced was less than usual .
constipation started on the sixth day , and the anal sphincter was weaker than usual .
on the eighth day , a skin rash , several blisters the size of rice grains , and calluses were observed on both sides of the anus ( fig .
three days after the administration , the constipation improved , and from the fourth day the urinary dysfunction improved to the point of waiting a few seconds before urination and not needing to suck in the stomach anymore .
the results of a blood test were as follows : white blood cell count , 4,930/mm ; hemoglobin , 14.1 g / dl ; platelet count , 179,000/mm ; aspartate aminotransferase , 56 iu / l ; alanine aminotransferase , 43 iu / l ; blood urea nitrogen , 17.35 mg / dl ; creatinine , 0.97 mg / dl ; albumin , 4.43 g / dl ; c - reactive protein , 7.9 mg / dl ; and erythrocyte sedimentation rate , 26 mm / hour . the immunofluorescence assay titer ( green cross labs , yongin , korea ) against orientia tsutsugamushi increased 4-fold or more .
the fact that the blisters appeared bilaterally on each buttock simultaneously without any previous history , the size of the blister , intensity of pain , and the depth of the ulcer all indicated the diagnosis more likely as herpes zoster than herpes simplex .
acyclovir ( 10 mg / kg every 8 hours ) was administered for seven days , and dexamethasone ( 10 mg / day ) was administered for six days and changed to prednisolone ( 100 mg orally ) .
although urinary dysfunction due to herpes zoster is rare , it can happen if the virus affects related nerves , particularly s2 and s3 of the sacral nerve ; this affects the motor and sensory nerves of the bladder .
chen , et al . reported that herpes zoster causes urinary dysfunction in only 4% of patients , but the ratio was 29% for patients who had the virus in the lumbosacral dermatome . regarding age and gender , herpes zoster accompanied by urinary dysfunction was mostly observed in middle - aged men , and the rate increased with age .
richmond reported that 78% of herpes zoster patients who experienced urinary retention had the virus in the sacral nerve , 11% had the virus in the thoracolumbar nerve , and 11% had the virus in the upper sternal nerve . in cases where the virus affected the sacral nerve , many patients also experienced constipation or bowel incontinence .
there are three types of urinary dysfunction caused by herpes zoster : cystitis , neuritis , and myelitis .
vzv directly attacks the bladder wall to cause urinary dysfunction , and inflammation of the mucous membrane or blisters are observed upon cystoscopy . in neuritis , vzv lurks in a posterior ganglion and then affects the motor nerve of the sacral nerve .
this disrupts the reflex of the detrusor muscle and weakens the bladder . in myelitis ,
the upper sacral nerve of the spinal cord is damaged , which causes a rigid bladder . voiding dysfunction caused by herpes
zoster responds well to treatment and usually improves along with the disappearance of skin rashes .
however , in some cases , acute urinary retention requires intermittent catheterization or an in - dwelling catheter .
chen , et al . reported cases of catheter insertion or urethral catheterization for patients with urinary dysfunction caused by herpes zoster .
the recovery took as little as two days to as long as eight weeks , but the average was 16 days .
yamanashi , et al . reported an average recovery period of 5.4 weeks for seven patients , and park , et al . reported an average recovery period of 12 days for 20 patients .
cases of urinary dysfunction caused by herpes zoster reported so far have included symptoms such as acute urinary retention , pain upon urination , urinary incontinence , cystitis , and neurogenic bladders .
all the patients had either an impaired immune system or were elderly people aged 60 or older .
there has been no reported case where a young adult with a normal immune system and without a previous illness had a dual infection of vzv and o. tsutsugamushi .
there was a case of a 75-year - old female patient who had a hip replacement surgery , followed by a knee replacement surgery .
she was affected by vzv in the sacral nerve , showing that in elderly people , multiple joint replacement surgeries can reactivate vzv .
cases of the simultaneous outbreak of o. tsutsugamushi and rickettsia typhi , leptospira , or malaria have been reported in the past , but no case involving dual infection with herpes zoster has been recorded .
one report discussed o. tsutsugamushi , a microorganism that causes monocyte - derived cells to trigger replication and activation of the cell , which in turn activates cd4 positive t lymphocyte .
both vzv and o. tsutsugamushi cause infection within cells , but their interaction mechanisms have yet to be determined .
this case study examined a patient who had voiding dysfunction and pain upon urination as the first symptoms of herpes zoster without dermatome lesions .
his symptoms improved with the administration of acyclovir and steroids without catheter insertion , intermittent catheterization , or administration of an -blocker or anticholinergic drugs . in conclusion , when patients under treatment for acute infection report sudden urinary retention , the possibility of voiding dysfunction caused by herpes zoster needs to be considered .
skin rashes on the bottom or lower limbs and changes in sensory nerves need to be checked .
early administration of acyclovir or steroids might help to reduce the duration of urinary dysfunction . | when a patient presents with acute voiding dysfunction without a typical skin rash , it may be difficult to make a diagnosis of herpes zoster . here , we present a case of scrub typhus in a 25-year - old man with the complication of urinary dysfunction .
the patient complained of loss of urinary voiding sensation and constipation .
after eight days , he had typical herpes zoster eruptions on the sacral dermatomes and hypalgesia of the s1-s5 dermatomes .
no cases of dual infection with varicella zoster virus and orientia tsutsugamushi were found in the literature . in the described case ,
scrub typhus probably induced sufficient stress to reactivate the varicella zoster virus .
early recognition of this problem is imperative for prompt and appropriate management , as misdiagnosis can lead to long - term urinary dysfunction .
it is important that a diagnosis of herpes zoster be considered , especially in patients with sudden onset urinary retention . |
the development of techniques for phalloplasty has paralleled the evolution of flap development in reconstructive surgery itself .
gillies and harrison , in 1948 , were amongst the first to take up the challenge .
the aims of penile reconstruction are to enable urination in the standing position , to enable sexual intercourse and to reconstruct an aesthetically acceptable penis with adequate tactile , protective and ideally erogenous sensation .
construction of a phallus with the above criteria has always been a formidable surgical challenge and many problems have not been solved satisfactorily , even with the introduction of free flap transfer .
a 21-year - old male sustained severe electrical burns on lower abdomen , genitalia , lower and upper limbs following accidental exposure to high tension electricity ( 12,000 v ) .
the lesions covered 25% of his total body surface area with 20% third degree burns and 5% second degree burns .
he presented with gangrene of right upper limb in its entirety , carbonization of external genitalia with subsequent complete loss of penis and left hemi scrotum and testis [ figure 1 ] .
the evolution of his general condition was very difficult as a result of haemodynamic and infection problems , which required intensive care .
locally , debridements of necrotic tissue were immediately performed , followed by a tensor fascia lata flap to protect the left femoral vessels and a local transposition flap to protect the brachial vessels in the left forearm .
( b ) penile gangrene disarticulation of right upper limb at the shoulder level done with primary closure of the defect .
the right hemiscrotum with testis was mobilized to cover the defect in the genital region with a perineal urethrostomy .
penis was planned to be reconstructed in stages . in the first stage , the right paragenital non - hairy skin was tubularized to create a neourethra from the perineal urethrostomy site to pubic region [ figure 2 ] .
six weeks later staged penile reconstruction proceeded using a right - side groin flap . with non - availability of other adjacent tissues and with the loss of right upper limb and entire left forearm being grafted with a transposition flap to protect brachial vessels , groin flap remained the option for reconstruction .
a tube within a tube was created from a single folded , 17 cm wide and 16 cm long groin flap [ figure 3 ] . a 4 cm wide area along the midline of flap , without
the presence of hair was tubed inward around a 16 french foley catheter to form the neourethra .
the adjacent skin of about 1 cm on either side was deepithelialized to allow the flap to be rolled onto itself .
the remaining 11 cm width of skin was wrapped around the inner tube portion to provide external cover . with the catheter introduced onto the pubic urethra , urethral repair and skin closure with a tumble of the groin flap done [ figure 4 ] .
about 3 weeks later , an axial delay of the flap done followed by flap division in another week 's time .
the flap provided a satisfactory aesthetic result and enabled urination in standing position after catheter removal at 15th day after flap division [ figure 5 ] .
advancements in microsurgical techniques have expanded the number of potential penile reconstructive procedures , and there is general agreement that they represent the method of choice for penile reconstruction . in phalloplasty , in female - to - male transsexuals and penile reconstruction in male patients following loss in trauma or malignancy ,
its main disadvantages of too hairy skin for urethral reconstruction and donor site morbidity can not be ignored . in this very complex surgical case , with loss of entire right upper limb and left forearm being grafted in its entirety and with the loss of majority of adjacent tissues , groin flap is probably the choice . however , the major difficulty was the staged nature of procedure .
the patient had a small fistula at the penoscrotal junction in the dorsal aspect which healed by conservative management .
after an 18 month follow - up , the patient was very pleased with the appearance of penis and his wish of male pattern urination being fulfilled [ figure 6 ] .
neither author has any financial conflict of interest with
any of the content discussed in this article . | plastic surgery continues to maintain a prominent presence in the evolution of male genital reconstruction . in this case report
, we are presenting a case of post - electric burn with a total loss of penis .
sustaining other major injuries following an electric burn with loss of right upper limb and extensive tissue damage to left upper limb , abdomen and both thighs , this young male patient was initially managed from life - threatening problems . with many options
closed following a major electric burn and its acute management , penile and urethral reconstruction was a unique and a great challenge in this patient . heeding to the patient 's wish of male pattern micturition , we had performed a successful reconstruction of urethra and entire phallus with groin flap . |
trauma remains a major health burden worldwide despite the various preventive measures that have been developed .
the major burden of trauma estimated at about 90% is borne by middle and low - income countries . during the trauma ; the maxillofacial region is highly vulnerable to injury either in isolation or in combination with other systems because of its exposure .
the changing etiological factors and patterns of maxillofacial injuries reported from different regions at different times is largely modulated by prevailing socioeconomic , cultural , environmental and legislative factors , as well as location of the trauma center .
recently , published literature shows that assault and inter - personal violence ( ipv ) are beginning to overtake road traffic crashes ( rtcs ) as the leading cause of maxillofacial injuries in the developed countries . on the other hand
, rtcs continues to be indicted as the leading cause of maxillofacial injuries from developing countries , although , significant changes in the etiology , mechanism and patterns of injury are being reported . over the last two decades ,
the increase in the use of motorcycles as a mode of commercial transportation in developing countries including nigeria has significantly affected the pattern of maxillofacial injuries .
studies have shown significant reduction in maxillofacial injuries in states where preventive measures have been effectively implemented . a clear understanding of the mechanisms of injury from other studies will help evaluate the effectiveness of current safety and preventive measures , and also the pave way for newer innovations in this area .
this information could also be used as a guide to the development and future funding of public health preventive programs .
this study aims to prospectively evaluate the patterns , etiological factors , severity , management and the outcomes of maxillofacial trauma at a tertiary maxillofacial center .
this was a prospective descriptive cohort study of consecutive patients with maxillofacial injuries that presented and were managed at our center between january and december 2013 .
data collected relates to patients demography , the mechanisms of injuries , patterns of trauma , soft tissue and bony structural classification , associated systemic injuries , treatment , length of hospital stay , associated complications and outcomes .
these data were collected using a well - designed data sheet before entering into the database for analysis .
the causes of the reported injuries were classified based on their mechanisms into rtcs , falls , assault , gunshot and sports related .
information relating to the use of seatbelts / helmets at the time of the incident was also obtained where applicable .
anatomic locations of mandibular fractures were classified according to ivy and curtis system while maxillary fractures were classified using the le fort system .
data were analyzed using statistical package for the social sciences ( spss ) version 17.0 for windows ( spss , chicago il , usa ) .
a total of 70 patients presented with 79 maxillofacial fractures , 49 soft tissues and 32 concomitant systemic injuries during the study period .
the mean age was 30.11 years ( 9 months60 years , standard deviation = 14.97 ) .
the peak age of incidence was in the 3 decade of life ( n = 20 , 28.6% ) .
males accounted for 80% of the patients in this study with a male to female ratio of 4:1 .
twenty - two ( 31.4% ) of the patients had isolated mandibular fracture , 11 ( 15.7% ) had isolated middle - third fractures while 9 ( 12.9% ) had combined middle third and mandibular fractures . in
all there were 40 ( 57.1% ) mandibular fractures and 39 ( 55.7% ) middle - third fractures . with regards to mandibular fractures ,
the body ( 20% ) was the most common site while the ramus and symphysis ( 1.4% each ) were least affected . in the middle third ,
zygomatic bone fracture ( 18.6% ) was the commonest while naso - orbito - ethmoidal complex fracture ( 1.4% ) accounted for the least fracture seen in this study .
fifty - two ( 74.3% ) patients sustained associated soft tissue injuries , and these were lacerations in 70% of the cases .
other associated systemic injuries were recorded in 26 ( 37.1% ) patients with orthopedic injuries accounting for the majority of the cases .
etiological factors of maxillofacial injuries distribution of associated systemic injuries only 8.6% and 4.3% of the patients that sustained injuries from vehicular and motorcycle road traffic crashes were seatbelt restrained and used crash helmets at the time of the accident respectively .
majority of the patients ( 98.6% ) denied being under the influence of alcohol at the time of the crash . in this study , closed reduction with maxillo - mandibular fixation was the major method of treatment of facial fractures ( 35.7% ) and only 2 ( 2.9% ) patients were managed by an open reduction with internal fixation .
distribution of maxillofacial injuries these delays were mainly due to the lack of funds for accessing healthcare ( 4.3% ) and logistic delay in accessing the operating theatre space ( 4.3% ) .
epidemiological differences in the patterns and occurrence of maxillofacial injuries have been well reported as a result of socio - economic , cultural , legislative and environmental influences . however , despite the changing patterns , global epidemiological surveys have revealed that some aspects of the facial fracture patterns remain similar among the various nations .
the results of this study are largely in agreement with those of previous reports , particularly regarding age incidence and gender distribution of patients . in this study ,
however , the gender ratio in the present study is much lower compared to the 23:1 reported in a previous study from the northern part of nigeria .
females in the southern part of nigeria participate more in social , public , work and sports related activities than their northern counterparts who are restricted for cultural and religious reasons and may not be as vulnerable to trauma .
our findings of the highest frequency of injuries in the 3 and 4 decades of life is in agreement with those reported elsewhere .
this is attributed to the fact that this age group is the most active phase of life .
road traffic crashes accounted for the majority ( 70% ) of maxillofacial injuries in this study , similar to previous reports .
rtcs remain the leading cause of trauma in the developing world because of non - observance and lack of strict enforcement of speed limits , use of restraining seatbelts and wearing of protective crash helmets .
furthermore , several roads are in bad repair , vehicles are poorly maintained and have minimal safety features .
conversely , in several developed countries , there is a downward trend of rtcs related maxillofacial injuries with ipv and assault becoming the leading etiological factors .
this decrease has been attributed to strict enforcement of protective measures and various traffic legislations .
the recent increase of commercial motorcycle transportation has also been blamed for the increase in rtcs in nigeria .
this is also shown in the current study as half of the motor - vehicular rtcs causing maxillofacial injuries were due to motorcycle incidents .
we noted that during the period of this study , no tricycle related maxillofacial injury was observed among our patients .
indeed , several governmental agencies in nigeria have introduced the tricycle to replace commercial motorcycle transportation ; to reduce the incidence of motorcycle related injuries .
global reports show a low incidence of tricycle related - crashes even in countries where it forms a major mode of commercial transportation .
the low incidence of tricycle crashes is due to a number of factors ; the speed that the tricycle can attain is limited , the design of three wheels gives it better stability compared to the motorcycle , the tricycle has a housing protecting the occupants unlike the motorcycle that completely exposes the driver and passenger to the environment .
the motorcycle driver can easily make risky maneuverings because of its narrow design and this is less likely with a tricycle .
one of the patients in the current study sustained a deep laceration of the tongue following epileptic fits requiring suturing .
a study from nigeria reported the incidence of orofacial injuries among enclamptic patients to be 42% .
the low incidence reported in our study could be due to the patients presenting at other units .
alcohol abuse has been reported to be linked to maxillofacial injuries from violence in several studies , the incidence ranging between 20% and 56% .
this has been attributed to the capacity of alcohol to impair judgment and fuel violence .
a study from the northern part of nigeria observed an increase in the prevalence of alcohol - related maxillofacial fractures after annual periods of religious fasting . in the present study ,
alcohol - related maxillofacial injury only accounted for 1.4% of our cases . in the current study
, there was a slight preponderance of mandibular fractures ( 57.1% ) compared to midface fractures ( 55.7% ) .
the small sample size recorded in this study might have accounted for this disparity . in the agreement with previously published data , however , the body of the mandible was the most common site of fracture , closely followed by the parasymphysis .
furthermore , fractures of the body of the mandible were the most frequent fractures due to road traffic accidents while the angle was most affected due to assaults and these findings agree with previous studies .
considerable variations exist in the rate of systemic injuries associated with maxillofacial fractures ranging from 12% to 46% .
these variations are due to lack of standardized definitions and classification of such injuries worldwide .
developing countries have recorded orthopedic injuries as the most frequent associated trauma but reports from the developed world support cranial injuries . about of the patients ( 37.1% ) in this study
these differences could be a reflection of the variation in etiology , poor utilization of protective devices as well as poor pre - hospital management in our environment , leading to more non - survivable fatal craniofacial injuries arriving at the hospital . because motorcycle is the major etiology , most of the patients surviving to the hospital are more likely to sustain orthopedic injuries because they are exposed on the bike .
open reduction and internal fixation ( orif ) is fast becoming the gold standard for management of maxillofacial fractures . however ,
the closed method of fracture reduction was employed in most ( 38.6% ) of the patients in this study , and this pattern is similar to those of previous reports from developing nations .
only 2.9% of our patients had open reduction with trans - osseous wiring at the fronto - zygomatic region
. the low rate of orif in this study was because of the unaffordability of such treatment in an environment where patients are responsible for the costs .
the rate of complication in our study was 11.4% , similar to a norwegian study , higher than 7.5% reported by a chinese study but much lower than the 25.26% reported in another study .
infection and malocclusion ( 2.9% each ) were the commonest complications , similar to the trend reported in an india study .
the infection in our study was mainly seen in patients that were not so compliant with the post - operative oral care instructions .
this was particularly more relevant as mandibullo - maxillary fixation needed a high degree of diligence and compliance to keep a good oral hygiene and prevent infection .
achieving a perfect occlusal harmony without open reduction in some old fracture cases is difficult , and this may explain the few cases of malocclusion recorded in this study .
this study shows that motorcycle related crashes are the leading etiological factor of maxillofacial injuries in our center .
the demographic patterns in this study mirror the one reported in the literature . the mandibular bony frame is the most fractured bone of the face in a similar version to that reported in the published series .
there is an urgent need for strict enforcement of adherence to speed limits , the use of the seatbelt and the wearing of safety helmets by motorcyclists .
use of tricycles to replace commercial motorcycle transportation may help to reduce rtcs in our environment . | background : trauma remains a leading cause of maxillofacial injury globally . changing etiological factors and patterns of maxillofacial injury continue to be reported and are largely modulated by socio - geographic and environmental factors .
it is important to have an in - depth understanding of the pattern and etiology in a particular region before effective preventive measures can be developed.aim:the aim was to evaluate the patterns , etiological factors , and management of maxillofacial injuries in ogun state , nigeria.materials and methods : a prospective descriptive cohort study of all consecutive patients that presented with maxillofacial injuries at our center between january and december 2013 .
information about demographic data , types of maxillofacial and associated injury , etiology of injury , treatment received and complications were collected and analyzed.results:seventy patients presented with maxillofacial injury during the study period with a male to female ratio of 4:1 .
the age range was 9 months to 60 years with a mean of 30.11 standard deviation 14.97 years .
majority of the facial fractures were due to motorcycle related crashes .
there were 57.1% mandibular fractures and 55.7% middle third fractures . closed reduction with maxillo - mandibular fixation was the major method of treatment of facial fractures .
postoperative complications were observed in 11.4% of patients.conclusion:road traffic crashes ( rtcs ) remain the leading etiological factor of maxillofacial injuries in our center .
enforcement of stricter traffic regulations and possibly replacement of motorcycles with tricycles for commercial transportation may help to reduce the incidence of rtcs . |
the goal of explaining the origin of species has inspired more than two centuries of scientific inquiry , involving early naturalists through to modern evolutionary biologists .
though only hinted at by darwin , the idea that the evolution of reproductive isolation is central to the process of speciation is now widely recognized [ 3 - 5 ] .
reproductive isolating mechanisms that restrict gene exchange between diverging species include prezygotic barriers that limit the potential for mating or zygote formation ( e.g. habitat and flowering time differences or pollen - pistil incompatibilities ) and postzygotic barriers that reduce the viability or fertility of hybrid offspring if interbreeding does occur .
whereas the evolution of prezygotic isolation is often easily understood as a byproduct of differential adaptation to variable ecological conditions or of selection to reduce the production of low - fitness hybrids , the evolution of postzygotic isolation has been a long - standing mystery because the production of dead or sterile hybrids can not be favored by natural selection .
dobzhansky and muller outlined a solution to this puzzle , explaining that if postzygotic isolation is caused by incompatible gene interactions between diverging species then natural selection need not oppose its evolution .
a new mutation might function perfectly well in the context of its native genetic background , increasing in frequency until it becomes fixed within the species .
but because the fitness of this new variant has only been tested against its own genetic background , it might be functionally incompatible with divergent alleles present in foreign genetic backgrounds , resulting in hybrid sterility or inviability . the key insight of the so - called dobzhansky - muller model is that , if hybrid incompatibilities are caused by two or more mutational differences between species , there can be transitional genotypes that are adaptive or neutral in ancestral populations and , therefore , not eliminated by natural selection . in contrast , if reduced hybrid fitness is caused by a single mutational step ( resulting in heterozygote disadvantage ) , natural selection is expected to impede the initial spread of the mutation within species .
nevertheless , in some situations , random genetic drift can overwhelm selection and lead to the fixation of such a mutation , particularly if the reduction in fitness is modest .
indeed , it is not uncommon for partial hybrid sterility in plants to be caused by heterozygous chromosomal rearrangements that evolved in a single mutational step . differentiating between these alternative genetic pathways to postzygotic isolation
one versus multiple mutational steps can help identify which evolutionary processes contribute to species divergence . to determine whether hybrid incompatibilities evolve in multiple steps , as predicted by the dobzhansky - muller model , it is necessary to pinpoint the causal genetic changes at the molecular level .
but even if the dobzhansky - muller model is confirmed , it provides no expectation as to which kinds of molecular genetic changes cause postzygotic isolation .
the dobzhansky - muller model is also agnostic about the evolutionary processes that cause divergence within species .
do the initial mutations increase in frequency by random genetic drift or by natural selection because they benefit the native species for some reason that is incidental to their eventual contribution to reproductive isolation ?
in several plant systems , the molecular variants that cause hybrid inviability and sterility have recently been identified , providing some of the first hints at answers to these fundamental evolutionary questions . here
, we focus on exclusively nuclear - encoded hybrid incompatibility genes but point the reader to several excellent reviews of cytoplasmic male sterility and the population genetic theory for how cytonuclear incompatibilities might evolve .
dobzhansky and muller outlined a solution to this puzzle , explaining that if postzygotic isolation is caused by incompatible gene interactions between diverging species then natural selection need not oppose its evolution .
a new mutation might function perfectly well in the context of its native genetic background , increasing in frequency until it becomes fixed within the species .
but because the fitness of this new variant has only been tested against its own genetic background , it might be functionally incompatible with divergent alleles present in foreign genetic backgrounds , resulting in hybrid sterility or inviability . the key insight of the so - called dobzhansky - muller model is that , if hybrid incompatibilities are caused by two or more mutational differences between species , there can be transitional genotypes that are adaptive or neutral in ancestral populations and , therefore , not eliminated by natural selection .
in contrast , if reduced hybrid fitness is caused by a single mutational step ( resulting in heterozygote disadvantage ) , natural selection is expected to impede the initial spread of the mutation within species .
nevertheless , in some situations , random genetic drift can overwhelm selection and lead to the fixation of such a mutation , particularly if the reduction in fitness is modest .
indeed , it is not uncommon for partial hybrid sterility in plants to be caused by heterozygous chromosomal rearrangements that evolved in a single mutational step . differentiating between these alternative genetic pathways to postzygotic isolation
one versus multiple mutational steps can help identify which evolutionary processes contribute to species divergence . to determine whether hybrid incompatibilities evolve in multiple steps , as predicted by the dobzhansky - muller model , it is necessary to pinpoint the causal genetic changes at the molecular level .
but even if the dobzhansky - muller model is confirmed , it provides no expectation as to which kinds of molecular genetic changes cause postzygotic isolation .
the dobzhansky - muller model is also agnostic about the evolutionary processes that cause divergence within species .
do the initial mutations increase in frequency by random genetic drift or by natural selection because they benefit the native species for some reason that is incidental to their eventual contribution to reproductive isolation ?
in several plant systems , the molecular variants that cause hybrid inviability and sterility have recently been identified , providing some of the first hints at answers to these fundamental evolutionary questions . here
, we focus on exclusively nuclear - encoded hybrid incompatibility genes but point the reader to several excellent reviews of cytoplasmic male sterility and the population genetic theory for how cytonuclear incompatibilities might evolve .
in the classic model of speciation , hybrid incompatibilities are thought to evolve as a by - product of adaptation to different environments .
some of the best evidence for this idea comes from recent work on hybrid necrosis , a form of plant hybrid inviability characterized by necrotic lesions , wilting , and inhibited growth .
a remarkable commonality has emerged among cloned hybrid necrosis genes : all encode proteins involved in plant defense against bacterial or fungal pathogens , and incompatible allelic combinations appear to induce autoimmune - like responses .
the plant immune system is a tightly coordinated network of proteins that recognize pathogen invasions and elicit a suite of cellular defense responses .
one might imagine that plant - pathogen molecular coevolution , occurring in independent populations with unique pathogen communities , could lead to hybrid mismatches between interacting components of the immune system that result in hyperactivation of defense responses in hybrid offspring . indeed , two - locus incompatibilities between pathogen resistance genes and their interacting partners have been implicated in hybrid necrosis between divergent strains of arabidopsis thaliana [ 18 - 20 ] , between the indica and japonica subspecies of asian cultivated rice , and between wild and domesticated species of tomato and lettuce .
for a few of these loci , alleles that confer necrosis differ by several amino acid changes from ancestral alleles , perhaps consistent with a sustained fine - tuning of these proteins to improve pathogen recognition .
other recent studies suggest that two - locus hybrid incompatibilities can also readily evolve in the absence of natural selection . based on his early genetic analyses of hybrid sterility between the indica and japonica varieties of oryza sativa
, oka suggested that defects in pollen development might be caused by loss - of - function alleles at different duplicate genes in each parent .
postzygotic isolation might evolve passively then , due to degenerative mutations and genetic drift acting on different paralogs ( genes related by duplication within a genome ) in diverging populations .
only recently has the cloning of several plant hybrid incompatibility genes provided empirical evidence for this idea .
indeed , hybrids between different strains of a. thaliana arrest as early embryos when they carry loss - of - function alleles at duplicate copies of the essential histidinol - phosphate amino - transferase gene ( hpa1 and hpa2 ) . likewise ,
in two distinct oryza crosses , null alleles at duplicate genes confer gametic male sterility .
. showed that pollen from indica - japonica hybrids fails to germinate when it carries loss - of - function alleles one from each parent at two duplicate genes of unknown function , doppelganger1 and 2 ( dpl1 and dpl2 ) .
similarly , pollen sterility in hybrids between o. sativa japonica and an amazonian wild rice , oryza glumaepatula , is caused by nonfunctional alleles at duplicate loci s27 and s28 that both encode the mitochondrial ribosomal l27 protein ( mtrpl27 ) . in this case ,
o. glumaepatula lacks the duplication ; pollen is defective when it carries a loss - of - function allele at s28 from o. sativa and is missing the s27 paralog ( by virtue of inheriting the corresponding genomic region from o. glumaepatula ) .
an extraordinary feature of each of these systems is that loss - of - function alleles have evolved repeatedly . in a. thaliana ,
roughly three - quarters of plant collections carry one of six possible loss - of - function alleles at either hpa1 or hpa2 . in oryza ,
several different disruptive mutations have arisen in closely related species for both the dpl1/dpl2 and s27/s28 incompatibilities .
these findings , along with the rich history of whole genome duplication in plants , suggest that the divergent resolution of gene duplicates via mutation and genetic drift might be a common source of postzygotic isolation in plants .
in addition to the cases described above , studies have occasionally discovered hybrid incompatibilities that map to single genetic loci , which might be taken as prima facie evidence that postzygotic isolation can evolve via a single mutational step . like the fixation of an underdominant chromosomal rearrangement , the establishment of a new sterility- or inviability - causing allele requires a hefty contribution from genetic drift to outweigh selection against unfit heterozygotes .
but , if instead , a single - locus hybrid incompatibility is conferred by two or more alleles that evolve independently implying multiple mutational steps it is possible to circumvent any initial fitness valley within species ( figure 1 ) .
the recent cloning of the single - locus hybrid incompatibility gene oak , which encodes a receptor - like kinase ( rlk ) and causes reduced growth and developmental abnormalities in interpopulational hybrids of a. thaliana , supports just such a multiple - step scenario .
indeed , the two incompatible oak alleles are 9% divergent at the amino acid level , with 55 of 152 residues differing in a key malectin - like domain . with such extreme levels of sequence diversity
, it is reasonable to infer that intermediate , viable genotypes predated the current incompatible pair of alleles .
although its adaptive significance is unclear , the occurrence of oak within a highly variable tandem array of rlk genes suggests that high rates of gene conversion and illegitimate recombination may have contributed to its evolution .
an ancestral population splits into two geographically isolated populations that diverge genetically and eventually fix different alleles ( red or blue ) at the same locus . in the f1 hybrid ,
note that hybrid incompatibilities can just as easily arise between ancestral and derived alleles if two or more rounds of mutation and fixation occur within a lineage .
the molecular characterization of three additional , single - locus incompatibilities between indica and japonica rice varieties also suggests the involvement of multiple mutational steps .
the s5 incompatibility locus causes embryo sac abortion in indica - japonica heterozygotes , but certain wide - compatibility varieties carry neutral alleles and can , therefore , produce fertile progeny when crossed to either subspecies . originally described by ikehashi and araki as a simple , three - allele system , a reasonable assumption has been that the neutral allele represents an intermediate step that allowed the indica - japonica incompatibility to evolve without a fitness cost . upon closer inspection , however
, genetic mapping of the s5 locus has revealed that hybrid female sterility is regulated by three , tightly linked genes
orf3 , orf4 , and orf5 and that haplotypes vary substantially among strains of indica , japonica , and wild rice . during female sporogenesis , killer
alleles at orf4 and orf5 cause endoplasmic reticulum stress that results in premature programmed cell death and embryo sac abortion , but these effects can be rescued by a protector
this killer - protector combination appears to be the ancestral genotype and is at high frequency in the wild species oryza rufipogon and oryza nivara , as well as in the outgroup species o. glumaepatula .
in contrast , the typical indica s5 haplotype has a deletion in orf4 that incapacitates its killing function , and the typical japonica haplotype carries mutations that disable both the orf5 killer and orf3 protector .
the s5 incompatibility causes an aberrant , gain - of - function phenotype in indica - japonica hybrids that carry killer alleles at orf4 and orf5 but lack the protector allele at orf3 . in the second case ,
long et al . fine - mapped the sa locus to two distinct , tightly linked loci : sam encodes a small ubiquitin - like modifier ( sumo ) e3 ligase - like protein , and saf encodes an f - box protein .
hybrid plants that are heterozygous at sam and also carry indica alleles at saf are semi - sterile because haploid pollen that inherits the japonica sam allele fails to develop properly .
to explain the surprising result that epistasis among three alleles is required for abortion of japonica sam - carrying microspores , the authors propose a complicated model in which indica - encoded sam and saf proteins are transported between haploid microspores during early pollen development to kill gametes that inherit the japonica sam allele .
the idea is that the japonica sam protein is unable to move between microspores , so pollen grains that carry indica alleles at sam and saf but do not receive the interacting japonica sam protein remain viable . for this incompatibility system to have evolved unopposed by natural selection
, the japonica sam allele must have spread after a permissive allele at saf had fixed initially ( figure 2 ) .
indeed , the current geographic distribution of sam / f haplotypes in o. sativa and its wild ancestor , o. rufipogon , is consistent with this scenario .
moreover , patterns of sam / f haplotype variation particularly , the finding that japonica - like haplotypes segregate in o. rufipogon but not indica populations support the hypothesis of independent origins of domestication for indica and japonica .
an important , unanswered question is what caused the japonica haplotype to become fixed . a classic ,
single - locus incompatibility between oryza sativa indica and oryza sativa japonica is conferred by two adjacent genes ( depicted here as striped and solid ) .
semi - sterility occurs in f1 hybrids that carry blue alleles at the striped and solid genes ( indica haplotype ) in combination with a green allele at the solid gene ( japonica haplotype ) .
the wild progenitor oryza rufipogon carries all three haplotypes : ancestral ( blue , blue ) , permissive ( red , blue ) , and japonica - like ( red , green ) .
mapped the s24 pollen sterility locus to an ankyrin-3 ( ank-3 ) gene , which kubo et al . later showed is dependent on an additional , unlinked locus , epistatic factor for s24 ( efs ) . like s5 and sa ,
the s24-efs hybrid incompatibility is a gain - of - function : pollen grains with japonica s24 alleles are aborted in s24-heterozygotes , but only when the plant is also homozygous for japonica alleles at efs . as with sa
, it is possible that the otherwise deleterious japonica s24 allele was only able to spread in a permissive , indica - like efs background , but the necessary phylogeographic analyses have not yet been done .
it is also worth noting that for both sa and s24 , certain wide compatibility varieties carry neutral alleles that rescue pollen sterility , indicating that the evolutionary histories of these incompatibilities might include alternate mutational routes .
in the classic model of speciation , hybrid incompatibilities are thought to evolve as a by - product of adaptation to different environments .
some of the best evidence for this idea comes from recent work on hybrid necrosis , a form of plant hybrid inviability characterized by necrotic lesions , wilting , and inhibited growth .
a remarkable commonality has emerged among cloned hybrid necrosis genes : all encode proteins involved in plant defense against bacterial or fungal pathogens , and incompatible allelic combinations appear to induce autoimmune - like responses .
the plant immune system is a tightly coordinated network of proteins that recognize pathogen invasions and elicit a suite of cellular defense responses .
one might imagine that plant - pathogen molecular coevolution , occurring in independent populations with unique pathogen communities , could lead to hybrid mismatches between interacting components of the immune system that result in hyperactivation of defense responses in hybrid offspring . indeed , two - locus incompatibilities between pathogen resistance genes and their interacting partners have been implicated in hybrid necrosis between divergent strains of arabidopsis thaliana [ 18 - 20 ] , between the indica and japonica subspecies of asian cultivated rice , and between wild and domesticated species of tomato and lettuce .
for a few of these loci , alleles that confer necrosis differ by several amino acid changes from ancestral alleles , perhaps consistent with a sustained fine - tuning of these proteins to improve pathogen recognition .
other recent studies suggest that two - locus hybrid incompatibilities can also readily evolve in the absence of natural selection . based on his early genetic analyses of hybrid sterility between the indica and japonica varieties of oryza sativa
, oka suggested that defects in pollen development might be caused by loss - of - function alleles at different duplicate genes in each parent .
postzygotic isolation might evolve passively then , due to degenerative mutations and genetic drift acting on different paralogs ( genes related by duplication within a genome ) in diverging populations .
only recently has the cloning of several plant hybrid incompatibility genes provided empirical evidence for this idea .
indeed , hybrids between different strains of a. thaliana arrest as early embryos when they carry loss - of - function alleles at duplicate copies of the essential histidinol - phosphate amino - transferase gene ( hpa1 and hpa2 ) . likewise ,
in two distinct oryza crosses , null alleles at duplicate genes confer gametic male sterility .
. showed that pollen from indica - japonica hybrids fails to germinate when it carries loss - of - function alleles one from each parent at two duplicate genes of unknown function , doppelganger1 and 2 ( dpl1 and dpl2 ) .
similarly , pollen sterility in hybrids between o. sativa japonica and an amazonian wild rice , oryza glumaepatula , is caused by nonfunctional alleles at duplicate loci s27 and s28 that both encode the mitochondrial ribosomal l27 protein ( mtrpl27 ) . in this case ,
o. glumaepatula lacks the duplication ; pollen is defective when it carries a loss - of - function allele at s28 from o. sativa and is missing the s27 paralog ( by virtue of inheriting the corresponding genomic region from o. glumaepatula ) .
an extraordinary feature of each of these systems is that loss - of - function alleles have evolved repeatedly . in a. thaliana ,
roughly three - quarters of plant collections carry one of six possible loss - of - function alleles at either hpa1 or hpa2 . in oryza ,
several different disruptive mutations have arisen in closely related species for both the dpl1/dpl2 and s27/s28 incompatibilities .
these findings , along with the rich history of whole genome duplication in plants , suggest that the divergent resolution of gene duplicates via mutation and genetic drift might be a common source of postzygotic isolation in plants .
in addition to the cases described above , studies have occasionally discovered hybrid incompatibilities that map to single genetic loci , which might be taken as prima facie evidence that postzygotic isolation can evolve via a single mutational step . like the fixation of an underdominant chromosomal rearrangement , the establishment of a new sterility- or inviability - causing allele requires a hefty contribution from genetic drift to outweigh selection against unfit heterozygotes .
but , if instead , a single - locus hybrid incompatibility is conferred by two or more alleles that evolve independently implying multiple mutational steps it is possible to circumvent any initial fitness valley within species ( figure 1 ) .
the recent cloning of the single - locus hybrid incompatibility gene oak , which encodes a receptor - like kinase ( rlk ) and causes reduced growth and developmental abnormalities in interpopulational hybrids of a. thaliana , supports just such a multiple - step scenario .
indeed , the two incompatible oak alleles are 9% divergent at the amino acid level , with 55 of 152 residues differing in a key malectin - like domain . with such extreme levels of sequence diversity
, it is reasonable to infer that intermediate , viable genotypes predated the current incompatible pair of alleles .
although its adaptive significance is unclear , the occurrence of oak within a highly variable tandem array of rlk genes suggests that high rates of gene conversion and illegitimate recombination may have contributed to its evolution .
an ancestral population splits into two geographically isolated populations that diverge genetically and eventually fix different alleles ( red or blue ) at the same locus . in the f1 hybrid ,
note that hybrid incompatibilities can just as easily arise between ancestral and derived alleles if two or more rounds of mutation and fixation occur within a lineage .
the molecular characterization of three additional , single - locus incompatibilities between indica and japonica rice varieties also suggests the involvement of multiple mutational steps .
the s5 incompatibility locus causes embryo sac abortion in indica - japonica heterozygotes , but certain wide - compatibility varieties carry neutral alleles and can , therefore , produce fertile progeny when crossed to either subspecies .
originally described by ikehashi and araki as a simple , three - allele system , a reasonable assumption has been that the neutral allele represents an intermediate step that allowed the indica - japonica incompatibility to evolve without a fitness cost . upon closer inspection , however
, genetic mapping of the s5 locus has revealed that hybrid female sterility is regulated by three , tightly linked genes
orf3 , orf4 , and orf5 and that haplotypes vary substantially among strains of indica , japonica , and wild rice . during female sporogenesis , killer alleles at orf4 and orf5 cause endoplasmic reticulum
stress that results in premature programmed cell death and embryo sac abortion , but these effects can be rescued by a protector
this killer - protector combination appears to be the ancestral genotype and is at high frequency in the wild species oryza rufipogon and oryza nivara , as well as in the outgroup species o. glumaepatula .
in contrast , the typical indica s5 haplotype has a deletion in orf4 that incapacitates its killing function , and the typical japonica haplotype carries mutations that disable both the orf5 killer and orf3 protector .
the s5 incompatibility causes an aberrant , gain - of - function phenotype in indica - japonica hybrids that carry killer alleles at orf4 and orf5 but lack the protector allele at orf3 . in the second case ,
long et al . fine - mapped the sa locus to two distinct , tightly linked loci : sam encodes a small ubiquitin - like modifier ( sumo ) e3 ligase - like protein , and saf encodes an f - box protein .
hybrid plants that are heterozygous at sam and also carry indica alleles at saf are semi - sterile because haploid pollen that inherits the japonica sam allele fails to develop properly .
to explain the surprising result that epistasis among three alleles is required for abortion of japonica sam - carrying microspores , the authors propose a complicated model in which indica - encoded sam and saf proteins are transported between haploid microspores during early pollen development to kill gametes that inherit the japonica sam allele .
the idea is that the japonica sam protein is unable to move between microspores , so pollen grains that carry indica alleles at sam and saf but do not receive the interacting japonica sam protein
remain viable . for this incompatibility system to have evolved unopposed by natural selection , the japonica sam allele must have spread after a permissive allele at saf had fixed initially ( figure 2 ) .
indeed , the current geographic distribution of sam / f haplotypes in o. sativa and its wild ancestor , o. rufipogon , is consistent with this scenario .
moreover , patterns of sam / f haplotype variation particularly , the finding that japonica - like haplotypes segregate in o. rufipogon but not indica populations support the hypothesis of independent origins of domestication for indica and japonica .
an important , unanswered question is what caused the japonica haplotype to become fixed . a classic ,
single - locus incompatibility between oryza sativa indica and oryza sativa japonica is conferred by two adjacent genes ( depicted here as striped and solid ) .
semi - sterility occurs in f1 hybrids that carry blue alleles at the striped and solid genes ( indica haplotype ) in combination with a green allele at the solid gene ( japonica haplotype ) .
the wild progenitor oryza rufipogon carries all three haplotypes : ancestral ( blue , blue ) , permissive ( red , blue ) , and japonica - like ( red , green ) .
figure adapted from . in the third case , zhao et al . mapped the s24 pollen sterility locus to an ankyrin-3 ( ank-3 ) gene , which kubo et al
. later showed is dependent on an additional , unlinked locus , epistatic factor for s24 ( efs ) . like s5 and sa ,
the s24-efs hybrid incompatibility is a gain - of - function : pollen grains with japonica s24 alleles are aborted in s24-heterozygotes , but only when the plant is also homozygous for japonica alleles at efs . as with sa , it is possible that the otherwise deleterious japonica s24 allele was only able to spread in a permissive , indica - like efs background , but the necessary phylogeographic analyses have not yet been done .
it is also worth noting that for both sa and s24 , certain wide compatibility varieties carry neutral alleles that rescue pollen sterility , indicating that the evolutionary histories of these incompatibilities might include alternate mutational routes .
in just the last few years , plant geneticists have made tremendous progress in identifying the molecular genetic basis of postzygotic reproductive isolation . with more than a dozen genes
now cloned , it is clear that plant hybrid incompatibilities usually evolve via two or more mutational steps , as is predicted by the dobzhansky - muller model .
this conclusion flows rather easily from those cases in which hybrid dysfunction is caused by epistasis between two or more unlinked genes , but it has been less obvious for a number of classic , single - locus incompatibilities . because of several recent detailed genetic studies , however , we now know that even these single - locus incompatibilities arose by mutations in two tightly linked genes , or , at the very least , two or more amino acid changes encoded within the same gene .
thus , it is possible that most of these incompatibility alleles appeared without any reductions in fitness within species . of course
, the key question for speciation is which evolutionary forces allow incompatibility alleles to increase in frequency and eventually become fixed within species . for a few of the cases discussed above there is evidence that natural selection ( e.g. hybrid necrosis ) or random genetic drift ( e.g. divergent resolution of duplicate genes ) may have played a role .
as we have seen , it is even possible for the very same gene to be involved in multiple cases of hybrid dysfunction : divergent duplicate loci carry a striking number of independently derived , loss - of - function mutations . yet
, there are many hybrid incompatibilities that do not involve disease resistance or duplicate genes , and , for these systems , we are only beginning to understand the evolutionary causes of divergence .
remarkably , at least 50 loci contribute to hybrid sterility between the closely related subspecies oryza sativa indica and oryza sativa japonica .
so does this large number reflect a special propensity in rice species for evolving hybrid sterility ? given that oryza flowers have six anthers with many pollen grains per ovule , a gametic pollen killer that lowers fertility by 50% might entail little or no reduction in heterozygote seed fertility . although our current sample of plant hybrid incompatibility genes comes from only two genera ( oryza and arabidopsis ) , there are a number of classic and more recently identified [ 48 - 51 ] incompatibility systems that are not yet molecularly defined . as advances in genomics and dna sequencing technologies enable rigorous genetic analysis of reproductive isolation in a number of emerging model systems , we should gain new insight into which factors affect the number and nature of hybrid incompatibilities that accumulate between plant species .
| in just the last few years , plant geneticists have made tremendous progress in identifying the molecular genetic basis of postzygotic reproductive isolation . with more than a dozen genes
now cloned , it is clear that plant hybrid incompatibilities usually evolve via two or more mutational steps , as is predicted by the dobzhansky - muller model .
there is evidence that natural selection or random genetic drift can be responsible for these incompatibilities . |
williams beuren syndrome ( wbs ) is one of the most common recurrent microdeletion syndromes , with an estimated incidence varying from 1 in 7500 to 1 in 20,000 births 1 , 2 .
patients can be recognized clinically , given their characteristic features , encompassing congenital heart defect , typically supravalvular aortic stenosis ( svas ) and/or peripheral pulmonic stenosis , short stature / growth retardation , recognizable facial dysmorphism , developmental delay and intellectual disability , distinct behavioral phenotype ; and , in some instances , hypercalcemia .
facial dysmorphism includes a wide mouth , short nose with bulbous or upturned nasal tip , long philtrum , thick everted vermillion of the lips , bitemporal narrowing , periorbital fullness , full cheeks , small jaw , and the characteristic stellate pattern of the iris 3 , 4 .
the cognitive profile in wbs ranges from mild to severe i d 3 , 5 , 6 .
although acquisition of language is often delayed in wbs patients , they later develop good language skills and become talkative 7 , 8 .
behavioral issues in wbs comprise anxiety , hyperacousis , attention problems , hypersociability or friendly behavior , and strong affinity and interest to music 7 , 9 , 10 .
some cases are more difficult to diagnose , since they present with atypical features 11 , or because they have a different ethnicity where the features may be different from those observed in caucasians . for instance , african patients with wbs from cameroon have been reported with incomplete wbs facial dysmorphism 8 .
finally , cases with a deletion smaller or larger than the recurrent 1.5 mb 7q11.23 microdeletion may also present with a milder or more severe phenotype 12 .
we here report a wbs patient from central africa carrying the recurrent wbs microdeletion who presented with a typical facial dysmorphism but more severe intellectual disability and atypical behavioral phenotype due to a confounding factor , that is , meningitis with coma supposedly resulting in brain damage .
an 8.2 years old congolese boy was referred by his pediatrician to the genetic clinic at the university hospitals of the university of kinshasa , dr congo , for evaluation of intellectual disability , allegedly resulting from an episode of meningitis and coma .
he was born from unrelated , healthy parents after an uneventful pregnancy and delivery . at the age of 2 years , he was n't able to standup without support or build simple sentences although he could call names of his relatives .
four months later , he suffered from meningitis and remained in coma for 2 days .
his behavior changed , including hyperactivity , irritability , hypersensitivity to noise and episodes of aggressiveness against strangers .
in contrast , he was friendly toward familiar persons and demonstrated a strong attraction to music and musical sounds .
he was diagnosed with sequels from meningitis and was followed by a neurologist until the age of 8 years , when he was referred for a genetic evaluation . at that time , he had severe global growth delay with microcephaly ( ofc 47 cm ; 3.82 sd ) , short stature ( length 109 cm ; 3.43 sd ) and low weight ( 17 kg ; < 3 sd ) . family history revealed that two siblings of the patient ( fig
there were two other males with i d in the pedigree , related through their mothers .
patient 's phenotype consisted of a triangular face with low set ears , long philtrum , flat nose bridge , bulbous nose tip , anteverted nostrils , pronounced nasolabial folds , large mouth , thick and everted vermillion of the upper and lower lip , and a small jaw ( fig .
1 , lower panel ) . he also had widely spaced nipples , a small penis , bilateral cryptorchidism , and deep palmar creases .
( a ) pedigree showing three males with i d , consistent with the xlinked inheritance ; ( b ) clinical features .
note triangular face with low set ears , long philtrum , flat nasal bridge , bulbous nose tip , anteverted nostrils , pronounced nasolabial folds , large mouth , thick and everted vermillion of the upper and lower lip and a small jaw , widely spaced nipples , and deep palmar creases .
although the pedigree was consistent with xlinked i d , a clinical diagnosis of wbs was retained .
fluorescent in situ hybridization ( fish ) was performed on nuclei from peripheral white blood cells using the commercially available vysis lsi eln kit ( vysis ; abbott laboratories , abbott park , il ) .
a total of 100 nuclei were examined and a single eln was observed in all of them , whereas the control probe always showed two signals .
microarraycgh was performed using a stripped cytosure isca v2 array 8 60 k format ( ogt , oxford uk ) slide , the cytosure genomic dna labelling kits ( oxford , uk ) , and the oligo acgh & chiponchip hybridization kits ( agilent , santa clara , ca ) following manufacturers procedures .
this revealed the presence of a recurrent 1.57 mb deletion arr[hg19]7q11.23 ( 72,634,87474,203,685)1 ( fig .
( a ) two interphase nuclei showing a single signal for the elastin probe ( red ) and two signals for the control probe ( green ) ; ( b ) chromosome icon and the zoomin on the deleted region .
we report on a central african patient with typical gestalt for wbs . retrospectively , we uploaded facial photographs of the patient to face2gene ( http://www.fdna.com/face2gene/ )
. basically , this tool captures gestalt signatures from patient 's facial photograph and compares these to patients in the database , then reports the 10 most likely matches .
it is surprising that the patient was only diagnosed at age 8.2 years , despite the typical facial appearance .
mean age of diagnosis for wbs in western countries is 3.5 years 13 , 14 .
first , the facial dysmorphism in wbs evolves over age and may be discrete in infancy and early childhood 4 , 13 , 14 .
second , our patient had no svas , one of the most characteristic features often leading to the diagnosis 13 , 15 .
finally , severe phenotypes have been associated with larger deletions . as the deletion in our patient corresponds to the classical wbs microdeletion
, we assume that the severe speech deficit and profound intellectual disability in the patient are more likely the complications of the meningitis and coma .
it is thus undeniable that an acquired condition modified the clinical course of the patient toward the more severe and atypical phenotype for wbs causing the delay in diagnosis .
such overlap of genetic disease with an infectious disease is expected to be frequent in the developing world , where infections represent the major medical problem . as previously reported , infections are major cause for misdiagnosis of genetic diseases in developing countries 16 .
the present report supports the assumption that infections constitutes a major pitfall for genetic diagnosis and warns for clinicians working in developing world that a genetic disease may be masked by a intercurrent infectious disease . to date
given the incidence of 1/7500 for wbs 2 , this suggests that most cases remain undiagnosed .
this is most likely due to a lack of training in the field of dysmorphology and lack of access to genetic facilities in that part of the world .
| key clinical messagepatients with williams beuren syndrome can be recognized clinically , given the characteristic dysmorphism , intellectual disability , and behavior .
we report on a congolese boy with typical wbs facial characteristics .
he suffered meningitis and coma at the age of 2 years then subsequently presented with profound intellectual disability and atypical behavior .
the wbs was only made at age 8.2 years and confirmed with fish testing and microarraycgh .
the present report aims to warn clinicians that infections may associate and/or modify a genetic disease as this may be observed in developing countries given the prevalence of infectious diseases . |
a hypersensitivity reaction ( hsr ) is a host - dependent idiosyncratic adverse drug reaction ( adr ) that can not be predicted by the dose , frequency , or length of the treatment .
hsr is defined by the triad of fever , rash , and internal organ involvement [ 16 ] .
hsrs occur with an incidence of 1 in 1000 to 1 in 10000 drug exposures , with a mortality rate approaching 10% [ 58 ] .
symptoms of hsr can range from general manifestations , such as morbiliform rash , urticaria , angioedema , fever , malaise , anaphylaxis , bronchospasm , and erythema multiforme , to severe cutaneous adverse reactions ( scar ) such as drug - induced hypersensitivity syndrome ( dihs)/drug reaction with eosinophilia and systemic symptoms ( dress ) , and stevens - johnson syndrome ( sjs)/toxic epidermal necrolysis ( ten ) [ 2 , 47 , 911 ] .
cutaneous eruptions are reported in the vast majority of dihs / dress cases , often presenting as maculopapular rash or generalized erythematous rash [ 7 , 8 ] . the internal organ most often affected is the liver , with drug - induced liver injury ( dili ) presenting as anomalies in liver function tests or the presence of hepatomegaly .
aspartate aminotransferase ( ast ) and alanine aminotransferase ( alt ) were found to be increased an average of 9 - 10-fold above the upper normal limits in a literature review of 172 dress cases .
the presence of serum bilirubin raised > 3 times over the upper limit of normal along with aminotransferase elevations is associated with a more severe set of symptoms than isolated aminotransferase abnormalities alone , an observation known as hy 's law [ 12 , 13 ] .
other internal organs , such as kidneys , are less often affected [ 4 , 7 ] .
skin rash , liver involvement , high - grade fever , hypereosinophilia , lymphadenopathy , and the increased presence of atypical lymphocytes are symptoms associated with dihs / dress cases classified as
maculopapular rash , erythema multiforme , exfoliative dermatitis , acute generalized exanthematous pustular dermatosis - like eruptions , and erythroderma are types of cutaneous manifestations associated with dihs , while mucosal involvement is rare . on the other hand , skin and mucosal involvement characterizes sjs / ten .
a further distinction between dihs / dress and sjs / ten is the time to onset of symptoms , which is often delayed ( 4 - 5 weeks ) in the former , while it occurs in the early stages of drug exposure ( approximately 3 weeks ) in the latter .
carbamazepine was the drug most often associated with dress among 44 drugs linked to this reaction in 172 cases reported over a period of 12 years .
anticonvulsants such as carbamazepine , phenytoin , phenobarbital , lamotrigine , zonisamide , and sodium valproate are the class of drugs most often related to dihs / dress .
other pharmaceutical agents , including allopurinol , nonsteroidal anti - inflammatory drugs , chlormezanone , aminopenicillins , cephalosporins , quinolones cycline antibiotics , and antiretrovirals , as well as anti - infective sulfonamides , have also been implicated [ 68 , 11 , 1416 ] .
scar development results in patient hospitalization , and the culprit drug is interrupted on the first day of hospitalization .
the mean time to recovery was 6.4 weeks in 172 dress cases , while death was recorded in 9 ( 5.2% ) patients .
death was connected with a more severe course of reaction , particularly observed in older patients , and often due to liver failure .
while the precise mechanism of hsr is unknown , various theories involve the interplay between metabolic and immunologic factors [ 2 , 4 , 6 ] , genetic predisposition , and , more recently , infection with human herpes viruses ( hhv ) , particularly hhv-6 .
drug metabolites bind to cellular macromolecules such as proteins , creating covalent adducts that can serve as antigenic stimuli for the immune system [ 4 , 6 ] .
several genetic factors are believed to predispose certain individuals to this type of adverse reaction .
the subset of the population that is susceptible to hsrs carries defects in drug detoxifying pathways ( e.g. , epoxide hydrolase ) , such that a greater amount of a reactive drug metabolite is produced than that which can be detoxified [ 2 , 4 , 6 ] .
additional genetic predispositions that render certain individuals susceptible to hsrs include human leukocyte antigen ( hla ) alleles , which are part of the major histocompatibility complex ( discussed by neuman et al . ) .
reactivation of latent viral infections has been linked to the development of more severe hsr symptoms , particularly in the context of dihs / dress .
for example , hhv-6 reactivation was associated with more severe organ involvement and a prolonged course of disease in 62 of 100 dihs patients , compared with the remaining 38 patients who did not experience hhv-6 reactivation . in the same study , all five deaths and ten cases of renal failure were observed among the 62 patients with hhv-6 dna detected in the serum .
hhv-6 is a lymphotropic dna virus belonging to the betaherpesviridae subfamily [ 19 , 20 ] .
while little is known about hhv-6a , hhv-6b infection often occurs during the first 2 years of life and is associated with acute febrile illness in young children and exanthem subitum in infants [ 1926 ] .
hhv-6 has been increasingly recognized as a cause of hepatitis and liver failure , as well as increased graft rejection and consequent decreased patient survival [ 2026 ] .
following the initial infection , hhv-6b remains present in a latent phase , and infection can be reactivated during episodes of immunosuppression [ 20 , 22 , 24 , 26 ] . inherited forms of both hhv-6a and hhv-6b
this condition , known as chromosomally integrated hhv-6 ( cihhv-6 ) , occurs in 0.8% of control populations but is found in approximately 2% of patient populations in europe , united states , and the united kingdom , with a lower rate in japan .
cihhv-6 is characterized by the complete integration of the hhv-6 genome into the host germ line genome , such that cihhv-6 dna is found in every nucleated cell in the body [ 24 , 27 ] .
persistently high viral copy numbers in whole blood or serum are observed in individuals with this condition [ 28 , 29 ] .
the viral genome of cihhv-6 is transmitted vertically in the germline in a mendelian manner .
approximately 1% of newborns are diagnosed with congenital hhv-6 , all of which are infected with cihhv-6 , directly or indirectly . in a sample of 43 infants with congenital infection , the majority ( 86.0% ) were born with inherited cihhv-6 , while the remaining ( 14.0% ) were not born with cihhv-6 but became infected with hhv-6 transplacentally from their cihhv-6-positive mothers .
the cellular dna damage machinery responds to virus infection and the foreign genomes that accumulate in the nuclei of infected cells .
many dna viruses have been shown to manipulate the cellular dna damage response pathways in order to create environments conducive to their own replication .
arbuckle et al . showed that hhv-6 dna integrates specifically and efficiently into the telomere sequences of chromosomes .
the integrated virus can be chemically activated with trichostatin a , a stimulatory compound known to reactivate latent herpesviruses .
active hhv-6 replication is determined by the presence of hhv-6 dna in serum , plasma , or cerebrospinal fluid , or by reverse - transcriptase polymerase chain reaction ( rt - pcr ) in peripheral blood mononuclear cells ( pbmc ) .
rt - pcr is the sole technique that allows active hhv-6 replication to be differentiated from inactive cihhv-6 .
the present paper reviews recently published case reports of scar ( dihs / dress and sjs / ten ) and dili developed secondary to drug exposure , with a particular focus on the role of hhv-6 reactivation and cihhv-6 presence in the clinical outcome of the reaction . our principal aim is to address the role of hhv-6 reactivation in the course of hsrs , particularly in dili and dihs / dress .
in addition , we will focus on the complex cellular responses triggered by hhv-6 reactivation .
studies discussed in this paper were selected based on a pubmed search of english language papers published in the last 15 years ( 19972011 ) using keywords such as hhv-6 and hypersensitivity ,
hhv-6 reactivation and drug , hhv-6 and drug rash , and chromosomally integrated hhv-6 .
the selected case reports are part of a larger database of hsr cases with hhv-6 reactivation . in addition
, our laboratory has studied hsrs and their mechanisms for the last 25 years , and we discuss the cases based on our experience related to the laboratory and clinical manifestations .
following initial infection in early childhood , hhv-6 continues to exist in a latent phase in the body .
consequently , hhv-6 dna can be measured in different body compartments in hhv-6-positive individuals without active infection .
trace amounts of hhv-6 viral dna were also detected by real - time quantitative pcr in 91 ( 26.8% ) of 339 pediatric patients diagnosed and treated for acute lymphoblastic leukemia .
low rates of hhv-6b dna were detected in whole blood ( 8.0% ) , pbmcs ( 16.5% ) , and polymorphonuclear leukocytes ( 10.5% ) belonging to 200 volunteers .
the mean observed hhv-6b viral loads were 81 copies/10 cells in whole blood , 62 copies/10 cells in pbmcs , and 34.5 copies/10 cells in polymorphonuclear leukocytes .
hhv-6-dna was positive in only 27 ( 39.1% ) blood samples from 69 children undergoing elective tonsillectomy for moderate tonsillar hyperplasia or recurrent streptococcal infection without evidence of acute hhv-6 infection , while evidence of hhv-6 dna was found in 100% of tonsil samples in the same population .
the common features of dihs identified in a sample of 7 patients were high fever ( 39c ) in all patients , facial edema in all patients , diffuse lymphadenopathy in 5 ( 71.4% ) patients , hypereosinophilia ( > 0.5 10/l ) in 4 ( 57.1% ) patients , atypical circulating lymphocytes in 4 ( 57.1% ) patients , alt elevated > 3 times the reference level in 5 ( 71.4% ) patients , and hypogammaglobulinemia in 4 ( 57.1% ) patients .
the onset of dress took place after a mean period of 25 days ( 5 days6 weeks ) of therapy with the incriminated drug .
similar findings are reported in other cohorts of dihs patients [ 15 , 56 ] .
several studies suggest that hhv-6 reactivation can only occur in susceptible individuals under conditions of transient immunosuppression , such as those transiently associated with the onset of dihs [ 56 , 57 ] .
lower serum igg levels were observed in 10 adult anticonvulsant - induced hsr patients ( mean 745 mg / dl ) compared to 15 controls ( p < 0.001 ) .
similarly , circulating levels of b cells were also decreased in dihs patients , compared to controls [ 15 , 57 ] . in susceptible individuals , continuous therapy with anticonvulsants , which have been associated with dihs ,
moreover , hhv-6 reactivation may occur at the same time , but it can not always be detected in blood , since it takes 24 weeks for antibodies to appear after infection or reactivation . because decreases in circulating igg and b cell levels are not observed with other hypersensitivity conditions such as sjs / ten , depressed levels of igg and b cells can be used as biomarkers for the onset of dihs .
hhv-6 reactivation may be partially explained by the immunosuppressive effects of dihs - related drugs .
the magnitude of inflammation is often proportional to the clinical manifestations , while hypogammaglobulinemia and hhv-6 reactivation worsen the clinical course of the disease .
a more detailed description of disease progression is presented in case reports , some of which are summarized in table 1 [ 3552 ] .
edema , predominantly on the face , was observed with a relatively high frequency [ 35 , 37 , 3944 , 4749 , 52 ] , as were lymphadenopathy [ 35 , 36 , 39 , 4143 , 45 , 46 , 4951 ] , hypereosinophilia [ 3540 , 4252 ] , and atypical lymphocytes [ 35 , 36 , 3840 , 42 , 44 , 46 , 47 , 52 ] .
internal organ involvement manifested itself largely as liver dysfunction , with elevated levels of liver enzymes [ 3546 , 4850 , 52 ] and cholestatic hepatitis with hepatocellular insufficiency .
multiple organ failure was observed in a couple of studies [ 47 , 51 ] , one of which resulted in death .
hypogammaglobulinemia , another symptom of dihs , was observed in a number of studies as well [ 35 , 38 , 39 , 45 , 49 ] . the offensive drug
is immediately interrupted in dihs patients , and treatment with prednisolone is often used to reverse the condition .
symptoms begin to improve gradually , but both fever and skin manifestations often relapse approximately 2 - 3 weeks after the onset of dihs . there is a delay until antibodies to the virus are generated .
symptoms relapse coincides with detection of hhv-6 reactivation , measured by circulating anti - hhv-6 antibodies and hhv-6 dna [ 15 , 56 ] .
anti - hhv-6 igg levels are elevated after 3 weeks following the onset of the reaction ( time of drug withdrawal ) , but not after 2 weeks [ 58 , 59 ] . increases in hhv-6 dna in pbmcs were found to precede the increases in anti - hhv-6 igg levels in anticonvulsant hsr patients and were absent in controls .
the presence of hhv-6 dna in pbmcs was found to correlate with the magnitude of anti - hhv-6 antibody titers ( p = 0.0002 ) .
intermittent detection of hhv-6 dna in pbmcs was not indicative of illness , such that hhv-6 dna can be found in the pbmcs at low levels in immunocompetent children , with and without symptoms .
significant increases in anti - hhv-6 igg titers were noted in all 6 patients included in a study .
hhv-6 reactivation was observed between 17 and 27 days after the onset of the reaction .
the presence of hhv-6 dna in plasma or serum is evidence of active hhv-6 infection .
in addition , the presence of serum anti - hhv-6 igm and increases in the levels of anti - hhv-6 igg levels are used to differentiate primary hhv-6 infection ( i.e. , seroconversion ) from hhv-6 reactivation .
serum anti - hhv-6 igg was detected in 7 dress patients analyzed , with serum anti - hhv-6 igm detected in only 4 ( 57.1% ) patients .
seroconversion was diagnosed in 2 patients , in which serum anti - hhv-6 igm titers were initially undetectable and became detectable in subsequent serum samples , coinciding with increases in anti - hhv-6 igg titers .
seroconversion is described in detail in a 56-year - old male patient with aromatic anticonvulsant - induced hsr . aside from the presentation of fever and cutaneous symptoms , other features of hhv-6 reactivation
can include lymphadenopathy , hepatosplenomegaly , encephalitis caused by the virus , and severe lymphocytopenia .
hhv-6 replication in hepatocytes led to severe hepatitis , associated with elevated enzyme levels , in an 18-year - old female .
higher odds of detecting hhv-6 dna in pbmcs and liver biopsies were found in cases of fulminant hepatic failure or acute decompensation of chronic liver disease , compared to chronic liver disease ( p = 0.02 ) , in a sample of 23 children ( median age 24 months ) with acute liver failure .
similarly , higher hhv-6 dna levels were found in cases of fulminant hepatic failure or acute decompensation of chronic liver disease , compared to chronic liver disease cases ( p = 0.0043 ) .
tumor necrosis factor- ( tnf ) and interleukin- ( il ) 6 levels were initially elevated prior to hhv-6 reactivation in 4 ( 66.7% ) of 7 patients in a study .
il-6 levels were once again elevated after hhv-6 infection in 5 ( 83.3% ) of 6 patients . il-6 and tnf- are produced primarily by monocytes and macrophages , the likely reservoir cells of latent hhv-6 .
these cytokines likely play an important role in viral reactivation , yet the low number of patients analyzed in this study makes a definite answer impossible .
serum levels of il-5 , interferon ( ifn)- , and eosinophil cationic protein were increased on day 29 and decreased on day 39 during an episode of carbamazepine dihs .
an interesting case of drug sensitization was observed in a patient who developed symptoms of dihs within a few days of being exposed to the antibiotic cefaclor .
this patient was previously exposed to cefaclor during an episode of carbamazepine - induced dihs .
slight increases in il-5 , il-6 , il-10 , ifn- , and eosinophil cationic protein were elevated in sera on day 6 of cefaclor dihs .
a drug - induced lymphocyte stimulation test using pbmcs showed that the patient was sensitized to carbamazepine during the first episode , but not to cefaclor .
results of the test showed sensitivity to cefaclor during the second episode . during the first episode , carbamazepine - induced hsr was diagnosed 17 days after the start of carbamazepine , consistent with dihs .
also , symptoms persisted after the drug was interrupted , and the patient responded to pulsed intravenous ( iv ) methylprednisolone .
in contrast , the patient developed the symptoms much quicker during the second episode , during which time the symptoms were milder and disappeared faster after interruption of cefaclor .
the involvement of hhv-6 during the first episode was established with anti - hhv-6 igg titers and hhv-6 dna .
the magnitude of inflammatory cytokine increase was higher , and eosinophilia was more pronounced during the more severe carbamazepine - induced dihs , prompting aihara et al . to argue that hsr is mediated by both elevated inflammatory cytokine levels and activated eosinophils .
it is also possible that corticosteroids administered as initial treatment for drug toxicity are involved in the reactivation of hhv-6 , epstein - barr virus ( ebv ) , and cytomegalovirus ( cmv ) , which became active in the later stage of the disease .
anti - hhv-6 igg titers were elevated in 7 dihs patients 2138 days after the onset of the reaction .
an elevation in anti - cmv igg was observed in all patients 3554 days after the onset of the reaction .
only 2 patients experienced an elevation in anti - ebv igg , 37 and 48 days after the onset of the reaction .
antibodies to herpes simplex virus , varicella - zoster virus , human parvovirus b19 , rubella , and measles were not changed .
viral dna was detected in whole blood in all patients for whom anti - viral igg was observed , and in serum for 6 of 7 hhv-6 patients , 5 of 6 hhv-7 patients , and all cmv and ebv patients .
there appears to be a correlation between relapse of symptoms and detection of increased viral loads for the virus that gets reactivated first ( i.e. , hhv-6 or hhv-7 ) .
further immune system perturbations brought about by the initial hhv reactivation are believed to be triggers for subsequent reactivation of other hhvs .
cihhv-6 was described and quantified in healthy blood donors and various hospital populations ( e.g. , organ transplant recipients and immunosuppressed patients ) [ 28 , 6167 ] .
the mean viral loads in whole blood ranged from 10 copies / ml to 10 copies / ml [ 28 , 62 , 64 ] . vertical transmission of inherited chromosomal integration
in addition , there are reports of cihhv-6 acquisition through bone marrow transplant , followed by the presence of hhv-6 in every cell derived from hematopoietic stem cells [ 24 , 6971 ] .
it is not known if cihhv-6 can activate from its integrated state in vivo , but several lines of evidence suggest that this is a significant possibility .
integrated hhv-6 has been shown to be activated in vitro using histone deacetylase inhibitors and other compounds known to reactivate herpesviruses from latency .
as previously discussed , transmission of cihhv-6 from infected mothers to their non - cihhv-6 infants , via the placenta , is another indicator that cihhv-6 may be activated from its integrated state in vivo .
children suspected of encephalitis in the united kingdom had a four times greater rate of cihhv-6 than the general population , and liver transplant patients with cihhv-6 have higher rates of graft rejections and opportunistic infections .
marek 's disease virus , a herpesvirus associated with tumors in chickens , can reactivate from its integrated state in vitro .
if specific drugs can cause integrated virus to activate , then individuals with cihhv-6 may be at an increased risk for drug - induced hsr .
report the case of a 47-year - old japanese male with fever ( 38.8c ) and generalized erythematous rash 78 days after carbamazepine exposure .
mild hepatotoxicity ( alt 70 iu / l , ast 73 iu / l , -glutamyl transpeptidase 129 iu / l , and lactate dehydrogenase 686 u / l ) and hypogammaglobulinemia ( igg 609 mg / dl , iga 34 mg / dl and igm 27 mg / dl ) were measured .
an abdominal skin biopsy revealed hydropic and vacuolar degeneration of epidermal basal cells , lymphocytic infiltration in the epidermis , and a dense upper dermal infiltrate consisting mainly of mononuclear cells . anti - hhv-6 igg titers were increased 128-fold 6 weeks after the reaction onset , while anti - hhv-6 igm titers remained unchanged throughout the course of the reaction , pointing towards hhv-6 reactivation .
dna was persistently high in serum ( > 10000 copies / ml ) and whole blood .
hhv-6 dna levels in primary infection and acute reactivation are typically below 5.5 10 copies .
of interest , the only cases reported of viral loads above this level ( or in the same range as individuals with cihhv-6 ) appear to be those with graft - versus - host disease or dihs , two conditions that have very similar symptoms and courses [ 18 , 27 , 74 ] .
in the present paper , we review cases of hsr associated with hhv-6 reactivation . in the vast majority of patients ,
hhv-6 infection occurs during infancy or early childhood . following resolution of the primary infection ,
the virus remains latent in salivary glands , pbmcs , and the central nervous system .
reactivates in immunosuppressed individuals and is associated with fever , rash , encephalitis , bone marrow suppression , and transplanted organ rejection .
primary hhv-6 infection can be identified by the presence of hhv-6 dna in the serum , plasma , or cerebrospinal fluid , or reverse transcription pcr in the absence of elevated antibody titers .
reactivation is marked by detection of both viral dna and elevated anti - hhv-6 antibody titers .
as anti - hhv-6 igg levels increase during the course of the reaction , anti - hhv-6 igm levels remain constant around < 1 : 20 in patients with hhv-6 reactivation [ 42 , 75 ] .
hsrs seem to occur with almost a thousandfold higher incidence in aids patients compared to immunocompetent individuals exposed to the same medication , as there is a much higher exposure to drugs in these patients , as well as a much higher incidence of viral infections [ 76 , 77 ] . aside from a higher incidence of hsr
, hiv positivity can also lead to more severe symptoms , compared to hiv - negative patients .
in addition , an in vitro study showed increased dna viral replication in pbmcs belonging to dihs patients that were depleted for natural killer cells ( nk ) .
these findings point toward an extensive immune barrier aimed against dihs development , while immune defects in components of these pathways can explain why only a relatively small amount of the population exposed to a drug will develop the reaction .
suggest that hhv-6 reactivation is not a consequence , but rather a prerequisite for anticonvulsant hsr .
the expansion of cd4 t cells and cd8 t cells in response to hhv-6 reactivation seems to be of extreme importance in the development of multiple organ failure during the course of anticonvulsant hsr . from their immunological findings ,
believe that continuous anticonvulsant therapy leads to transient immune dysfunction , marked by decreased igg production .
while t cells protect against reactivation of viruses from a latent stage , antibodies prevent the dissemination of reactivating lytic virus .
therefore , dihs may occur when transient drops in b cell counts and immunoglobulin production allow hhv-6 to be reactivated from latency , in conjunction with the presence of drug - specific t cells .
the likely explanation for the lag between start of anticonvulsant therapy and onset of disease is that time is required for immunoglobulin levels to drop below a certain threshold .
an in vitro study using the human t cell line mt4 found that therapeutic doses of both sodium valproate and carbamazepine led to enhanced hhv-6b replication in a dose - independent manner , whereas phenytoin and sulfasalazine had no effects on hhv-6b replication or cell proliferation . in vivo ,
there have been reports of phenytoin and sulfonamide antibiotics leading to hhv-6 reactivation in dihs patients [ 42 , 43 , 47 , 52 ] . as such , while widespread , hhv-6 infection is not encountered in all dihs cases .
for example , hhv-6 dna was detected by pcr in blood or serum in a 25-year - old female treated with phenobarbital , while it was absent in a 21-year - old female treated with phenytoin .
matsuda et al . describe 2 cases of carbamazepine - induced dihs in which no viral reactivation was observed .
symptoms , including fever , skin eruptions , and liver involvement , occurred between 22 days and 4 weeks after carbamazepine initiation in the two patients , which is consistent with dihs
. furthermore , eosinophilia and a drop in white blood cell count were observed in both patients .
hhv-6 reactivation typically occurs 2 - 3 weeks after the development of rash in dihs patients [ 8 , 76 ] .
hhv-6 reactivation is often accompanied by relapse of fever and hepatitis . a cascade of viral reactivation
can then be started , with hhv-6 , ebv , and/or hhv-7 at the top , followed , with some delay , by other herpes viruses , particularly cmv .
reactivation of these viruses is followed by development or exacerbation of clinical symptoms of dihs , including various organ failures [ 8 , 76 , 80 ] .
of particular interest is cmv reactivation , which can be followed by transient fever , skin rash , myocarditis , pneumonia , or gastrointestinal bleeding .
clinical symptoms ( fever , hepatitis , and/or skin rash ) correspond to the appearance of detectable hhv-6 dna levels .
fever and/or hepatitis are the most common clinical features of hhv-6 reactivation in dihs patients .
based on these findings , hhv-6 reactivation leads to a more severe course of disease in patients with dihs .
hhv-6 reactivation is associated with more severe organ involvement and a prolonged course of the disease in patients with drug rash and systemic symptoms , compared with the remaining patients who did not experience hhv-6 reactivation .
significant detection of hhv-6 dna corresponds to the presence of anti - hhv-6 igg titers , and there is a positive correlation between hhv-6 dna detection and the development of fever and hepatitis . while hhv-6 reactivation is a common feature of dihs / dress , hhv-6 dna was not detected in 15 patients with generalized morbilliform / maculopapular drug reactions .
hhv-7 , cmv , or ebv dna was not detected in any of the patients included in this study . in situ hybridization
revealed hhv-6 mrna in a small subset of infiltrating mononuclear cells around blood vessels and appendages in dihs patients only .
similarly , only 1 ( 25.0% ) of 4 sjs / ten patients for whom data was available showed an increase in anti - hhv-6 igg titers from 1 : 10 to 1 : 1280 . aside from a lower likelihood of hhv-6 reactivation
, sjs / ten is marked by a quicker onset of reaction than dihs / dress ( 2 - 3 weeks versus a mean 34 days ) .
however , fever ( > 38.5c ) , leukocytosis , eosinophilia , and atypical lymphocytosis were common features of both sjs / ten and dihs / dress , whereas liver dysfunction was observed predominantly in sjs / ten patients and lymph node enlargement was observed predominantly in dihs / dress patients .
treatments for sjs / ten included systemic corticosteroids in 7 ( 87.5% ) patients and iv immunoglobulin in 1 ( 12.5% ) patient .
furthermore , teraki et al . warn that some of the clinical features of anticonvulsant - induced sjs / ten may differ from sjs / ten induced by other drugs , such as a more delayed onset of symptoms in the former category . additionally , the rate of hepatic dysfunction and hematological anomalies was also higher than that described with sjs / ten induced by other drugs .
hhv-6 reactivation and atypical lymphocytosis were observed in dihs patients predominantly , compared to sjs / ten patients .
the precise morphological nature of the skin reaction is another criterion used to differentiate dihs from sjs / ten .
carbamazepine was associated with 12 ( 52.2% ) of 23 anticonvulsant hsr cases reported in a cohort study .
maculopapular exanthematous eruptions were observed in 15 patients , erythroderma in 11 patients , exfoliative dermatitis in 3 patients , bullous eruption in 1 patients , urticaria in 1 patient , and purpura ( vasculitis ) in 2 patients .
there was no relationship between the drug used and the length or severity of the reaction .
in addition to the skin , 20 ( 87.0% ) patients had at least one other organ affected by the reaction , while 8 ( 34.8% ) patients had at least two other organs affected by the reaction .
dili was observed in 12 ( 52.2% ) patients , and kidney involvement was observed in 8 ( 34.8% ) patients .
other affected organs include the spleen ( splenomegaly ) , the lung , the thyroid ( hypothyroidism ) , the heart , and the pancreas .
drug - drug interactions are particularly problematic when several pharmaceutical agents capable of inducting hsr are administered simultaneously . for example , conilleau et al .
report the case of a child exposed intermittently to two anticonvulsants prior to developing dihs . despite ethosuximide
being interrupted prior to hospitalization and sodium valproate being associated more often with dihs , it appears that the reaction was caused by the combination of these two structurally unrelated anticovulsants , as a patch test was positive for both ethosuximide and sodium valproate . in a separate case , an infant that was exposed to carbamazepine , phenytoin , and phenobarbital presented with initial hhv-6 infection . despite the patient being diagnosed with phenobarbital hypersensitivity , all three of these aromatic antiepileptic drugs have the potential to cause hsr .
minocycline - induced dihs with ebv reactivation was reported in a 24-year - old african american female .
sequential activation of hhv-6 , hhv-7 , herpes simplex virus , and cmv was observed in a 46-year - old male who developed dihs after being exposed to cyanamide , a drug used to control alcoholism .
hhv-6 reactivation was observed in 2 elderly patients with allopurinol dihs [ 26 , 79 ] , and ebv reactivation was observed in a 40-year - old black male with allopurinol dihs and pancreatitis .
the cause of the reaction was unknown in an 18-year - old female with sjs / ten exposed to valacyclovir , promethazine , synthroid , belladonna , and orthotricycline .
in addition to anticonvulsants , patients can be exposed to additional medication for the treatment of comorbid diseases , opportunistic infections , or even for the management of hsr symptoms ( e.g. , antibiotics or nonsteroidal inflammatory drugs administered for the treatment of fever ) .
furthermore , epilepsy patients are often exposed to more than one anticonvulsant , most of which are capable of causing an hsr . while medication is interrupted at the time of hsr diagnosis and the patient is encouraged to avoid rechallenge with the drug that was deemed to have initiated the reaction , it is still important that all medication is considered and tested individually for its capacity to initiate an hsr .
investigators seldom check to see if there is an additive effect between the incriminated anticonvulsant and other medication , or if the anticonvulsant caused the reaction on its own . also , it is unlikely that hhv-6 reactivation is measured upon admission . as a result , it is difficult to establish whether the drug or the virus initiates the reaction .
the same investigators report the case of a 46-year - old woman who developed dihs 4.5 months after initiating therapy with zonisamide and corticosteroids .
since the reaction occurred well outside the time frame characteristic of dihs and since reactivation of herpes viruses was not measured , it is possible that this phenomenon could have created the conditions necessary for dihs to occur in this patient with long - term exposure to anticonvulsant therapy .
the presence of proinflammatory cytokines during dili and scar is supported by findings that a significantly higher number of eosinophils was found both in the blood and tissue of patients with a drug - induced maculopapular exanthems , compared to control subjects with normal skin and skin from patients with psoriasis .
the presence of hla - a*3101 allele and homozygosity for epoxide hydrolase 1 ( ephx1 ) single nucleotide polymorphism rs1051740 ( t - c ) in exon 3 , associated with modifications in epoxide hydrolase activity , were found in a patient with anticonvulsant hsr , possibly triggered by hhv reactivation , suggesting a genetic predisposition to hsr upon hhv reactivation .
hhv-6 reactivation can be further observed in the absence of an hsr and is primarily associated with hepatotoxicity .
phillips et al . were able to show with the help of transmission electron microscopy that full and empty virus capsids accumulate in the nucleus of hepatocytes , while viral particles bud out into the nuclear envelope and the cytoplasm .
over the years , several drugs were used to treat hhv reactivations per se , as well as opportunistic infections in hsr patients with hhv reactivation , including idoxuridine , cytarabine , and vidarabine .
vancomycin was used to treat an opportunistic infection with staphylococcus aureus in a dihs patient with hhv-6 reactivation . however , treatment against hhv-6 reactivation is often withheld out of fear of worsening the condition due to an increasing number of drugs that the patient is exposed to .
drug - induced adverse reactions represent a concern for patients , clinicians , the pharmaceutical industry , and health providers .
interestingly , very little mention of hhv-6 reactivation is made in a comprehensive article discussing dili , including acute liver failure in major dili registries from europe and the united states . in conclusion
, this paper provides a multifaceted assessment of drugs implicated in hsr that have been reported to induce hhv-6 reactivation .
this information may facilitate the possible link between the diagnosis of scar and dili , and hhv-6 reactivation . | background .
there are challenges in the clinical diagnosis of drug - induced injury and in obtaining information on the reactivation of human herpes viruses ( hhv ) during idiosyncratic adverse drug reactions
. objectives .
( i ) to develop a unified list of drugs incriminated in drug - induced hepatotoxicity and severe cutaneous reactions , in which drug hypersensitivity leads to hhv-6 reactivation and further complication of therapy and recovery and ( ii ) to supplement the already available data on reporting frequencies of liver- or skin - induced cases with knowledge of individual case reports , including hhv-6 reactivation and briefly introducing chromosomally integrated hhv-6 .
data sources and extraction .
drugs identified as causes of ( i ) idiosyncratic reactions , ( ii ) drug - induced hypersensitivity , drug - induced hepatotoxicity , acute liver failure , and stevens - johnson syndrome , and ( iii ) human herpes virus reactivation in pubmed since 1997 have been collected and discussed .
results .
data presented in this paper show that hhv-6 reactivation is associated with more severe organ involvement and a prolonged course of disease .
conclusion . this analysis of hhv-6 reactivation associated with drug - induced severe cutaneous reactions and hepatotoxicity will aid in causality assessment and clinical diagnosis of possible life - threatening events and will provide a basis for further patient characterization and therapy . |
animal testing procedures were approved by the animal care and use committee of the national institute on aging .
methods are described in detail in the supplementary material , available in an online appendix at http://diabetes.diabetesjournals.org/cgi/content/full/db09-0807/dc1 .
serial sections ( 10-m thickness ) were cut from tissues containing circumvallate , foliate , and fungiform papillae , using a cryostat ( microm ; thermo scientific ) . to obtain systematic samples without bias throughout the papillae ,
taste buds are 80100 m in length , therefore sampling every tenth section ensures that no two sections will be from the same taste bud .
approximately 100120 taste buds per group were analyzed immunohistochemically as described previously ( 11,29 ) . only nuclear profile
the total number of cells in the section was determined by counting the number of to - pro-3stained nuclei present in each taste bud .
the percentage of immunoreactive taste cells was calculated by : number of immunoreactive taste cells / total number of taste cells in each taste bud .
plasma levels of gut and appetite hormones were measured using a mouse multiplex assay kit ( millipore ) according to the manufacturer 's instructions .
quantification of pancreatic immunohistochemistry images was performed in matlab ( mathworks ) as described previously ( 30 ) .
hypothalamic protein levels of various factors involved in energy regulation were determined by western blot analyses .
vip knockout and wild - type ( n = 8) mice were habituated to the laboratory environment for 30 min each day prior to testing .
the same animals were used for both the taste behavioral tests and analyses of physiological parameters ( pancreatic function , gut / appetite plasma hormone analyses ) .
test stimuli consisted of various concentrations of sucrose ( 25 , 75 , 150 , 300 , and 600 mmol / l ; fisher scientific ) , sodium chloride ( nacl : 100 , 200 , 400 , 600 , 800 , and 1,000 mmol / l ; sigma ) , denatonium benzoate ( 0.001 , 0.01 , 0.1 , 0.3 , 1 , 5 , and 10
mmol / l ; sigma ) , and citric acid ( 0.01 , 0.1 , 1 , 3 , 5 , 10 , 30 , and 100 mmol / l ; fisher scientific ) .
brief - access taste testing took place in a davis ms-160 gustometer ( dilog instruments ) as previously described ( 11,3134 ) .
brief - access procedures minimize postingestive effects that may confound other assays such as intake tests ( 33 ) . to test salty , sour , and bitter stimuli responses , mice were water deprived prior to testing .
for testing of the sweet stimulus , mice were both food and water restricted ( 2.5 ml of water , 1 g of food ) prior to testing .
all mice were tested with a single tastant at various concentrations per trial , to prevent the mice from being affected by prior experience .
animal testing procedures were approved by the animal care and use committee of the national institute on aging .
methods are described in detail in the supplementary material , available in an online appendix at http://diabetes.diabetesjournals.org/cgi/content/full/db09-0807/dc1 .
methods are described in detail in the supplementary material . serial sections ( 10-m thickness )
were cut from tissues containing circumvallate , foliate , and fungiform papillae , using a cryostat ( microm ; thermo scientific ) . to obtain systematic samples without bias throughout the papillae ,
taste buds are 80100 m in length , therefore sampling every tenth section ensures that no two sections will be from the same taste bud .
approximately 100120 taste buds per group were analyzed immunohistochemically as described previously ( 11,29 ) . only nuclear profile
the total number of cells in the section was determined by counting the number of to - pro-3stained nuclei present in each taste bud .
the percentage of immunoreactive taste cells was calculated by : number of immunoreactive taste cells / total number of taste cells in each taste bud .
plasma levels of gut and appetite hormones were measured using a mouse multiplex assay kit ( millipore ) according to the manufacturer 's instructions .
quantification of pancreatic immunohistochemistry images was performed in matlab ( mathworks ) as described previously ( 30 ) .
hypothalamic protein levels of various factors involved in energy regulation were determined by western blot analyses .
vip knockout and wild - type ( n = 8) mice were habituated to the laboratory environment for 30 min each day prior to testing .
the same animals were used for both the taste behavioral tests and analyses of physiological parameters ( pancreatic function , gut / appetite plasma hormone analyses ) .
test stimuli consisted of various concentrations of sucrose ( 25 , 75 , 150 , 300 , and 600 mmol / l ; fisher scientific ) , sodium chloride ( nacl : 100 , 200 , 400 , 600 , 800 , and 1,000 mmol / l ; sigma ) , denatonium benzoate ( 0.001 , 0.01 , 0.1 , 0.3 , 1 , 5 , and 10
mmol / l ; sigma ) , and citric acid ( 0.01 , 0.1 , 1 , 3 , 5 , 10 , 30 , and 100 mmol / l ; fisher scientific ) .
brief - access taste testing took place in a davis ms-160 gustometer ( dilog instruments ) as previously described ( 11,3134 ) .
brief - access procedures minimize postingestive effects that may confound other assays such as intake tests ( 33 ) . to test salty , sour , and bitter stimuli responses , mice were water deprived prior to testing .
for testing of the sweet stimulus , mice were both food and water restricted ( 2.5 ml of water , 1 g of food ) prior to testing .
all mice were tested with a single tastant at various concentrations per trial , to prevent the mice from being affected by prior experience .
gross taste bud morphology and taste cell number were assessed in wild - type and vip knockout mice using classical taste cell type specific immunocytochemical markers ( supplementary tables 1 and 2 ) .
compared with wild - type mice , vip knockout mice showed no gross taste bud abnormalities and expressed similar distribution patterns of taste cell specific markers ( fig .
vip immunoreactivity was present in the taste cells of wild - type animals ( fig .
the lack of vip , therefore , did not significantly affect initial taste cell development and number .
expression of taste cell markers and vip in circumvallate papillae of wild - type ( wt ) and vip knockout ( ko ) mice .
type ii cell marker ( plc2 ) ( a and b ) , type iii cell marker ( ncam ) ( c and d ) , and type iv cell marker ( shh ) ( e and f ) are expressed in taste cells of both wild - type and vip knockout mice .
g and h : vip ( red ) is expressed in a subset of taste cells of wild - type mice .
i and j : vip is not expressed within the vip knockout mouse taste bud .
( a high - quality digital representation of this figure is available in the online issue . ) both vpac1/2 receptors were primarily expressed in phospholipase c2 ( plc2)-positive taste cells ( type ii cells : fig . 2 ; supplementary figs . 2 and 3 ) .
the presence of vip and its cognate receptors in type ii taste cells suggests that vip signaling is local to the taste bud and could likely affect tongue - localized gustation .
coexpression of vip receptors ( vpac1/vpac2 ) and plc2 in circumvallate papillae of wild - type ( wt ) and vip knockout ( ko ) mice .
a c : vpac1 ( red ) and plc2 ( green ) are colocalized in a subset of plc2-positive cells ( yellow cells ) in wild - type mice .
d f : vpac1 ( red ) and plc2 ( green ) are colocalized in a subset of plc2-positive cells ( yellow cells ) in vip knockout mice .
i : vpac2 ( red ) and plc2 ( green ) are colocalized in a subset of plc2-positive cells ( yellow cells ) in wild - type mice .
l : vpac2 ( red ) and plc2 ( green ) are colocalized in a subset of plc2-positive cells ( yellow cells ) in vip knockout mice .
( a high - quality digital representation of this figure is available in the online issue . )
vip is coexpressed with the taste transduction marker -gustducin and with the t1r2 gpcr ( 20 ) .
the majority of vip - immunoreactive taste cells also coexpresses -gustducin , although a subset also expresses t1r2 . -gustducin and t1r2 are markers for type ii cells , and based on this specific expression pattern , we hypothesized that vip could be involved in the transduction of sweet and bitter stimuli . to investigate this
, we first determined the expression patterns in the wild - type and vip knockout mice of additional hormones and receptors known to play a functional role in sweet taste coding , that is , the t1r3 sweet stimulant receptor , glp-1 , and leptin receptor ( ob - rb ) ( fig .
f ) . both wild - type and vip knockout mice showed similar expression patterns of t1r3 in the circumvallate papillae ( fig .
interestingly , we found that glp-1 expression was significantly increased in the taste cells of vip knockout mice compared with wild type ( fig .
leptin receptor expression was markedly reduced in vip knockout mice compared with wild type ( fig .
peripheral leptin has been shown to suppress sweet taste preference through its action on the leptin receptors in the taste cells ( 2628 ) .
elevation in glp-1 expression and the reduction in leptin receptor expression strongly indicates that vip could play a role in modulating sweet taste . taken together ,
it is apparent that there are significant differences in the numbers of glp-1 and ob - rb immunopositive cells between wild - type and vip knockout mice ( fig .
3 g , supplementary table 2 ) . to enhance our understanding of these alterations in the taste cells of vip knockout mice
, we next determined whether other systemic alterations in appetite / energy - regulatory hormones occur in vip knockout mice .
expression of taste - related proteins in circumvallate papillae of wild - type ( wt ) and vip knockout ( ko ) mice .
a and b : sweet taste receptor ( t1r3 , green ) is expressed in both wild - type and vip knockout mice .
c and d : glp-1 ( green ) is expressed in wild - type and vip knockout mice .
e and f : leptin receptor ( green ) is expressed in wild - type and vip knockout mice .
g : the percentage of immunopositive cells quantified in both wild - type and vip knockout mice for the following markers : plc2 , ncam , shh , t1r3 , glp-1 , and leptin receptor .
cells were scored as immunoreactive only if a nuclear profile was present within the cell .
( a high - quality digital representation of this figure is available in the online issue . )
as we detected reduced leptin receptor expression in vip knockout mouse taste cells compared with wild type , we measured plasma leptin levels in these mice .
circulating leptin can bind to and activate the leptin receptor on taste cells , where it acts to specifically inhibit gustatory responses to sweet substances , without affecting responses to sour , salty , and bitter substances in lean mice ( 2628 ) . in vip knockout mice , both nonfasting and fasting glucose levels
in addition , we noted a significant elevation in fasting insulin in vip knockout mice compared with wild type ( fig .
we also found that plasma leptin levels were significantly elevated in vip knockout mice compared with wild type ( fig .
4e ) were not different between wild - type and vip knockout mice , whereas vip knockout mice possessed considerably higher plasma gastrointestinal polypeptide ( fig .
4h ) . plasma pancreatic polypeptide ( pp ) levels between wild - type and vip knockout mice were not significantly different despite a trend for lower pp levels in the vip knockout mice ( fig .
4i ) . vip knockout ( ko ) mice possess alterations in multiple hormones controlling appetite and energy balance .
multiplex hormone analysis from plasma samples of wild - type ( wt ) and vip knockout mice reveal multiple distinctions between the two animal groups .
a and b : significantly higher nonfasting and fasting glucose occurs in the vip knockout mice .
the vip knockout mice also possessed significantly higher plasma insulin ( c ) and leptin ( d ) levels .
no significant differences between wild - type and vip knockout for amylin were noted ( e ) .
vip knockout mice demonstrated higher levels of plasma gastrointestinal polypeptide ( f ) , glp-1 ( g ) , and peptide tyrosine tyrosine ( pyy ) ( h ) .
no significant difference in the plasma levels of pp were noted between wild - type and vip knockout mice ( i ) .
each panel depicts means sem from eight animals in each group . * p < 0.05 , * * p < 0.01 . as vip knockout mice possessed significantly elevated plasma insulin levels
compared with wild - type mice ( p < 0.01 ) , we next investigated pancreatic islet size and distribution of -cells and -cells .
vip knockout mice showed similar total -cell area compared with wild - type ( fig .
no significant difference in the total percentage of - or -cells in the islets ( fig .
5d ) and the number of small- , middle- , and large - sized islets between wild - type and vip knockout mice was observed ( fig .
pancreatic islet sizing in vip knockout ( ko ) and wild - type ( wt ) mice .
representative pancreatic islets from wild - type ( a ) or vip knockout ( b ) mice demonstrate similar insulin ( red fluorescence ) and glucagon ( green fluorescence ) expression profiles .
c : mean islet area ( mm ) measured in wild - type or vip knockout pancreata .
d : similar mean -cell ( ) and -cell ( ) percentage in the islets of vip knockout and wild - type mice .
e : relative distribution of the different islet sizes ( 1 = small ; 2 = middle ; 3 = large ) for vip knockout and wild - type mice .
( a high - quality digital representation of this figure is available in the online issue . ) as a considerable amount of our molecular / immunocytochemical data suggested a role for vip in taste modulation , we next investigated vip 's role in taste perception .
we tested the ability of wild - type and vip knockout mice to detect prototypical taste stimuli , that is , sweet ( sucrose ) , sour ( citric acid ) , salty ( sodium chloride ) , and bitter ( denatonium benzoate ) .
vip knockout mice displayed significantly altered concentration - dependent licking for sucrose ( p = 0.0108 between mean half - maximal effective concentration [ ec50 ] values ) and denatonium benzoate ( p = 0.0003 between mean ec50 values ) compared with wild - type mice , demonstrated by their lower ec50 values compared with wild type ( fig .
vip knockout mice also showed concentration - dependent changes in licking for citric acid ( p = 0.023 between mean ec50 values ) ( fig .
6b ) , but there was no significant alteration for sodium chloride in the vip knockout mice compared with wild - type mice ( fig .
these results indicate that vip plays a specific functional role in modulating taste perception of sweet , bitter , and sour stimuli .
altered sweet , bitter , and sour taste responses of wild - type ( wt ) and vip knockout ( ko ) mice in brief - access taste tests ( a d ) .
taste responses , expressed as tastant / water lick ratios and as a function of stimulus concentration , of vip knockout ( closed triangle , dotted line ) and wild - type ( open square , solid line ) to sucrose ( a ) , citric acid ( ca ; b ) , denatonium benzoate ( db ; c ) , and sodium chloride ( nacl ; d ) .
our data indicate that there is an alteration in the biochemical , but not structural , nature of the tongue of vip knockout mice , as well as complex alterations in circulating metabolic hormones . one paradoxical finding , however , was the reduction in leptin receptor levels in the tongue .
vip knockout mice demonstrate several features reminiscent of insulin resistance ( e.g. , high fasting glucose and insulin levels ) .
perhaps this change in taste preference induced by increased tongue glp-1 levels and reduced leptin receptor levels could be a compensatory mechanism engendered to ameliorate their dysglycemia by attempting to reduce sugar intake .
high circulating levels of leptin would be expected to reduce sweet taste preference ( 25 ) , but with a significant ablation of the lingual ob - rb , this would be prevented . however ,
if this ob - rb ablation was universal , especially in the hypothalamus , then the animal would likely consume even more food and thus exacerbate its dysglycemic state .
we , therefore , assessed the expression level of ob - rb in the hypothalami of wild - type and vip knockout mice .
in contrast to the tongue , no difference in ob - rb expression between wild - type and vip knockout hypothalami was observed ( fig .
in addition , we found no difference in insulin receptor substrate 2 ( irs2 ) , t1r3 receptor , or glp-1 expression in the hypothalami of wild - type or vip knockout mice ( fig .
vip knockout mice showed a small reduction in food intake compared with wild - type mice ( fig .
7f ) and there was no significant difference in body weight between vip knockout and wild - type mice ( fig .
thus , it appears that the vip knockout mice possess a complex physiological reaction to the imposed loss of vip , one that may , in part , attempt to ameliorate their dysglycemia .
vip knockout mice show an increase in sweet taste preference and have elevated circulating leptin levels , which together could potentially be a mechanism to prevent additional excessive sugary food intake ( summarized in fig .
however , this mechanism would succeed only if there was localized lingual loss of the ob - rb , which was observed in the vip - null mice .
normal leptin receptor and irs2 expression in the hypothalami of wild - type ( wt ) and vip knockout ( ko ) mice .
a : representative western blots of three wild - type ( 13 ) or vip knockout ( 13 ) mice hypothalami .
the tissue extracts were probed with specific antisera for the leptin receptor ( ob - rb ) , irs2 , and -actin as a loading control . for the ob - rb blot ,
a plasma membrane extract was used , whereas cytoplasmic tissue extracts were used for the irs2 and actin blots .
b e : mean sem of the relative band intensities for the specific western blots measured as relative absorbance units minus background absorbance per square pixel ( au - b / px ) .
f : mean food intake ( g ) in wild - type and vip knockout mice .
values are expressed as means sem . a diagrammatic summary of the complex physiological parameters observed in the vip knockout ( ko ) mice compared with the wild - type ( wt ) controls . the vip knockout mice present with a diabetic - like state , and they actively attempt to ameliorate this condition with a series of physiological alterations ( high circulating leptin , low tongue ob - rb expression , high tongue glp-1 expression , normal hypothalamic ob - rb expression ) that in concert could attenuate the animals ' euglycemic disruption by enhancing their sweet taste and potentially reducing their desire to consume sweet foods .
gross taste bud morphology and taste cell number were assessed in wild - type and vip knockout mice using classical taste cell type specific immunocytochemical markers ( supplementary tables 1 and 2 ) .
compared with wild - type mice , vip knockout mice showed no gross taste bud abnormalities and expressed similar distribution patterns of taste cell specific markers ( fig .
vip immunoreactivity was present in the taste cells of wild - type animals ( fig .
the lack of vip , therefore , did not significantly affect initial taste cell development and number .
expression of taste cell markers and vip in circumvallate papillae of wild - type ( wt ) and vip knockout ( ko ) mice .
type ii cell marker ( plc2 ) ( a and b ) , type iii cell marker ( ncam ) ( c and d ) , and type iv cell marker ( shh ) ( e and f ) are expressed in taste cells of both wild - type and vip knockout mice .
g and h : vip ( red ) is expressed in a subset of taste cells of wild - type mice .
i and j : vip is not expressed within the vip knockout mouse taste bud .
( a high - quality digital representation of this figure is available in the online issue . )
both vpac1/2 receptors were primarily expressed in phospholipase c2 ( plc2)-positive taste cells ( type ii cells : fig . 2 ; supplementary figs . 2 and 3 ) .
the presence of vip and its cognate receptors in type ii taste cells suggests that vip signaling is local to the taste bud and could likely affect tongue - localized gustation .
coexpression of vip receptors ( vpac1/vpac2 ) and plc2 in circumvallate papillae of wild - type ( wt ) and vip knockout ( ko ) mice .
a c : vpac1 ( red ) and plc2 ( green ) are colocalized in a subset of plc2-positive cells ( yellow cells ) in wild - type mice .
d f : vpac1 ( red ) and plc2 ( green ) are colocalized in a subset of plc2-positive cells ( yellow cells ) in vip knockout mice .
i : vpac2 ( red ) and plc2 ( green ) are colocalized in a subset of plc2-positive cells ( yellow cells ) in wild - type mice .
l : vpac2 ( red ) and plc2 ( green ) are colocalized in a subset of plc2-positive cells ( yellow cells ) in vip knockout mice .
( a high - quality digital representation of this figure is available in the online issue . )
vip is coexpressed with the taste transduction marker -gustducin and with the t1r2 gpcr ( 20 ) .
the majority of vip - immunoreactive taste cells also coexpresses -gustducin , although a subset also expresses t1r2 . -gustducin and t1r2 are markers for type ii cells , and based on this specific expression pattern , we hypothesized that vip could be involved in the transduction of sweet and bitter stimuli . to investigate this
, we first determined the expression patterns in the wild - type and vip knockout mice of additional hormones and receptors known to play a functional role in sweet taste coding , that is , the t1r3 sweet stimulant receptor , glp-1 , and leptin receptor ( ob - rb ) ( fig . 3a
f ) . both wild - type and vip knockout mice showed similar expression patterns of t1r3 in the circumvallate papillae ( fig .
interestingly , we found that glp-1 expression was significantly increased in the taste cells of vip knockout mice compared with wild type ( fig .
leptin receptor expression was markedly reduced in vip knockout mice compared with wild type ( fig .
peripheral leptin has been shown to suppress sweet taste preference through its action on the leptin receptors in the taste cells ( 2628 ) .
elevation in glp-1 expression and the reduction in leptin receptor expression strongly indicates that vip could play a role in modulating sweet taste . taken together ,
it is apparent that there are significant differences in the numbers of glp-1 and ob - rb immunopositive cells between wild - type and vip knockout mice ( fig .
3 g , supplementary table 2 ) . to enhance our understanding of these alterations in the taste cells of vip knockout mice , we next determined whether other systemic alterations in appetite / energy - regulatory hormones occur in vip knockout mice .
expression of taste - related proteins in circumvallate papillae of wild - type ( wt ) and vip knockout ( ko ) mice .
a and b : sweet taste receptor ( t1r3 , green ) is expressed in both wild - type and vip knockout mice .
c and d : glp-1 ( green ) is expressed in wild - type and vip knockout mice .
e and f : leptin receptor ( green ) is expressed in wild - type and vip knockout mice .
g : the percentage of immunopositive cells quantified in both wild - type and vip knockout mice for the following markers : plc2 , ncam , shh , t1r3 , glp-1 , and leptin receptor .
cells were scored as immunoreactive only if a nuclear profile was present within the cell .
values are the means sem . * * p < 0.01 , * * * p < 0.001 .
( a high - quality digital representation of this figure is available in the online issue . )
as we detected reduced leptin receptor expression in vip knockout mouse taste cells compared with wild type , we measured plasma leptin levels in these mice .
circulating leptin can bind to and activate the leptin receptor on taste cells , where it acts to specifically inhibit gustatory responses to sweet substances , without affecting responses to sour , salty , and bitter substances in lean mice ( 2628 ) . in vip knockout mice , both nonfasting and fasting glucose levels
in addition , we noted a significant elevation in fasting insulin in vip knockout mice compared with wild type ( fig .
we also found that plasma leptin levels were significantly elevated in vip knockout mice compared with wild type ( fig .
4e ) were not different between wild - type and vip knockout mice , whereas vip knockout mice possessed considerably higher plasma gastrointestinal polypeptide ( fig .
4h ) . plasma pancreatic polypeptide ( pp ) levels between wild - type and vip knockout mice were not significantly different despite a trend for lower pp levels in the vip knockout mice ( fig .
4i ) . vip knockout ( ko ) mice possess alterations in multiple hormones controlling appetite and energy balance .
multiplex hormone analysis from plasma samples of wild - type ( wt ) and vip knockout mice reveal multiple distinctions between the two animal groups .
a and b : significantly higher nonfasting and fasting glucose occurs in the vip knockout mice .
the vip knockout mice also possessed significantly higher plasma insulin ( c ) and leptin ( d ) levels .
no significant differences between wild - type and vip knockout for amylin were noted ( e ) .
vip knockout mice demonstrated higher levels of plasma gastrointestinal polypeptide ( f ) , glp-1 ( g ) , and peptide tyrosine tyrosine ( pyy ) ( h ) .
no significant difference in the plasma levels of pp were noted between wild - type and vip knockout mice ( i ) .
as vip knockout mice possessed significantly elevated plasma insulin levels compared with wild - type mice ( p < 0.01 ) , we next investigated pancreatic islet size and distribution of -cells and -cells .
vip knockout mice showed similar total -cell area compared with wild - type ( fig .
no significant difference in the total percentage of - or -cells in the islets ( fig .
5d ) and the number of small- , middle- , and large - sized islets between wild - type and vip knockout mice was observed ( fig .
pancreatic islet sizing in vip knockout ( ko ) and wild - type ( wt ) mice .
representative pancreatic islets from wild - type ( a ) or vip knockout ( b ) mice demonstrate similar insulin ( red fluorescence ) and glucagon ( green fluorescence ) expression profiles .
c : mean islet area ( mm ) measured in wild - type or vip knockout pancreata .
d : similar mean -cell ( ) and -cell ( ) percentage in the islets of vip knockout and wild - type mice .
e : relative distribution of the different islet sizes ( 1 = small ; 2 = middle ; 3 = large ) for vip knockout and wild - type mice .
( a high - quality digital representation of this figure is available in the online issue . )
as a considerable amount of our molecular / immunocytochemical data suggested a role for vip in taste modulation , we next investigated vip 's role in taste perception .
we tested the ability of wild - type and vip knockout mice to detect prototypical taste stimuli , that is , sweet ( sucrose ) , sour ( citric acid ) , salty ( sodium chloride ) , and bitter ( denatonium benzoate ) .
vip knockout mice displayed significantly altered concentration - dependent licking for sucrose ( p = 0.0108 between mean half - maximal effective concentration [ ec50 ] values ) and denatonium benzoate ( p = 0.0003 between mean ec50 values ) compared with wild - type mice , demonstrated by their lower ec50 values compared with wild type ( fig .
vip knockout mice also showed concentration - dependent changes in licking for citric acid ( p = 0.023 between mean ec50 values ) ( fig .
6b ) , but there was no significant alteration for sodium chloride in the vip knockout mice compared with wild - type mice ( fig .
taken together , these results indicate that vip plays a specific functional role in modulating taste perception of sweet , bitter , and sour stimuli . altered sweet , bitter , and sour taste responses of wild - type ( wt ) and vip knockout ( ko ) mice in brief - access taste tests ( a d ) .
taste responses , expressed as tastant / water lick ratios and as a function of stimulus concentration , of vip knockout ( closed triangle , dotted line ) and wild - type ( open square , solid line ) to sucrose ( a ) , citric acid ( ca ; b ) , denatonium benzoate ( db ; c ) , and sodium chloride ( nacl ; d ) .
our data indicate that there is an alteration in the biochemical , but not structural , nature of the tongue of vip knockout mice , as well as complex alterations in circulating metabolic hormones . one paradoxical finding , however , was the reduction in leptin receptor levels in the tongue .
vip knockout mice demonstrate several features reminiscent of insulin resistance ( e.g. , high fasting glucose and insulin levels ) .
perhaps this change in taste preference induced by increased tongue glp-1 levels and reduced leptin receptor levels could be a compensatory mechanism engendered to ameliorate their dysglycemia by attempting to reduce sugar intake .
high circulating levels of leptin would be expected to reduce sweet taste preference ( 25 ) , but with a significant ablation of the lingual ob - rb , this would be prevented .
however , if this ob - rb ablation was universal , especially in the hypothalamus , then the animal would likely consume even more food and thus exacerbate its dysglycemic state .
we , therefore , assessed the expression level of ob - rb in the hypothalami of wild - type and vip knockout mice .
in contrast to the tongue , no difference in ob - rb expression between wild - type and vip knockout hypothalami was observed ( fig .
in addition , we found no difference in insulin receptor substrate 2 ( irs2 ) , t1r3 receptor , or glp-1 expression in the hypothalami of wild - type or vip knockout mice ( fig .
vip knockout mice showed a small reduction in food intake compared with wild - type mice ( fig .
7f ) and there was no significant difference in body weight between vip knockout and wild - type mice ( fig .
thus , it appears that the vip knockout mice possess a complex physiological reaction to the imposed loss of vip , one that may , in part , attempt to ameliorate their dysglycemia .
vip knockout mice show an increase in sweet taste preference and have elevated circulating leptin levels , which together could potentially be a mechanism to prevent additional excessive sugary food intake ( summarized in fig .
however , this mechanism would succeed only if there was localized lingual loss of the ob - rb , which was observed in the vip - null mice .
normal leptin receptor and irs2 expression in the hypothalami of wild - type ( wt ) and vip knockout ( ko ) mice .
a : representative western blots of three wild - type ( 13 ) or vip knockout ( 13 ) mice hypothalami .
the tissue extracts were probed with specific antisera for the leptin receptor ( ob - rb ) , irs2 , and -actin as a loading control . for the ob - rb blot ,
a plasma membrane extract was used , whereas cytoplasmic tissue extracts were used for the irs2 and actin blots .
b e : mean sem of the relative band intensities for the specific western blots measured as relative absorbance units minus background absorbance per square pixel ( au - b / px ) .
f : mean food intake ( g ) in wild - type and vip knockout mice .
values are expressed as means sem . a diagrammatic summary of the complex physiological parameters observed in the vip knockout ( ko ) mice compared with the wild - type ( wt ) controls . the vip knockout mice present with a diabetic - like state , and they actively attempt to ameliorate this condition with a series of physiological alterations ( high circulating leptin , low tongue ob - rb expression , high tongue glp-1 expression , normal hypothalamic ob - rb expression ) that in concert could attenuate the animals ' euglycemic disruption by enhancing their sweet taste and potentially reducing their desire to consume sweet foods .
we have demonstrated that vip knockout mice possess alterations in their taste cells , circulating metabolic hormones , and taste preferences .
vip and its cognate receptors are expressed in the type ii taste cells of the circumvallate papillae .
vip knockout mice demonstrated normal gross taste bud morphology ( figs . 1 and 2 )
however , several key biochemical and pharmacologic alterations in their tongues were seen , that is , vip knockout compared with wild - type mice exhibited significant increases and decreases in glp-1 and leptin receptor , respectively , in taste cells ( fig .
that glp-1 in taste cells enhances sweet taste perception , this strongly indicated to us that taste cell vip signaling may be involved in sweet taste perception .
we demonstrated that vip knockouts possess a greater preference for sweet compounds compared with wild - type mice ( fig .
vip knockout mice also exhibited concentration - dependent changes in licking bitter stimuli and sour stimuli , but there was no change in salt perception compared with wild - type mice .
it is interesting to note that vpac1/2 receptors were expressed on type ii taste cells .
receptor systems responsive to bitter , sweet , and umami stimuli use similar signal transduction systems that themselves are narrowly tuned and achieve functional specificity by expression in nonoverlapping subsets of type ii cells .
interestingly , in this study , compared with our previous study ( 11 ) , both vip knockout and wild - type mice presented much higher sucrose / water lick ratios than glp-1 receptor knockout ( glp-1r knockout ) mice and their wild - type controls .
it is unsurprising that distinct strains of mice possess different tastant responses , and this observation may be important for future experimental consideration of specific strains to facilitate efficient and sensitive detection of alterations in different taste modalities .
one unexpected finding in our vip knockout mice was the significant reduction in leptin receptor ( ob - rb ) expression in the taste cells ( fig . 3f and g ) .
circulating leptin can activate leptin receptors on taste cells and specifically inhibit gustatory responses to sweet compounds ( 25 ) .
hence , the significant reduction in taste cell leptin receptor expression in vip knockout mice may , in part , account for their heightened preference for sweet tastants .
loss of the ob - rb would reduce any inhibitory action that circulating leptin would have upon sweet taste .
this finding was interesting in light of our data showing that circulating levels of leptin in vip knockout mice were considerably higher than those in wild type .
therefore , despite the presence of high concentrations of this sweet taste suppressor , the reduction of taste cell leptin receptor was able to facilitate enhanced sweet sensation by preventing inhibitory leptin signaling activity in the tongue .
the elevated sweet preference of the vip knockout mice may also stem from the elevated levels of taste cell glp-1 .
we have previously shown that glp-1 's ability to activate the glp-1r in the tongue strongly modulates sweet taste in mice ( 11 ) . increased levels of glp-1 could , therefore , potentiate glp-1r activity in the tongue , especially considering our previous findings that the enzyme that degrades glp-1 in the peripheral circulation ( dipeptidyl peptidase-4 ) is not present in taste cells of mice ( 11 ) .
the precise temporal nature of ob - rb loss , peripheral increase in leptin levels , or elevations in taste cell glp-1 remains to be determined , but our data suggest that there is a strong connection between peripheral energy regulatory hormones and lingual preference for foodstuffs . from our data ,
because of the multiple sites and mechanisms of action of vip in mammals , it is unsurprising that the metabolic phenotype of vip knockout mice is complex . hence , despite increased leptin and pyy levels in vip knockout mice there was no dramatic diminution of body weight or food intake .
this may , in part , be due to removal of an inhibitory action of vip upon food intake , demonstrated in multiple species including mice ( 35 ) .
therefore , the resultant phenotype of the vip knockout mice may be truly an intricate mixture of alterations of metabolic food desire and digestion . in our study ,
several lines of evidence indicate a strong local taste cell action of vip . first , vip knockout mice demonstrated concentration - dependent changes in licking for sweet , bitter , and sour tastants , but not salt , indicating that there was not a generalized , widespread positive effect on all taste modalities .
second , vip knockout and wild - type mice responded similarly to the salty taste stimulus ; therefore , vip knockout mice had no difficulty in learning and completing the taste test .
lastly , vip and vip receptors are specifically localized within sweet- and bitter - sensitive taste cells , consistent with the specific taste alterations we observed . further reinforcing a selective local action of vip , we found no vip immunoreactivity in type i cells .
it has been demonstrated that amiloride - sensitive na channels are expressed in type i cells of the fungiform papillae , suggesting that these taste cells could play a role in transducing salty taste stimuli ( 36 ) . in line with this finding
, we found that there was no difference in salt perception between vip knockout and wild - type mice .
thus , it would be reasonable to propose that vip signaling in the taste bud plays a local role in modulating sweet , bitter , and sour taste stimuli .
there are numerous potential mechanisms by which vip may affect both sweet and bitter stimuli .
sweet and bitter signal transduction is commonly mediated by plc mechanisms involving taste receptor activation , as well as through adenylate cyclase
vpac1/2 receptors are coupled to multiple g - protein effectors to activate phospholipase d , plc , and adenylate cyclase .
vip stimulation of vpac receptors would therefore likely compete for availability of the plc and adenylate cyclase signaling pathways , potentially diluting the functional signal transduction efficacy of the primary taste receptors .
we have previously demonstrated that receptor stimulation of multiple related receptor systems can result in strong inhibitory effects that are profound and long - lasting ( 37 )
. genetic removal of the stimulating ligand will result in a diminution in the number of active vpac1/2 receptors , reducing any diluting competitive actions upon the taste receptor 's signaling capacity .
future experimentation upon the signaling efficiency of taste receptors in such an environment may shed light on this hypothesis .
the ability of vip ablation to affect not only sweet and bitter taste perception , but also the response to a sour stimulus may be , in part , due to a role of vip in controlling cell - cell signaling between taste cells and the type iii ( presynaptic ) cells possessing the putative sour receptor ( pkd2l1 ) .
recent data have shown that although presynaptic cells do not possess tastant - sensitive gpcrs ( 38 ) , they still respond to tastant stimulation ( 5 ) .
communication between taste cells and presynaptic cells is thought to involve atp and serotonin secretion from the taste cells ( 39 ) . in many areas of the gut
, vip is coreleased with both atp and nitric oxide ( no ) to exert relaxatory effects on smooth muscle ( 40 ) .
no is commonly secreted from postsynaptic nervous tissue to act upon presynaptic neurons to control neurotransmitter release , and no has also been associated with taste transduction ( 41 ) .
therefore , it may be possible that in the tongue vip may act in an analogous manner as in other parts of the digestive tract ( i.e. , in a concerted manner with no and atp ) .
presynaptic cell communication , resulting in the reduction in sour taste we observed in vip knockout mice .
we have demonstrated a complex physiological pattern of changes in vip knockout mice that superficially may seem unconnected but together form a coherent and rational reactive mechanism to maintain energy homeostasis .
they also possess significantly higher levels of circulating leptin but a striking reduction in ob - rb expression in the taste cells , while maintaining normal levels of ob - rb and other energy - regulatory factors ( glp-1 , t1r3 ) in the hypothalamus .
the loss of vip in our mice induces a dysglycemic state , that is , high resting glucose paired with a high insulin level . with knockout animals that survive birth , it is likely that they have physiologically adapted to counteract their deficits . for example , the lack of alteration of islet -cell mass in the vip knockout mice may be compensated for by the nonselective activity of structurally similar peptides , for example , pituitary adenylate cyclase activating polypeptide , which can activate vpac2 receptors in the vip knockout pancreata , substituting for vip activity ( 18 ) .
it would be interesting to speculate that reactive actions in vip knockout mice have attempted to ameliorate their diabetic - like pathology by increasing their sweet preference ( through reduced inhibitory leptin receptor activity and increased glp-1 expression in the tongue ) so as not to overconsume carbohydrates , while also trying to dampen their overall appetite ( increased peripheral circulating leptin with normal hypothalamic ob - rb expression ) ( fig .
central nervous system vip is antagonistic to dopamine and serotonin nervous activity ( 42,43 ) .
both of these transmitters control food reward responses ( 44,45 ) , and therefore vip knockout mice may possess an elevated reward response , curtailing their intake and preventing weight increases .
human analogy of vip knockout mice may be seen in supertasters ( possessing increased sweet and bitter sensitivity ) who tend to be more lean and
perhaps eat less than nonsupertasters ( 46 ) . however , it is unclear whether the vip knockout mice would not have gained weight if their chow was sweetened with sucrose , and more studies are needed to explore this further .
thus , an interesting hypothesis would be that these multiple concerted responses in the vip knockout mice suggest that the tongue could potentially act as a vessel for the animal to modulate its energy intake to correct any endogenous glycemic imbalances . in this way
, we could potentially view the tongue as a sensory mechanism that is bidirectionally regulated , and thus forms a bridge between available foodstuffs and the intricate metabolic hormonal balance in the animal itself .
, it is becoming apparent that there is a strong link between gustation and peripheral energy balance .
hormonal signaling within the taste buds of the tongue is likely to influence food intake and peripheral glucose homeostasis ( 15 ) . for example , hormones such as glp-1 and leptin , crucial to the control of peripheral somatic energy balance / appetite , are now thought to exert a strong local effect on the tongue with respect to food perception .
carbohydrates are among the most vital food stuffs for mammalian survival , and it would make evolutionary sense for mammals to have evolved multimodal carbohydrate sensing mechanisms .
we feel that gaining a greater appreciation for which endocrine factors are present within taste cells and their putative roles in taste perception will shed much - needed light on how taste perception is linked to peripheral somatic energy balance
. this knowledge will be invaluable for finding novel therapeutic targets and agents for the treatment of disorders such as metabolic syndrome and type 2 diabetes . | objectiveit is becoming apparent that there is a strong link between taste perception and energy homeostasis .
recent evidence implicates gut - related hormones in taste perception , including glucagon - like peptide 1 and vasoactive intestinal peptide ( vip ) .
we used vip knockout mice to investigate vip 's specific role in taste perception and connection to energy regulation.research design and methodsbody weight , food intake , and plasma levels of multiple energy - regulating hormones were measured and pancreatic morphology was determined .
in addition , the immunocytochemical profile of taste cells and gustatory behavior were examined in wild - type and vip knockout mice.resultsvip knockout mice demonstrate elevated plasma glucose , insulin , and leptin levels , with no islet -cell number / topography alteration .
vip and its receptors ( vpac1 , vpac2 ) were identified in type ii taste cells of the taste bud , and vip knockout mice exhibit enhanced taste preference to sweet tastants .
vip knockout mouse taste cells show a significant decrease in leptin receptor expression and elevated expression of glucagon - like peptide 1 , which may explain sweet taste preference of vip knockout mice.conclusionsthis study suggests that the tongue can play a direct role in modulating energy intake to correct peripheral glycemic imbalances . in this way
, we could view the tongue as a sensory mechanism that is bidirectionally regulated and thus forms a bridge between available foodstuffs and the intricate hormonal balance in the animal itself . |
parkinson s disease ( pd ) is the most common neurodegenerative movement disorder characterized by tremor , rigidity , bradykinesia and is likely to increase due to the aging populations .
pd involves principally the death of dopamine ( da ) neurons in the substantia nigra pars compacta ( snc ) but other neurotransmitters and neuro- modulators are also affected .
treatment of motor symptoms of pd with the da precursor , l-3,4-dihydroxyphenylalanine ( l - dopa ) , introduced 50 years ago still remains the gold standard for pd treatment .
however , various complications including motor fluctuations and abnormal involuntary movements , such as l - dopa - induced dyskinesias ( lid ) , limit the quality of life in pd patients and can be very difficult to manage .
lid increases with the duration of l - dopa treatment and up to 95% of pd patients become afflicted after 15 years [ 5 , 6 ] .
although the mechanisms of these involuntary movements are not well understood , they are paralleled by changes in various neurotransmitter systems and intracellular signaling pathways .
glutamate neurotransmission is reported to be increased in the basal ganglia in pd and lid [ 8 , 9 ] .
amantadine , a noncompetitive antagonist at n - methyl - d - aspartate ( nmda ) ionotropic glutamate receptors and , to a lesser extent clozapine , are presently the only drug used in the clinic to reduce lid in some pd patients without worsening parkinsonian symptoms [ 10 - 14 ] .
however , the antidyskinetic effect of amantadine may be transient and high doses may not be tolerated in some pd patients because of cognitive impairment , thus limiting its use . on the basis of these considerations , combined with the rich distribution and diverse physiological roles of metabotropic glutamate ( mglu ) receptors within the basal ganglia , recent
attention has been placed on these receptors as alternative targets to modulate glutamate hyperactivity in pd and lid .
studies in animal models and pd patients indicate that antagonists of group i mglu receptor , especially mglu5 receptor , could be considered as a suitable therapeutic approach in pd and lid .
mglu5 receptor specific binding was reported to be increased in the basal ganglia of parkinsonian monkeys with lid and in parkinsonian patients with motor complications [ 17 - 21 ] . in the 6-hydroxydopamine ( 6-ohda)-lesioned rat model , mglu5 receptor negative allosteric modulators 2-methyl-6-(phenylethynyl)pyridine ( mpep ) ,
3-[(2-methyl-1,3-thiazol-4-yl)ethynyl]pyridine ( mtep ) and mrz-8676 administered acutely inhibit lid [ 22 - 26 ] .
the mglu5 receptor negative allosteric modulators mpep , mtep , fenobam and afq056 ( mavoglurant ) were found to acutely reduce lid in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine ( mptp)-lesioned monkeys [ 27 - 29 ] . in the light of these acute studies
, it becomes important to test the long - term effects of mglu5 receptor negative allosteric modulators behaviourally and their associated brain molecular changes .
sub - chronic and chronic administrations of mglu5 receptor negative allosteric modulators in mptp monkeys and 6-ohda rats reduce the development of l - dopa induced motor complications ( table 1 ) . in previous studies
, we reported that development of lid over a month of treatment were lower by overall 70% with addition of mpep to the l - dopa treatment in de novo mptp monkeys and this was associated with a normalization of glutamate receptors as shown for mglu5 , nmda containing nr2b subunit , ampa and mglu2/3 receptors ( table 2 ) . in 6-ohda rats the vesicular glutamate transporter 2 ( vglut2 )
was also normalized by the addition of mpep to the chronic treatment with l - dopa .
mglu5 receptor negative allosteric modulators , mavoglurant and adx-48621 ( dipraglurant ) , were shown to reduce lid in parkinsonian patients and were well tolerated without worsening motor symptoms [ 31 - 33 ] .
however , recent studies with mavoglurant did not show reduced lid in pd patients [ 34 , 35 ] .
the mechanisms underlying the development of lid remain unknown , but evidence suggests that lid is the result of maladaptive plasticity at striatal synapses [ 7 , 36 ] and in an altered activity of dopaminergic neurotransmission in the basal ganglia . da binds to five different subtypes of g protein - coupled da receptors divided in two classes , the d1 ( d1 and d5 ) and the d2 class ( d2 , d3 and d4 ) of receptors .
d1 receptors were shown to be expressed in neurons containing substance p and dynorphin , projecting to the substantia nigra pars reticulata ( snr ) and to the internal globus pallidus ( gpi ) , which constitute the direct striatal output pathway .
d2 receptors are predominantly localized in neurons expressing enkephalin , projecting to the external globus pallidus ( gpe ) , constituting the indirect pathway .
d2 receptors are found on postsynaptic and pre - synaptic nigrostriatal dopaminergic terminals , of the substantia nigra neurons and of presynaptic corticostriatal terminals where they can inhibit striatal glutamate release .
denervation - induced supersensitivity of d1 and d2 receptors was initially recognized as a plausible mechanism of lid [ 41 , 42 ] .
numerous studies measured the density of d1 and d2 receptors in the brain of human and animal models , but no general consensus emerged ; the wide methodological discrepancies , time to sacrifice , post - mortem delays , etc , may account for this lack of consensus .
post - mortem studies have shown that striatal da receptors particularly the d2 subtype were increased in pd patients [ 42 , 43 ] or unchanged [ 44 , 45 ] , while both d1 and d2 receptor subtypes were increased in mptp monkeys [ 46 - 48 ] .
administration of l - dopa was shown to reverse these increases in pd patients [ 42 , 43 ] and primates in many studies [ 47 - 49 ] .
hence , d1 mrna levels are reported to remain unchanged [ 50 - 52 ] or to be reduced after mptp lesion in monkeys [ 51 - 53 ] .
d2 receptors mrna levels were reported to be increased concomitantly with its corresponding protein levels in striatum with mptp [ 51 - 54 ] and returned to control levels when l - dopa was administered .
these reports support that lid are more complex than hypersensitivity due to a simple increase in the density of striatal da receptors and its mrna .
numerous interactions between mglu5 receptor and d1 , d2 , nmda , a2a adenosine receptors suggest that these receptors may function together as closely associated signaling partners in the development of lid [ 55 , 56 ] .
a chronic treatment with mpep in mptp monkeys treated with l - dopa that prevented the development of lid was shown to normalize changes produced by l - dopa on d2 receptor and its mrna , preproenkephalin ( ppe ) mrna , preprodynorphin ( ppd ) mrna , phosphorylated extracellular signal - regulated kinase 1 and 2 ( erk1/2 ) and phosphorylated akt / gsk3 signaling proteins , but not d1 and its mrna ( table 3 ) .
similar findings for ppe and ppd mrna levels in 6-ohda rats were observed with chronic mpep or mtep treatment with l - dopa associated with the prevention of development of abnormal involuntary movement scale ( aims ) ( table 3 ) .
mtep was also shown to normalize phosphorylated erk1/2 and phospho - mitogen- and stress - activated protein kinase-1 ( msk-1 ) in l - dopa treated 6-ohda rats ( table 3 ) . while both mpep and mtep have off - target activities these are at higher doses [ 57 - 61 ] and at the doses shown in table 3 the results reported are more likely due to mglu5 receptor negative allosteric modulator activity .
adenosine , a purinergic messenger , plays a crucial role in many physiological processes and is released by many cells including neurons and glia [ 62 , 63 ] . in the basal ganglia , adenosine interacts closely with da and is involved in the function of striatal gabaergic striatopallidal neurons projecting from the caudate nucleus and the putamen , mainly to the gpe [ 64 - 69 ] .
the implication of adenosine to regulate the excessive glutamate neurotransmission observed in pd and lid is also demonstrated [ 37 , 70 , 71 ] .
hence , adenosine has received increasing attention because of its interaction with da and glutamate receptors ; this could have major implications for the development of new pharmacological targets for the treatment of pd and lid .
adenosine binds to four classes of specific g - protein - coupled receptor subtypes named a1 , a2a , a2b and a3 .
adenosine receptor subtypes a1 and a2a are mainly localized in the basal ganglia , more precisely in the striatum , whereas a2b and a3 receptors are widely distributed in the brain [ 73 , 74 ] . in the basal ganglia ,
the stimulation of a1 receptors generally leads to inhibition of neurotransmitter release , such as the inhibition of glutamate release at corticostriatal terminals in the striatum [ 62 , 76 ] .
the a2a receptor is co - expressed at postsynaptic sites of the same medium spiny neurons as those bearing da d2 receptors [ 77 , 78 ] and projecting to the gpe of the indirect pathway .
the restricted and specific distribution of a2a receptors to the indirect pathway of the basal ganglia provide specificity that could lead to a reduced incidence of adverse effects .
presynaptic and glial a2a receptors are also localized in the caudate nucleus and the putamen .
a2a receptors are found on presynaptic glutamatergic corticostriatal terminals and these receptors can modulate positively the glutamatergic cortical input by stimulating glutamate release .
adenosine a2a antagonists can reduce the excessive striatopallidal and subthalamic neuronal activity and bring a new target in pd therapy [ 37 , 65 , 67 , 79 ] .
several behavioural analyses have shown the potential efficacy of adenosine receptor modulators in the treatment of pd and lid , such as a2a receptor antagonists [ 79 - 83 ] .
the a2a receptor antagonists preladenant and kw-6002 ( istradefylline ) , are reported to extend the duration of the antiparkinsonian effect of l - dopa and enhance the parkinsonian activity of low doses of da agonists ; this is without worsening dyskinesias showing the dopa - sparing activity of a2a receptor antagonists [ 81 , 84 - 87 ] ; reviewed in : [ 88 - 91 ] .
moreover a recent paper showed that the a2a receptor antagonist istradefylline did not increase lid of mptp - treated marmosets in a chronic treatment and at days 21 and 28 it slightly but significantly reduced dyskinesias .
presynaptically , a2a receptors can colocalize with a1 receptors in corticostriatal afferents , where they act together to modulate and regulate glutamate release .
experiments using isolated striatal nerve terminal preparations have shown that most of the striatal glutamatergic terminals contain both a1 and a2a receptors . in response to variation in adenosine concentrations , functional studies in striatal glutamatergic terminals
have shown that the a1a2a adenosine receptor heteromer provides a switch mechanism that can produce opposite effects on glutamate release [ 93 - 95 ] .
functional interactions between da and adenosine receptors are supported by the anatomic localization of these receptors in striatal projection neurons .
a1 receptors are mainly co - expressed with d1 da receptors on striatal neurons that project to the gpi and the substantia nigra .
d1 and adenosine a1 receptors are known to form functionally interacting complexes , the heteromer d1-a1 , in cortical neurons and basal ganglia [ 96 , 97 ] .
using co - immunoprecipitation and colocalization , as well as bioluminescence resonance energy transfer ( bret ) and fluorescence resonance energy transfer ( fret ) techniques , d2 and adenosine a2a receptors were shown to form functional homo- and hetero - oligomers [ 96 , 98 , 99 ] . moreover , in cell lines cotransfected with plasmids containing d2 and a2a receptors and in primary culture of striatal neurons , long - term administration of a2a or d2 agonists induces an internalization and desensitization of the d2-a2a complex [ 98 , 99 ] , whereas d2 antagonists trigger an increase in d2 and a2a immunoreactivity .
also , in sh - sy5y neuroblastoma cells cotransfected with the d2 receptor , a2a antagonists enhance striatal d2 receptor signaling and block increased a2a receptor signaling .
interestingly , a recent report in rats and monkeys has shown that d2-a2a - cannabinoid ( cb)1 receptor heteromers are present in the striatum of intact and hemiparkinsonian 6-ohda - lesioned rat as well as in intact and mptp parkinsonian monkeys [ 102 , 103 ] and that this heteromer expression is altered in lesioned striatum . also , acute or chronic l - dopa treatment induced disruption of d2-a2a - cb1 heteromers [ 102 , 103 ] .
these authors suggested that lid could be induced , at least in part , by an imbalance in the indirect pathway due to absence of cross - talk between d2-a2a - cb1 receptor heteromers functional components [ 102 , 103 ] .
in addition to adenosine a2a and da d2 receptors , mglu5 receptors are also known to interact and colocalize postsynaptically in the striatopallidal gabaergic efferent neurons [ 16 , 71 , 104 ] .
experiments using optical sectioning techniques found that a2a and mglu5 receptors are also co - localized in rat striatal cultures .
in addition , double labelling electron microscopy colocalization experiments in the putamen of monkey have shown a substantial degree of a2a and mglu5 receptor colocalization mainly in postsynaptic elements .
immuno- reactive dendrites or spines in the monkey putamen co - express mglu5 receptors .
accordingly , a2a receptors activation can increase the phosphorylation of darpp-32 at thr-34 via an erk pathway and the induction of c - fos expression is also increased in striatopallidal neurons when a2a and mglu5 receptors are co - activated [ 71 , 106 ] . an important increase in adenosine release was observed when glutamatergic neurotransmission becomes overactive [ 71 , 107 ] .
functional interaction of a2a and mglu5 receptors have also been demonstrated at the behavioural level , a synergistic effect on the induction of motor activity being observed following acute combined treatment with a low doses of mglu5 negative allosteric modulator , mpep , and a2a antagonist kw-6002 .
hence , the mglu5 receptor - mediated effect in the striatum was abolished by the blockade of a2a receptors .
this colocalization provides a structural framework for the existence of multiple functional interactions of a2a , d2 and mglu5 receptors . a better understanding of the functional interactions that exist between these receptor heteromers in normal and lesioned striatum as well as following acute or chronic l - dopa treatment foreshadow a potential therapy targeting mglu5-d2-a2a receptor heteromers in pd patients .
as presented in the previous section of this review , in de novo mptp monkeys , development of lid was shown to be lower with addition of mpep to the l - dopa treatment and to be associated with a normalization of glutamate and da receptors [ 18 , 109 ] .
therefore , we investigated a2a receptors in the brain of these de novo mptp monkeys treated with l - dopa and mpep where motor behaviour , glutamate and da receptors were previously reported .
a2a receptors were measured by in situ hybridization of their mrna levels using oligonucleotides probes corresponding to bases 593637 and 714757 of human a2a receptors cdna [ 110 , 111 ] under conditions we previously described for mptp monkeys and human post - mortem brains . fig .
1 shows representative a2a receptor mrna levels in brain slices of control and mptp monkeys treated with vehicle , l - dopa , and l - dopa+ mpep .
high expression of this receptor in control monkeys is observed in the caudate nucleus and putamen but not in the gpi and gpe as we previously reported . in the posterior caudate nucleus analyzed ( fig .
2 ) , no significant effect of lesion and treatment was measured on a2a receptor mrna levels ( one - way analysis of variance ( anova ) followed by post - hoc pairwise comparisons with fisher s least significant difference test , ( fisher s test ) f3,13 = 2.026 , p = 0.1600 ) consistent with our previous findings in another group of mptp monkeys . by contrast
, significant changes of a2a receptor mrna levels were measured in the lateral ( anova and fisher s test , f3,13 = 6.140 p = 0.0078 ) and medial ( anova and fisher s test , f3,14 = 3.740 , p = 0.0389 ) putamen .
the putamen showed an increase of a2a receptor mrna levels with the mptp lesion in its lateral and medial parts ( + 61% and + 43% in the lateral and medial parts respectively compared to intact - saline - treated monkeys ) ( fig .
2 ) . a2a receptor mrna levels remained elevated in mptp monkeys treated with l - dopa , those animals displaying dyskinesias . in mptp monkeys
treated with l - dopa and mpep that developed less dyskinesias than those treated with l - dopa , a2a receptor mrna levels were lower and this was significant compared to mptp saline treated animals in the lateral putamen a sub - region associated with motor control whereas in the medial part the difference was intermediate , neither significant compared to controls nor mptp + l - dopa treated mptp monkeys .
these results are consistent with our previous findings where a2a receptor mrna levels were elevated in mptp and dyskinetic mptp + l - dopa treated mptp monkeys and lower in mptp monkeys treated with l - dopa and ci-1041 , an nmda glutamate receptor antagonist , that prevented the development of dyskinesias .
however , we can not rule out the possibility that mpep or ci-1041 treatments could have a direct effect on the expression of a2a receptors thereby associating the change in levels of these receptors to the antagonist treatment themselves rather than to the dyskinetic condition .
indeed in mice , striatal binding of the a2a receptor antagonist ligand [ i]zm241385 was reduced in brain slices pre - incubated with mpep compared to untreated animals and this was attributed to a possible change of conformation of the mglu5-a2a heteromers and consequently the binding affinity of [ i]zm241385 for a2a receptors .
nevertheless , these findings in mptp monkeys model well the human condition where we showed that a2a receptor mrna levels were elevated in pd patients with dyskinesias compared to pd patients without dyskinesias and controls in the putamen while no significant effect was measured in the caudate nucleus . in this
regard , post - mortem studies have shown an increase of basal ganglia a2a adenosine receptors and its mrna levels in pd patients with lid as compared to pd patients without lid and controls .
as for post - mortem human brains , a2a receptor levels were only increased in dyskinetic monkeys as compared to untreated monkeys .
these results in pd patients were in accordance with a report by varani et al .
( 2010 ) showing elevated a2a receptor mrna levels ( 5.2 fold increase ) and an overexpression of a2a protein levels ( 280% ) , measured by western blot in the putamen of pd patients , when compared to healthy subjects . in the present study and in our previous ones in mptp monkeys and
pd patients similar changes were observed for a2a receptor mrna levels and [ h]-sch 58261 specific binding to a2a receptors supporting that changes of expression of this receptor in pd is the result of altered transcriptional activity and are functionally associated with changes of this receptor protein levels .
a2a receptors availability in pd patients with and without lid was evaluated by pet studies and higher levels of a2a receptor binding in pd patients with lid compared to those without lid were reported [ 115 , 116 ] .
this suggests that lid might be the result of altered adenosine transmission and that a2a receptor antagonists could produce potential beneficial antidyskinetic activity .
the molecular mechanisms involving a2a receptors in the induction of dyskinesia is not yet fully elucidated .
however , it is well known that activation of a2a receptors disrupts the inhibitory actions of d2 receptors within the indirect pathway resulting in a facilitation of da transmission . in the light of various biochemical and behavioural results showing the functional interaction between a2a and d2 receptors ,
it was postulated that altered striatal d2-a2a functional interaction in pd creates an imbalance in the activity of the two efferent pathways of the basal ganglia that could be associated with motor alterations inherent to dyskinesia [ 102 , 117 ] .
these changes suggest that both striatal a2a receptors and their mrna levels are modulated by chronic l - dopa treatment and might be related to the close interactions with da and glutamate systems in the basal ganglia .
these findings also suggest that the striatal mglu5mglu5 dimeric receptor complexes on the striato - pallidal efferents neurons as well as the putative mglu5-d2-a2a complexes on the same neuronal population , might be involved in striatal plasticity and hence could be relevant for the management of pd and lid [ 67 , 71 , 105 ] .
the mechanisms involved in the occurrence of lid are complex and have been investigated in numerous studies using animal models and pd patients .
multiple changes in the basal ganglia dopaminergic systems in d1 and d2 receptors and their signaling pathways have been observed such as modulation in the expression and the activity of subtypes of da receptors , g proteins , effectors , protein kinases , transcription factors , etc .
abnormal adaptation in the striatum leading to lid may also involve faulty interaction between glutamate and da inputs and da - glutamate signaling in the nigrostriatal pathway .
glutamate is the brain most abundant excitatory neuro- transmitter mediating as much as 70% of synaptic transmission in the central nervous system and its overactivity is well documented in pd and lid .
an attractive strategy to treat lid is to use anti - glutamatergic adjunct drugs that can modulate basal ganglia dopaminergic neurotransmission [ 120 - 122 ] .
the present review showed that a chronic treatment with mglu5 negative allosteric modulators in 6-ohda rats and mptp monkeys treated with l - dopa normalized changes produced by l - dopa on d2 receptor and its mrna , ppe mrna , ppd mrna , erk1/2 and akt / gsk3 signaling proteins , but not d1 and its mrna . in the basal ganglia of post - mortem pd patients with motor complications , changes of glutamate neurotransmission and its receptors such as nmda , ampa , mglu2 and
mglu5 receptors were reported [ 8 , 20 , 123 ] . in these same patients ,
a2a receptor levels were measured and they were increased in the basal ganglia and associated with lid .
these observations in human brains do not provide a causal link between glutamate and adenosine receptors in motor complications .
nevertheless , they show altered adenosine and glutamate receptors in human pd and that normalization of glutamatergic transmission in mptp monkeys with a mglu5 receptor negative allosteric modulator normalizes also a2a receptors suggesting possible interactions between these receptors . moreover , in mptp monkeys , a chronic treatment with l - dopa and a nmda receptor antagonist ci-1041 , that prevented lid , was shown to normalize basal ganglia a2a receptors as well as nmda receptors suggesting the close link between these neurotransmitters in dyskinesias .
the interactions of a2a receptors , with dopaminergic and glutamatergic receptors represent an interesting area of research . in the light of theses
interactions , new therapeutic approaches could include the combination of an a2a receptor antagonist with dopaminomimetic drugs or with anti- glutamatergic drug .
this combination could lead to the use of lower doses of each drug , especially l - dopa , and could have an impact on pd symptoms and the development of motor complication .
for example , a2a receptor antagonists could be combined to mglu5 receptor negative allosteric modulators and to lower doses of l - dopa or da agonists to increase the antiparkinsonian activity of the dopaminergic drugs without increasing or even decreasing dyskinesias .
the mglu5 receptor subtype is highly expressed in striatal medium spiny neurons and plays a key role in modulating the responses mediated by nmda receptors and l - type calcium channels .
in addition , an antagonistic interaction between the d2 receptor and mglu5 receptors is reported .
the response of the basal ganglia to a chronic treatment with a mglu5 receptor negative allosteric modulators in rodent models of pd show that striatal molecular changes relevant to lid are reversed by mpep or mtep , including delta fosb protein , prodynorphin mrna , glutamic acid decarboxylase ( gad65 and gad67 ) mrna and phospho - erk1/2 protein levels .
we showed that mpep prevented dyskinesias and the effects of chronic l - dopa on various ionotropic and metabotropic glutamate receptors in the basal ganglia of mptp monkeys thus showing the widespread activity of mglu5 receptor negative allosteric modulators in the basal ganglia .
mpep did not affect d1 receptor levels and its mrna but was associated with an increased in d2 receptors levels , its mrna and its associated signaling proteins .
in rodent and primate models of pd and lid , mglu5 receptor negative allosteric modulators are well documented to inhibit dyskinesias and have an extensive range of beneficial biochemical effects . in parkinsonian primate and rodent treated with l - dopa and mglu5 negative allosteric modulators ,
crosstalk between mglu5-d2-a2a receptors seems to be present whereas recent studies reported that l - dopa disrupts d2-a2a - cb1 receptor heteromers crosstalk in the striatum of hemiparkinsonian rats and primates in the caudate nucleus but not the putamen ( two levels were investigated , pre and post commissural ) .
the mglu5 negative allosteric modulator treatment in parkinsonian primates and rats affects not only glutamate receptors but also da and a2a receptors .
this may be indirect by restoring glutamate neurotransmission but could also involve the direct interactions of the trio mglu5-d2-a2a receptor crosstalk [ 96 , 129 ] .
moreover , supporting a close mglu5-a2a receptor interaction , mtep treatment was reported to decrease mice brain a2a receptor specific binding and regulate the conditioned effects of cocaine .
thus , mglu5 receptor negative allosteric modulators preventing the development of lid and inhibiting the expression of already developed lid not only affects several glutamate receptors but also d2 and a2a receptors , neuropeptides and akt / gsk3 and erk1/2 signaling . an abundant literature mainly from rodent models of pd supports the striatal overactivation of erk1/2 via da d1 receptors to be associated with dyskinetic behaviors ; reviewed in that is critically modulated by striatal mglu5 receptors .
thus mglu5 receptor negative allosteric modulators affect various markers of both the striatal direct and indirect output pathways .
the implication of these receptor interactions in mental and neurodegenerative diseases and more specifically in the development and expression of pd symptoms and lid needs to be considered and further investigated to find novel targets and ultimately , novel pharmacological treatments .
| patients with parkinson s disease ( pd ) receiving l-3,4-dihydroxyphenylalanine ( l - dopa , the gold - standard treatment for this disease ) frequently develop abnormal involuntary movements , termed l - dopa - induced dyskinesias ( lid ) .
glutamate overactivity is well documented in pd and lid .
an approach to manage lid is to add to l - dopa specific agents to reduce dyskinesias such as metabotropic glutamate receptor ( mglu receptor ) drugs .
this article reviews the contribution of mglu type 5 ( mglu5 ) receptors in animal models of pd.several mglu5 negative allosteric modulators acutely attenuate lid in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine ( mptp ) monkeys and 6-hydroxydopamine(6-ohda)-lesioned rats .
chronic administration of mglu5 negative allosteric modulators to mptp monkeys and 6-ohda rats also attenuates lid while maintaining the anti - parkinsonian effect of l-dopa.radioligand autoradiography shows an elevation of striatal mglu5 receptors of dyskinetic l - dopa - treated mptp monkeys but not in those without lid .
the brain molecular correlates of the long - term effect of mglu5 negative allosteric modulators treatments with l - dopa attenuating development of lid was shown to extend beyond mglu5 receptors with normalization of glutamate activity in the basal ganglia of l - dopa - induced changes of nmda , ampa , mglu2/3 receptors and vglut2 transporter.in the basal ganglia , mglu5 receptor negative allosteric modulators also normalize the l - dopa - induced changes of dopamine d2 receptors , their associated signaling proteins ( erk1/2 and akt / gsk3 ) and neuropeptides ( preproenkephalin , preprodynorphin ) as well as the adenosine a2a receptors expression.these results show in animal models of pd reduction of lid with mglu5 negative allosteric modulation associated with normalization of glutamate , dopamine and adenosine receptors suggesting a functional link of these receptors in chronic treatment with l - dopa . |
injuries to the hamstring musculature are commonplace in many mainstream sports and occupations involving physical activity [ 1 , 2 ] .
they have not declined in recent times and the high rate of recurrence suggests that the current understanding of such injuries remains incomplete .
hamstring muscle strains / tears account for 1315% of injuries in australian football [ 4 , 5 ] , 11% of injuries in elite new zealand cricketers , 1214% of injuries in professional soccer [ 7 , 8 ] , and up to 24% of injuries in gaelic football . many predisposing factors for hamstring injury
have been suggested within the literature , including insufficient warm - up , poor flexibility , muscle imbalances , neural tension , fatigue , and previous injuries .
inadequate flexibility within the posterior thigh compartment appears to be one of the more commonly accepted causes of hamstring injuries [ 2 , 16 ] ; however , the evidence for decreased hamstring flexibility as a risk factor remains equivocal [ 11 , 17 ] .
a recent cochrane review found no evidence for stretching as a sole intervention for prevention of hamstring injury , and this has led to the suggestion that decreased flexibility is but one factor in the multifactorial etiology of hamstring strain injury . despite varying theories for the observed increases in muscle flexibility after the application of stretching
, we are lacking in evidence for any credible explanation . in a recent review article , weppler and
magnusson suggested that such increases in tissue flexibility may result , not from affecting the mechanical properties of the muscle being stretched , but from changes in the individual 's perception of stretch or pain .
they suggested that the point of limitation in hamstring range may increase , not because of changes within the muscle structure itself but rather because the individual experiencing the stretching may adopt a new stop point for limitation in hamstring range based on altered perceptions of stretch and pain .
they referred to this as the sensory theory and proposed that increases in muscle flexibility after stretching were likely due to the modified sensation .
changes in the mobility of the nervous system ( neurodynamics ) achieved through movement and stretching could modify such sensations [ 2123 ] . decreased hamstring flexibility as evidenced by limited range in the passive straight leg
raise test ( slr ) could be due to altered neurodynamics affecting the sciatic , tibial , and common fibular nerves .
altered posterior lower extremity neurodynamics could arguably influence resting muscle length and lead to changes in the perception of stretch or pain .
providing movement or stretching could lead to changes in the neurodynamics and modification of sensation and could help to explain the observed increase in flexibility .
the mechanosensitivity of the neural structures in the posterior leg , thigh , buttock , and vertebral canal may play a part in determining the flexibility of the hamstring muscles
. protective muscle contraction of the hamstring muscles found in the presence of neural mechanosensitivity [ 26 , 27 ] may account for hamstring tightness and thereby predispose the muscle to subsequent strain injury .
neurodynamic sliding interventions are thought to decrease neural mechanosensitivity [ 26 , 28 , 29 ] and it is possible that the inclusion of these interventions in the management of hamstring flexibility could be beneficial . in a recent pilot study involving 28 male soccer players ,
our research team was able to demonstrate that a neurodynamic sliding intervention led to a short - term increase in hamstring flexibility .
findings from that study were limited by a small sample size , inclusion of young males only , and also because the experimental group was compared to a control group that received no intervention . despite these shortcomings
, the study did suggest that neurodynamic treatment can significantly increase hamstring flexibility in a young male athletic population and proposed that future research compares neurodynamic techniques with other interventions in a broader population of subjects .
therefore , the aim of this study was to examine the immediate effects of a neurodynamic sciatic sliding technique , hamstring stretching , and placebo control intervention in asymptomatic subjects with decreased hamstring flexibility or short hamstring syndrome .
we hypothesized that an isolated neurodynamic sciatic sliding technique would improve range of motion , assessed by passive straight leg raise test ( slr ) , greater than hamstring stretching or placebo in the short term .
findings from this study may provide further the evidence for the relevance of neural tissues in determining range of motion and may indicate benefits for adding neural mobilization techniques to the rehabilitation and/or prevention of hamstring injuries .
we recruited a sample of 120 subjects ( 60 female ; mean age 33.4 7.4 , range 2045 ) who exhibited bilateral short hamstring syndrome ( slr test = 80 or less ) [ 3133 ] .
sample size was calculated using ene 3.0 software ( autonomic university of barcelona , spain ) and calculations were based on detecting between - group mean differences of 7 at postdata . assuming a standard deviation of 8 when comparing 2 means , an alpha level of 0.05 , and desired power of 90% , a sample size of 29 subjects per group was generated .
we increased the sample size by 25% ( 40 per group ) to increase statistical power .
exclusion criteria were hamstring injury within the past year , exceeding 80 in the initial slr test , verbal report of performing regular lower extremity muscle stretching exercises , history of neck trauma ( whiplash ) , neck symptoms , history of fracture in any part of the body , history of growth disorders , history of neurological or orthopedic disorders , diagnosis of herniated disk , low back pain in the last 6 months , and body mass index ( bmi ) lower than 20 kg / cm or higher than 30 kg / cm .
we chose the bmi range as an exclusion criterion to allow for better identification of body landmarks and introduce some degree of homogeneity for subject body type .
all subjects signed an informed consent before they were included in the study , and all procedures were conducted according to the declaration of helsinki .
all physical measurements were obtained by a pair of trained examiners who were blinded to each subject 's group allocation .
the passive slr test was used to determine changes in hamstring muscle flexibility and has demonstrated high interobserver reliability ( 0.940.96 ) [ 34 , 35 ] .
each test was performed with the subject supine wearing shorts or underwear , and the following bony landmarks were identified and labeled with a marker : the anterior superior iliac spine ( asis ) , greater trochanter and lateral epicondyle of the femur , and the head of the fibula and the fibular malleolus .
the passive slr test was recorded 3 times for each subject using a universal goniometer .
one examiner performed the passive slr by keeping the knee in full extension and the ankle in neutral plantarflexion - dorsiflexion .
full ankle dorsiflexion was avoided to prevent calf muscle stiffness or pain ( gastrocnemius and soleus ) from confounding the sensation of hamstring stiffness and pain which would signal the limit of the slr test .
the examiner would hold the talus and avoid any hip rotation during flexion of the hip as they lifted the subject 's lower limb until he or she first complained of stiffness or pain in the region of the thigh , bent his / her knee , or began to swing into a posterior pelvic tilt ( noted as movement of the asis ) .
the second examiner placed the axis of the goniometer over the mark on the greater trochanter of the femur .
the stationary arm of the goniometer was placed parallel to the table and checked with a level , and the moving arm was placed in the line between the head of the fibula and the fibular malleolus , and the degree of elevation of the straight leg was then noted ( figure 2 ) . within - session intra - rater reliability was established on the first 10 subjects as sufficient for clinical measurement ( icc = 0.96 ) ; and although less relevant to calculation of responsiveness data and subsequent interpretation of the study findings , between - session intra - rater reliability was similarly established as sufficient ( icc = 0.94 ) .
the average of 3 measurements was obtained for each subject and used to determine changes in hamstring muscle flexibility .
subjects were randomly divided into 3 blocks of 40 , using a simple random distribution ( http://www.randomization.com/ ) into the 3 intervention groups : stretching , neurodynamic , and control .
the stretching group would receive static passive stretches to their hamstring muscles ; the neurodynamic group would receive neurodynamic sliders ; and the control group would receive passive mobilization of their intrinsic foot joints as placebo .
these interventions were performed on each subject 's dominant leg ; the duration of each intervention was standardized , and all interventions were provided by a therapist who remained blinded to the slr measurements .
subjects were informed that the intervention being provided to them was a relaxation technique that was thought to improve slr comfort and range .
subjects in the stretching group received passive stretching of the hamstring muscles in their dominant leg .
while lying supine , a researcher who was blinded to slr test measures would passively position the subject into the slr position ( hip in flexion , knee in extension , and ankle in neutral ) without pain / discomfort to the point where resistance to movement was first noted ( figure 3 ) .
this position was then maintained for 30 seconds [ 36 , 37 ] and repeated further 5 times . during the 30 second stretches , the therapist monitored the subjects to ensure they did not make any compensation that could modify the stretching position .
the objective of the technique is to produce a sliding movement of neural ( sciatic ) structures relative to their adjacent tissues [ 21 , 38 ] .
sliders involve the application of movement / stress to the nervous system proximally while releasing movement / stress distally and then reversing the sequence .
recent research has shown that sliders actually result in greater excursion than simply stretching the nerve .
subjects were supine with their neck and thoracic spine supported in a forward flexed position .
concurrent hip and knee flexion were alternated dynamically with concurrent hip and knee extension ( figure 4 ) .
subjects in the control group received passive mobilization of the intrinsic foot joints while being in the supine position .
this was chosen as placebo intervention due to the absence of anatomic and/or physiological relations between this region and technique and the hamstring muscles and their stretching positions .
passive movements applied in a randomized order were supination , pronation , abduction , adduction , flexion , and extension ( figure 5 ) .
as well as standardizing the total treatment times for subjects in each group , preservation of blinding for the study was maintained by standardizing the interval between the slr assessments for all subjects .
no subject reported any persistent discomfort or pain associated with their participation in the study .
descriptive statistics ( mean , standard deviation , 95% ci ) were calculated for pretest and posttest slr averaged values for the 3 groups .
slr responsiveness data were calculated using the between - session icc established in this study ( icc = 0.96 ) and the formulae sem = sd (1 icc ) and mdc95 = 1.96 2 sem . to analyze the difference in pre-/postintervention between groups on hamstring extensibility ( slr ) , a 3 2 mixed model anova was performed , with groups ( stretching , neurodynamic and control ) as the between - subjects variable , and time ( time : pre and post ) as the within - subjects variable .
simple main effects analyses with a bonferroni corrected alpha would be utilized if an interaction was observed . within - group
an effect size greater than 0.8 was considered large , around 0.5 was moderate , and less than 0.2 was small .
there were no significant differences in baseline characteristics between groups at the beginning of the study ( table 1 ) .
standard deviations for slr measurements ranged from 3.7 to 8.0. this allowed us to calculate a range of sem from 1.1 to 2.4 and subsequently a range for the mdc95 of 3.16.6. the neurodynamic group had a pretest mean range of 59.8 ( 95% ci : 58.161.3 ) and posttest mean range of 69.7 ( 95% ci : 68.570.9 ) .
the stretching group had a pretest mean range of 59.9 ( 95% ci : 57.762.2 ) and posttest mean range of 65.5 ( 95% ci : 62.968.0 ) .
finally , the control group had a pretest mean range of 59.4 ( 95% ci : 57.561.2 ) and posttest mean range of 59.4 ( 95% ci : 57.661.1 ) .
the before and after differences in mean slr values exceeded the upper limit of this mdc95 range for the neurodynamic group ( 9.9 ) but not for the stretching group ( 5.5 ) or the control group ( 0.03 ) ( table 2 ) .
a statistically significant interaction was observed , f(2,117 ) = 313.715 , p < 0.001 ( p = 0.843 ) ( figure 6 ) .
there was no difference between the 3 groups at the start , p = 0.893 ; however , at the end of the study , the groups were significantly different .
mean slr values were significantly higher for both the neurodynamic and stretching groups compared to the control group ( p < 0.001 ) and for the neurodynamic group compared to the stretching group ( p = 0.006 ) .
both the neurodynamic and stretching groups significantly improved rom compared to their baseline values ( p < 0.001 ) , whereas there was no significant improvement noted for the control group ( p = 0.800 ) .
the effect sizes were large for neurodynamic group ( d = 2.36 ) , moderate for stretching group ( d = 0.73 ) , and very small for control group ( d < 0.01 ) .
results from this study show that an isolated neurodynamic intervention provides a greater immediate increase in passive slr range of motion than static hamstring stretching in subjects with short hamstring syndrome .
although both interventions were more effective in increasing slr range than the placebo control , only the neurodynamic intervention group demonstrated before and after differences in mean slr which exceeded the mdc95 upper limit of 6.6. the results confirmed our initial hypothesis that an isolated neurodynamic sciatic sliding technique would provide a greater immediate improvement in hip flexion , assessed by passive slr , than hamstring stretching or placebo . increasing hamstring flexibility
has been suggested as an important factor in the treatment and prevention of lower extremity overuse injuries [ 44 , 45 ] .
much of the research on increasing hamstring flexibility has focused on the varying modes of stretching , such as proprioceptive neuromuscular facilitation ( pnf ) [ 46 , 47 ] , static stretching [ 4648 ] , plyometric stretching , and ballistic stretching .
very few studies have examined the effect of neurodynamic interventions on hamstring flexibility [ 24 , 30 , 40 ] and the results of this study can be seen as adding further evidence for the potential role of neural tissue mechanosensitivity in limiting the slr .
a new conceptual model showing an interrelationship between the different factors involved in hamstring strains may provide a better understanding of this multifactorial injury and therefore improve its prevention and prediction methods .
neural tissue mechanosensitivity presenting clinically as tightness in the hamstrings is a plausible yet only retrospectively studied potential risk factor for and/or potential differential diagnosis to be considered in , hamstring strain injury .
the sensory theory proposed by weppler and magnusson suggests that muscle flexibility and its response to sudden stretch have more to do with perceptions of stretch and pain ( sensation ) than the biomechanical effects on muscle tissue itself .
this proposal was supported in a study by aparicio and others which demonstrated that a suboccipital muscle inhibition technique altered hamstring muscle flexibility when compared to a placebo intervention .
the authors measured hamstring flexibility in 3 ways ( forward flexion distance test , straight leg raise test , and popliteal angle test ) and found significant before and after differences on all 3 measures for the suboccipital muscle inhibition technique but not for the placebo intervention .
the fact that such a distant technique ( suboccipital region ) could have an immediate effect on the flexibility in the hamstrings may lend support to the sensory theory limiting flexibility of the posterior thigh structures .
it seems reasonable to attribute the observed increase in hamstring tissue flexibility following the suboccipital muscle inhibition technique to changes in the subjects ' perceptions of stretch or pain associated with the 3 flexibility measures .
the results of this study demonstrate a mean increase in the hip flexion range of the slr for the neurodynamic group of 9.86 which compares favorably with other studies .
castellote - caballero and others reported mean increases in slr of 9.4 following a similar neurodynamic slider technique , and aparicio et al . reported mean increases of 5.9 for the right slr and 5.5 for the left slr following application of the suboccipital muscle inhibition technique .
mendez - sanchez and others completed a pilot study on young healthy male soccer players and found mean increases of only 3.7 in the right slr and 2.2 in the left slr after sustained hamstring stretching intervention .
when they added a neurodynamic slider technique to the sustained hamstring stretching intervention , they found greater mean increases of 6.2 in the right slr and 6.3 in the left slr .
finally , hopper et al . reported mean increases of only 4.7 in slr after the application of massage techniques to the hamstring musculature .
it has been suggested that stretching of the hamstring musculature to improve tissue flexibility may reduce the number of leg overuse injuries after exercise , although further high quality studies are needed . while some theories explaining the therapeutic effects of muscle stretching suggest there is alteration of the viscoelastic properties of muscles , studies have shown the importance of distinguishing between real and apparent increases in muscle flexibility [ 37 , 56 ] .
observed changes in slr following interventions may be more associated with increased tolerance to the uncomfortable stretch sensation rather than true changes to muscle elasticity .
although the results from this study do not provide information on the mechanisms for the observed changes , they do suggest that neurodynamic interventions can significantly increase slr more than static stretching in the short term in healthy subjects with short hamstring syndrome .
this study only examined immediate effects of a single episode and the lack of longer term follow - up should be considered .
it is not known how long the observed increase in hamstring flexibility might have lasted .
furthermore , it is not known if repetition and an appropriate dosage of the neurodynamic interventions over time might lead to longer lasting effects .
finally , we did not conduct any long - term follow - up to determine if the observed changes in flexibility might have resulted in any change in incidence of hamstring injuries in these subjects with short hamstring syndrome .
findings from this study indicate that a neurodynamic sliding intervention will increase short - term hamstring flexibility as measured by the passive slr to a greater degree than static hamstring stretching in healthy subjects with short hamstring syndrome .
future research should look at longer term results and assess the effect of combining neurodynamic techniques with other interventions . | background .
hamstring injuries continue to affect active individuals and although inadequate muscle extensibility remains a commonly accepted factor , little is known about the most effective method to improve flexibility . purpose . to determine if an isolated neurodynamic sciatic sliding technique would improve hamstring flexibility to a greater degree than stretching or a placebo intervention in asymptomatic subjects with short hamstring syndrome ( shs ) . study design .
randomized double - blinded controlled trial . methods .
one hundred and twenty subjects with shs were randomized to 1 of 3 groups : neurodynamic sliding , hamstring stretching , and placebo control .
each subject 's dominant leg was measured for straight leg raise ( slr ) range of motion ( rom ) before and after interventions .
data were analyzed with a 3 2 mixed model anova followed by simple main effects analyses .
results . at the end of the study , more rom was observed in the neurodynamic and stretching groups compared to the control group and more rom in the neurodynamic group compared to stretching group . conclusion .
findings suggest that a neurodynamic sliding technique will increase hamstring flexibility to a greater degree than static hamstring stretching in healthy subjects with shs
. clinical relevance .
the use of neurodynamic sliding techniques to improve hamstring flexibility in sports may lead to a decreased incidence in injuries ; however , this needs to be formally tested . |
during 20072014 , we collected 27 fecal and 24 intestinal samples from pigs with suspected pedv or tgev infections ; the pigs came from swine farms in northern italy ( po valley ) , which contains the regions of piemonte , lombardia , emilia romagna , and veneto ( technical appendix figure 1 ) .
clinical signs included watery diarrhea in sows and a death rate in piglets of 5%10% , lower than is typical with pedv or tgev infections .
samples were submitted for testing by electron microscopy , pedv elisa , viral isolation , pan - cov reverse transcription pcr ( rt - pcr ) , and rt - pcr for prcv and tgev ; selected positive pan - cov samples were sequenced ( 1214 ) ( technical appendix ) .
results of electron microscopy showed that 25 ( 49% ) of the 51 samples contained cov - like particles , but all samples were negative for viral isolation .
although only 38 samples ( 74% ) were positive by pan - cov rt - pcr , 47 ( 92% ) were positive by the pedv elisa ( table 1 ) ( 12,13 ) .
of the 38 pan - cov positive samples , 18 were selected for partial rna - dependent rna polymerase ( rdrp ) , spike ( s1 ) ( 14 ) , and membrane ( m ) sequencing ( table 1 ) .
all samples were negative for prcv and tgev by rt - pcr , ruling out co - infection with pedv and tgev or prcv ( 15 ) .
* cluster i represents strains circulating from 2007 through mid-2009 ; cluster ii represents strains circulating from mid-2009 through 2012 ; and cluster iii represents strains circulating since 2014 .
em , electron microscopy ; m , membrane ; pan - cov rt - pcr , pan - coronavirus reverse transcription pcr ; pedv , porcine epidemic diarrhea virus ; prcv , porcine respiratory coronavirus ; rdrp , rna - dependent rna polymerase ; s1 , spike ; tgev , transmissible gastroenteritis virus ; + , positive ; , negative ; na , not tested or sequenced .
samples selected for whole genome sequencing . on the basis of the partial sequences from rdrp and the s1 and m genes , the strains from italy clustered into 3 temporally divided groups , suggesting 3 independent virus entries .
cluster i represents strains circulating from 2007 through mid-2009 ; cluster ii represents strains circulating from mid-2009 through 2012 ; and cluster iii represents strains circulating since 2014 ( technical appendix figure 2 , panels a c ) .
cluster i was identified in emilia romagna ( n = 1 ) , lombardia ( n = 5 ) , and veneto ( n = 1 ) .
cluster ii was identified in emilia romagna ( n = 1 ) and lombardia ( n = 8) .
cluster iii was identified in emilia romagna at 2 swine farms . to help explain the temporal clustering ,
a single s1 gene segment was sequenced from clusters i and ii ( pedv / italy/7239/2009 and secov / italy/213306/2009 , respectively ) . because of the recent outbreak of pedv in europe , the 2 positive samples from cluster iii ( pedv / italy/178509/2014 and pedv / italy/200885/2014 ) were sequenced ( figure 1 , panel a ) .
a ) analysis performed on the basis of the nucleotide sequence of the complete spike ( s1 ) gene of 4 representative strains from the 3 clusters and b ) whole genome of 2 positive strains from clusters i and ii .
cluster i represents strains circulating from 2007 through mid-2009 ; cluster ii represents strains circulating from mid-2009 through 2012 ; and cluster iii represents strains circulating since 2014 .
the strains from this study are represented in blue ; strains from china are green ; strains from north america are red ; and strains from europe are purple .
pedv , porcine epidemic diarrhea virus ; prcv , porcine respiratory coronavirus ; tgev , transmissible gastroenteritis virus ; secov , swine enteric coronavirus .
one strain from each cluster was selected for whole genome sequencing ( technical appendix ) .
unfortunately , the whole genome was obtained from only clusters i and ii ( pedv / italy/7239/2009 and secov / italy/213306/2009 , respectively ; figure 1 , panel b ) .
recombination analysis was conducted on the 2 whole genomes and was not detected in pedv / italy/7239/2009 .
recombination was detected in secov / italy/213306/2009 at position 20636 and 24867 of pedv cv777 and at position 20366 and 24996 of tgev h16 ( figure 2 ) , suggesting the occurrence of a recombination event between a pedv and a tgev .
the complete s gene of secov / italy/213306/2009 shared 92% and 90% nt identity with the prototype european strain pedv cv777 and the original highly virulent north american strain colorado 2013 , respectively , and the remaining genome shared a 97% nt identity with the virulent strains tgev h16 and tgev miller m6 .
whole - genome analysis of pedv / italy/7239/2009 showed that it grouped with the global pedv strains ( 6 ) and shared 97% nt identity with pedv strains cv777 , dr13 virulent , and north american s - indel strain oh851 ( table 2 ) .
potential recombination points in the secov strains in study of swine enteric coronaviruses in italy .
the potential parent strains h16 ( tgev ) and cv777 ( pedv ) are shown in teal and purple , respectively .
pedv , porcine epidemic diarrhea virus ; tgev , transmissible gastroenteritis virus ; secov , swine enteric coronavirus .
* cluster i represents strains circulating from 2007 through mid-2009 ; cluster ii represents strains circulating from mid-2009 through 2012 .
ger , germany ; orf , open reading frame ; prcv , porcine respiratory coronavirus ; pedv , porcine epidemic diarrhea virus ; secov , swine enteric coronavirus ; tgev , transmissible gastroenteritis virus
during 20072014 , most ( 92% ) samples collected from the po valley in italy were positive for pedv by elisa ; only 72% were positive by pan - cov pcr .
however , because we were investigating the presence of pedv or tgev in samples with clinical signs of diarrhea , the high occurrence of pedv may not reflect the actual prevalence of pedv in italy . the increased percentage of pedv found in samples tested by elisa , compared with the proportion found by pcr , may be explained by the number of ambiguous bases in the pan - cov primers ; the ambiguous bases severely reduce the efficiency of the reaction .
the swine enteric cov strains from italy in our study , including the recombinant strain , were reported in pigs with mild clinical signs , indicating that pedv and secov have been circulating in italy and likely throughout europe for multiple years but were underestimated as a mild form of diarrhea . to understand the evolution of pedv in italy , the partial rdrp , s , and m genes
cluster i ( 2007mid 2009 ) resembles the oldest pedv strains ; cluster ii resembles a new tgev and pedv recombinant variant ; and cluster iii , identified from 2 pig farms in northern italy in 2014 , resembles the pedv s - indel strains identified in germany , france , belgium , and the united states .
the > 99.3% nt identity of the s1 gene within cluster iii and in previously identified strains could suggest the spread of the s - indel strain into europe .
however , directionality of spread can not be determined because of a lack of global and temporal pedv sequences .
although our findings could indicate 3 introductions of pedv in italy , the results more likely suggest the high ability of natural recombination among covs and the continued emergence of novel covs with distinct pathogenic properties . further investigation is needed to determine the ancestor of the secov strain or to verify whether the recombinant virus was introduced in italy .
recombinant secov was probably generated in a country in which both pedv and tgev are endemic , but because the presence of these viruses in europe is unclear and secov has not been previously described , it is difficult to determine the parental strains and geographic spread of secov .
future studies are required to describe the pathogenesis of secov and its prevalence in other countries .
technical appendix . additional methods , tables , and figures from study of swine enteric coronaviruses in italy . | porcine epidemic diarrhea virus ( pedv ) has been detected sporadically in italy since the 1990s .
we report the phylogenetic relationship of swine enteric coronaviruses collected in italy during 20072014 and identify a drastic shift in pedv strain variability and a new swine enteric coronavirus generated by recombination of transmissible gastroenteritis virus and pedv . |
to achieve the first objective , we conducted a cross - sectional study in the alpujarras from april through june 2006 .
we then compared current leishmaniasis seroprevalence data with data from 2 cross - sectional surveys conducted in 1984 and 1991 ( 35 ) .
the villages sampled for all 3 studies were similar and had been selected at random from within each of the 3 bioclimatic levels ( thermo- , meso- , and supra - mediterranean ) that comprise the inhabited zone of the alpujarras ( figure 1 ) ( 10 ) . for each level , respectively , altitudes are 0700 , 600900 , and 9001,800 m above sea level ; annual mean temperatures are 1719 , 1317 , and 815c ; and annual rainfalls are 200350 , 6001,000 , and 1,0001,600 inches . the dates for sample collection were set to coincide with organized events at which dogs were gathered ( e.g. , antirabies vaccination campaigns ) . all 3 surveys used indirect immunofluorescence for diagnosis ; protocols and positivity threshold were identical . dogs with a titer > 160 were considered positive . to determine and compare the existence or lack of statistically significant differences between present and past prevalence rates
the owners of the dogs included in the cross - sectional study conducted in the alpujarras from april to june 2006 were asked to complete an epidemiologic record for each dog tested ; data on the animal and its environment were recorded for subsequent use in univariate and multivariate logistic regression analyses ( tables 1 , 2 ) .
density data were included for 2 vectors , phlebotomus perniciosus and p. ariasi sandflies , captured with sticky traps in june 2006 in the same villages in which the surveys were conducted .
we investigated the existence of interaction and/or confusion between variables by constructing and comparing logistic regression models .
confusion was noted between the location during daytime and location at night , so location during daytime was excluded from the multivariate analysis .
guard dogs ( n = 34 ) , sheepdogs ( n = 8) , stray dogs ( n = 4 ) , dogs in kennel ( n = 2 ) . * determined by multivariate logistic regression . with age as continuous variable ( 0.125 years , average 4.7 years ) , odds ratio ( or ) = 1.142 ; with weight as continuous variable ( 260 kg , average = 17.7 ) , or = 1.047 .
guard dogs , sheepdogs , stray dogs , and dogs in kennels . in our 2006 survey , 57 ( 13.0% ) of 439 dogs had an antibody titer > 160 ( seroprevalence rate ) , 268 ( 61.0% ) dogs had titers 2080 , and 114 ( 26.0% ) had no titer . in terms of bioclimatic level ,
canine leishmaniasis seroprevalence was 20.1% at the meso - mediterranean , 13.3% at the thermo - mediterranean , and only 1.1% at the supra - mediterranean levels .
statistically significant differences ( test p<0.001 ) indicate that these differences are not random .
the evolution - in - time analysis ( figure 2 ) shows how over 22 years ( 19842006 ) , seroprevalence of canine leishmaniasis has progressively increased at the meso - mediterranean level , climbing from 9.2% in 1984 to 15.4% in 1991 and finally to 20.1% in 2006 ( p = 0.015 ) ; in contrast , no significant changes have taken place in global prevalence or in the other 2 bioclimatic levels studied ( 35 ; this study ) .
canine leishmaniasis seroprevalence rates in the alpujarras , spain , 19842006 , by time and bioclimatic level .
dogs at greatest risk for canine leishmaniasis in the disease - endemic region of the alpujarras were large dogs ( > 25 kg ) and older dogs ( > 4 years ) that worked as guard dogs or sheepdogs , slept outdoors , and lived at the thermo- or meso - mediterranean level in a village such as torvizcn , where the p. perniciosus density is > 4 sandflies / m .
risk for these dogs was 54,571 greater than for dogs that were kept as pets , were small , were < 4 years of age , and slept inside a house in a village such as mecina bombarn or prtugos at the supra - mediterranean level where p. perniciosus density is < 4 sandflies / m .
among the drivers of global change that have the potential to influence vector - borne diseases , climatic and nonclimatic ( socioeconomic , demographic , and environmental ) factors have been cited ( 1,2 ) . although it is not easy to attribute our findings progressive increase in seroprevalence at the meso - mediterranean level and drop and subsequent rise at the thermo - mediterranean level ( figure 2)to 1 or more drivers ,
we can attempt to find some form of association in the changes that have occurred in the alpujarras throughout these 2 decades . studies on climate change in spain confirm a warming tendency ( reflected at the global level ) , which provides evidence that temperatures have been increasing for a quarter of a century ( 11,12 ) .
thus , we must assume that changes in temperature , rainfall , or humidity will have equally affected the 3 bioclimatic levels researched in the alpujarras and may have influenced the spatial and temporal distribution and the seasonal dynamics of sandflies .
an increase was detected in the period of p. perniciosus activity in the region ; these effects were probably more notable at the meso - mediterranean level because this is where the density of this vector species is at its highest ( 6 ; martn - snchez et al . ,
data ) . during the time period researched , the human population remained constant in the alpujarras ; it has , however , progressively decreased in the disperse populations and increased in population centers . unfortunately , no official figures for the canine population are available , although we were informed by staff of local town halls that in 1991 , the 615 dogs analyzed accounted for 100% of the total number of dogs registered in the villages sampled ( 3 ) .
one change during the period studied was the gradual disappearance of livestock enclosures in rural population centers .
( 13,14 ) and are ideal places for sandfly blood sucking , mating , and oviposition ( 15 ) .
with respect to risk factors , the increase in canine leishmaniasis seroprevalence as animal s age increases seems logical because in a leishmaniasis - endemic area , the greater the age , the longer the animal will have been exposed to sandflies and the greater the probability of having been bitten by an infected female sandfly .
seroprevalence of canine leishmaniasis also increases gradually with weight , which could be attributed to the vector being more attracted to larger animals .
dogs that sleep outdoors are at greater risk than those sleeping indoors because the density of the vector ( p. perniciosus ) is greater outdoors than inside a house .
the association between p. perniciosus density and canine leishmaniasis seems logical , considering that p. perniciosus is the main vector species in the alpujarras .
this is the only species found to have been infected in this region ( 8,9 ) , although p. ariasi is a proven vector in the nearby region of the axarquia ( 13 ) . in the alpujarras ,
the percentage of dogs kept as pets has increased from 42% in 1991 ( 3 ) to 59% in 2006 ( p<0.001 ) .
this increase corresponds to a general increase in standard of living and , along with other factors such as the disappearance of livestock enclosures from within population centers , would have acted to reduce transmission .
only the extension of the vector s activity period , which was detected at some trap sites , would have acted to increase transmission .
such extension of activity may be related to the increase in temperature brought about by climate change ( 11,12 ) . | to examine prevalence changes and risk factors for canine leishmaniasis , we conducted a cross - sectional seroprevalence study and a survey during april
june 2006 .
seroprevalence had increased at the meso - mediterranean bioclimatic level over 22 years .
risk was highest for dogs that were older , large , lived outside , and lived at the meso - mediterranean level . |
les infections bordetella pertussis continuent dtre un important problme de sant publique au canada .
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une nouvelle mthode pcr en temps rel du b pertussis fonde sur le gne de porine a t mise lessai paralllement plusieurs mthodes dj publies qui ciblent des gnes comme lis481 , le promoteur de ptx , la pertactine et une thialase ventuelle .
les mthodes ont t values laide dun groupe de rfrence de bactries respiratoires communes , y compris diverses espces de bordetella et 107 chantillons nasopharyngs cliniques
la mthode visant llment is481 avait la sensibilit analytique la plus leve , mais manquait de spcificit pour le b pertussis dans le groupe de rfrence et les chantillons cliniques . les mthodes ciblant les gnes du promoteur de ptx et de la perctatine ont galement donn des rsultats faux positifs .
une mthode de pcr fonde sur le gne thialase tait hautement spcifique , mais ne dcelait pas toutes les souches de rfrence du b pertussis .
cependant , une nouvelle mthode ciblant le gne de porine a dmontr une forte spcificit au b pertussis , la fois dans le groupe de rfrence et dans les chantillons cliniques et , daprs les rsultats confirms par squenage , prdit correctement tous les cas positifs au b pertussis dans les chantillons cliniques .
daprs lanalyse de rgression probit , la limite de dtection de 95 % de la nouvelle mthode tait de quatre units formant colonies par raction .
une nouvelle mthode faisant appel la porine pour dceler le b pertussis donne un rendement suprieur et peut tre utile pour amliorer la dtection molculaire du b pertussis dans des chantillons cliniques .
the reference panel was comprised of b pertussis ( american type culture collection [ atcc ] baa-589 , 9340 and 9797 , and a clinical strain isolated at bc children s hospital [ vancouver , british columbia ] from a patient nasopharyngeal aspirate ) , b parapertussis ( atcc 15237 and a clinical strain isolated at the bc centre for disease control [ vancouver , british columbia ] ) , b holmesii ( atcc 51541 ) , b bronchiseptica ( atcc baa-588 ) , haemophilus influenzae ( atcc 10211 ) , pseudomonas aeruginosa ( atcc 27853 ) , klebsiella pneumoniae ( atcc 13882 ) , staphylococcus aureus ( atcc 43300 ) , staphylococcus epidermidis ( atcc 12228 ) , streptococcus agalactiae ( atcc 13813 ) , streptococcus mitis ( atcc 6249 ) , streptococcus pneumoniae ( atcc 49619 ) , streptococcus pyogenes ( atcc 19615 ) , corynebacterium pseudodiphtheriticum ( atcc 10700 ) , mycoplasma pneumoniae ( atcc 29342 ) , chlamydophila pneumoniae ( atcc vr-1310 ) and a clinical strain of neisseria mucosa isolated at bc children s hospital .
bordetella species were streaked on charcoal agar medium ( oxoid ) and grown at 37c for 72 h in a humidified environment .
bacterial suspension was prepared in phosphate - buffered saline to a turbidity equivalent to a 0.5 mcfarland standard , titred by colony counting and cryopreserved at 80c .
the titred bacterial stocks were diluted in te8 buffer ( 10 mm tris , 1 mm edta ; ph 8.0 ) and used directly in the pcr reactions .
other bacteria were grown on blood or chocolate agar plates ( oxoid ) overnight at 37c in a 5% co2 atmosphere , and a bacterial suspension was prepared in te8 buffer to a turbidity equivalent to a 0.5 mcfarland standard .
a total of 107 nasopharyngeal wash specimens were used in the present study , including selected pcr - positive ( by a sybr green pcr ) specimens since august 2009 , and specimens submitted between september 2011 to october 2012 to the microbiology and virology laboratory of bc children s hospital for b pertussis pcr .
testing was performed exclusively on retrospective , residual samples that were stored at 80c . to maintain patient anonymity
, each sample was coded and all patient identifiers were removed to ensure that personnel involved in the present study were unaware of any patient information .
ethics approval was not considered to be necessary because studies that involve the secondary use of anonymous human biological materials are exempted from review by the local research ethics board of the university of british columbia .
dna from 0.2 ml nasopharyngeal wash specimens was extracted using the qiasymphony virus / bacteria kit in an automated dna extraction platform qiasymphony sp ( qiagen , usa ) .
the titred bacterial suspensions and dna extracts from 107 nasopharyngeal wash specimens were analyzed using taqman real - time pcr or by sybr green pcr with the primers and probes shown in table 1 .
for taqman real - time pcr assays , 5 l of sample extract or diluted bacterial suspensions was mixed with 20 l of a master mix containing 12.5 l of taqman universal pcr master mix ( applied biosystems , usa ) and primers and probes to final concentrations shown in table 1 .
thermal cycling was performed in a abi7500 fast instrument ( applied biosystems , usa ) with one cycle of 95c for 10 min , followed by 45 cycles consisting of 95c for 15 s and 60c for 60 s. the sybr green pcr was performed with the same parameters except that the reaction mix contained 12.5 l 2xitaq sybr green pcr master mix ( bio - rad , usa ) and the thermal cycling was performed in a smartcycler ( cepheid , usa ) with one cycle of 95c for 2 min , followed by 45 cycles consisting of 95c for 10 s and 64c for 60 s. the sybr green pcr reactions were subjected to meltcurve analysis after the final amplification cycle .
samples with discrepant results from different assays were analyzed by sequencing pcr products using the bigdye terminator v3.1 cycle sequencing kit ( applied biosystems , usa ) in an abi 3130 genetic analyzer according to the manufacturer s instructions .
for probit regression analysis , b pertussis strain atcc baa-589 stock suspension was serially diluted to 10 , 50 , 100 , 200 , 500 and 1000 colony forming units ( cfu)/ml , and eight replicates of each dilution were tested by real - time pcr ; 95% detection limit was determined as described previously ( 16 ) .
the reference panel was comprised of b pertussis ( american type culture collection [ atcc ] baa-589 , 9340 and 9797 , and a clinical strain isolated at bc children s hospital [ vancouver , british columbia ] from a patient nasopharyngeal aspirate ) , b parapertussis ( atcc 15237 and a clinical strain isolated at the bc centre for disease control [ vancouver , british columbia ] ) , b holmesii ( atcc 51541 ) , b bronchiseptica ( atcc baa-588 ) , haemophilus influenzae ( atcc 10211 ) , pseudomonas aeruginosa ( atcc 27853 ) , klebsiella pneumoniae ( atcc 13882 ) , staphylococcus aureus ( atcc 43300 ) , staphylococcus epidermidis ( atcc 12228 ) , streptococcus agalactiae ( atcc 13813 ) , streptococcus mitis ( atcc 6249 ) , streptococcus pneumoniae ( atcc 49619 ) , streptococcus pyogenes ( atcc 19615 ) , corynebacterium pseudodiphtheriticum ( atcc 10700 ) , mycoplasma pneumoniae ( atcc 29342 ) , chlamydophila pneumoniae ( atcc vr-1310 ) and a clinical strain of neisseria mucosa isolated at bc children s hospital .
bordetella species were streaked on charcoal agar medium ( oxoid ) and grown at 37c for 72 h in a humidified environment .
bacterial suspension was prepared in phosphate - buffered saline to a turbidity equivalent to a 0.5 mcfarland standard , titred by colony counting and cryopreserved at 80c .
the titred bacterial stocks were diluted in te8 buffer ( 10 mm tris , 1 mm edta ; ph 8.0 ) and used directly in the pcr reactions .
other bacteria were grown on blood or chocolate agar plates ( oxoid ) overnight at 37c in a 5% co2 atmosphere , and a bacterial suspension was prepared in te8 buffer to a turbidity equivalent to a 0.5 mcfarland standard .
a total of 107 nasopharyngeal wash specimens were used in the present study , including selected pcr - positive ( by a sybr green pcr ) specimens since august 2009 , and specimens submitted between september 2011 to october 2012 to the microbiology and virology laboratory of bc children s hospital for b pertussis pcr .
testing was performed exclusively on retrospective , residual samples that were stored at 80c . to maintain patient anonymity , each sample was coded and all patient identifiers
were removed to ensure that personnel involved in the present study were unaware of any patient information .
ethics approval was not considered to be necessary because studies that involve the secondary use of anonymous human biological materials are exempted from review by the local research ethics board of the university of british columbia .
dna from 0.2 ml nasopharyngeal wash specimens was extracted using the qiasymphony virus / bacteria kit in an automated dna extraction platform qiasymphony sp ( qiagen , usa ) .
the titred bacterial suspensions and dna extracts from 107 nasopharyngeal wash specimens were analyzed using taqman real - time pcr or by sybr green pcr with the primers and probes shown in table 1 .
for taqman real - time pcr assays , 5 l of sample extract or diluted bacterial suspensions was mixed with 20 l of a master mix containing 12.5 l of taqman universal pcr master mix ( applied biosystems , usa ) and primers and probes to final concentrations shown in table 1 .
thermal cycling was performed in a abi7500 fast instrument ( applied biosystems , usa ) with one cycle of 95c for 10 min , followed by 45 cycles consisting of 95c for 15 s and 60c for 60 s. the sybr green pcr was performed with the same parameters except that the reaction mix contained 12.5 l 2xitaq sybr green pcr master mix ( bio - rad , usa ) and the thermal cycling was performed in a smartcycler ( cepheid , usa ) with one cycle of 95c for 2 min , followed by 45 cycles consisting of 95c for 10 s and 64c for 60 s. the sybr green pcr reactions were subjected to meltcurve analysis after the final amplification cycle .
samples with discrepant results from different assays were analyzed by sequencing pcr products using the bigdye terminator v3.1 cycle sequencing kit ( applied biosystems , usa ) in an abi 3130 genetic analyzer according to the manufacturer s instructions .
for probit regression analysis , b pertussis strain atcc baa-589 stock suspension was serially diluted to 10 , 50 , 100 , 200 , 500 and 1000 colony forming units ( cfu)/ml , and eight replicates of each dilution were tested by real - time pcr ; 95% detection limit was determined as described previously ( 16 ) .
real - time pcr assays targeting the is481 element ( is481 ) , b pertussis/ b parapertussis gene for an outer membrane porin protein ( omp ) , pertussis toxin ( ptx ) promoter ( bptp ) , pertactin gene ( prn ) , and a putative thialase gene ( bp283 ) ( table 1 ) ( 1115 ) were selected from the literature .
in addition , two new sets of primers and taqman probes were designed based on ptx promoter ( pt - p ) and b pertussis gene for a porin protein ( bptd_0837 ) ( por ) .
the new ptx promoter assay was designed through modification of the primer and probe sequences described by grogan et al ( 12 ) in an attempt to further improve the specificity of the assay . by aligning the genomes of several different bordetella species ,
it was noted that the published probe sequence has only a single nucleotide mismatch with the corresponding sequences of b bronchiseptica and b parapertussis genome .
in contrast , the newly designed probe has > 3 nucleotide differences with the corresponding sequences of b bronchiseptica and b parapertussis genome .
the new por assay was designed by exploiting the nucleotide sequence variation between the b pertussis gene for a porin protein and the corresponding sequences in the genomes of b parapertussis , b bronchiseptica and b holmesii ( figure 1 ) .
all pcr assays were first tested using a panel comprised of atcc strains and patient isolates of various bordetella species and other bacteria commonly found in the respiratory tract ( table 2 ) .
the omp assay ( 15 ) is a sybr green - based , shared primer assay designed to amplify both b pertussis and b parapertussis - specific dna , but with different size pcr products that can subsequently be differentiated by their dissociation curves .
when this pcr assay was applied to the panel of different bordetella species and other respiratory bacteria , false - positive amplification was observed with b holmesii , b bronchiseptica , s mitis and s pyogenes , but the dissociation curves were distinct from that of b pertussis and b parapertussis strains ( table 2 ) . apart from this assay , none of the other assays showed any cross - reactivity with other bacteria tested in the present study that do not belong to the genus bordetella .
the analytical sensitivity of is481 assay for b pertussis was much higher ( by cycle threshold > 7 ) than other assays .
the assay detected all b pertussis strains in the test panel , but also resulted in the false - positive detection of b parapertussis , b holmesii and b bronchiseptica strains , confirming the nonspecific nature of this target . among the single - target taqman assays , the bptp assay nonspecifically detected b bronchiseptica along with b pertussis strains , while the ptx - promoter assay , pt - p , designed in the present study lacked sensitivity and failed to detect all b pertussis strains .
the bp283 assay was specific to b pertussis but failed to detect two of the four b pertussis strains tested .
the prn assay detected all b pertussis strains but it weakly detected b bronchiseptica at a relatively high concentration ( 5.410 cfu / ml ) .
in contrast , the newly designed por assay did not exhibit any cross - reactivity with dna from any of the non - b pertussis strains .
based on these results , the is481 , bptp , bp283 , prn and por assays were selected for further analysis of clinical samples . among the 107 nasopharyngeal wash specimens , 21 ( 19.6% )
samples were positive according to the is481 assay , 20 ( 18.7% ) samples were positive according to the bptp and por assays , and 19 ( 17.7% ) samples were positive according to the bp283 and prn assays ( table 3 ) .
there were three discrepant samples : a sample that was positive according to all assays except the bp283 assay ; a sample that was missed by the prn and bptp assays ; and a sample that was only positive using the bptp and is481 assays .
sequencing of pcr products confirmed that the first two samples were , in fact , b pertussis , while the third sample was b bronchiseptica ( figure 2 ) .
taking into account the definitive sequence identification , the performances of all five b pertussis - specific assays were comparable , but the newly designed por assay correctly detected all samples with a clinical sensitivity equal to that of is481 assay . based on a probit regression analysis ,
the diagnosis of whooping cough caused by b pertussis could be difficult , particularly at the early stages because the signs and symptoms of the disease closely resemble those of other common respiratory illnesses , such as those caused by mycoplasma pneumoniae , corynebacterium pneumoniae and adenoviruses .
moreover , other related bordetella species , such as b parapertussis , b bronchiseptica and b holmesii , are also known to cause respiratory illness in humans , although generally with a milder clinical presentation ( 3,8 ) .
specificity of diagnostic and surveillance methodologies for b pertussis is , therefore , critical for the effective management and control of pertussis disease .
however , pcr testing for pertussis , which is now in widespread use , has variable specificity and needs further standardization and optimization ( 1,9 ) . in the present study
, we aimed to identify or develop a highly specific and sensitive , real - time pcr assay for b pertussis that can replace the pcr assays based on is481 , which is a repeat element present in the genomes of different bordetella species at variable copy numbers ( 5 ) .
we tested and compared the performance characteristics of both previously published and in - house developed pcr assays that target different genes and genomic regions specific to b pertussis , in addition to that of is481-based pcr assays .
we observed remarkable variation in the specificity of different pcr assays while assessing a sample panel , comprised of reference strains of various bordetella species .
however , the sensitivity and specificity of these assays remained approximately 95% in the clinical samples .
the discrepant clinical sample results obtained by the different b pertussis specific real - time pcr assays are consistent with those obtained using the atcc panel of different bordetella species .
the bp283 assay , which failed to detect two known atcc strains of b pertussis , also failed to detect one of the b pertussis - positive clinical samples .
the is481 and bptp assay , which detected a known atcc strain of b bronchiseptica , also produced one false - positive result among the clinical samples , which was eventually confirmed to be positive for b bronchiseptica by sequencing .
the results of using the prn assay on the atcc panel suggests that this assay could also falsely detect b bronchiseptica , particularly at bacterial concentrations > 10 cfu / ml in clinical samples .
the novel real - time pcr assay that targets the porin gene of b pertussis demonstrated superior performance over all other b pertussis pcr assays tested in the present study .
the higher analytical sensitivity , but poor specificity of the is481 pcr , is well described in the literature and is a generally accepted phenomenon ( 57,11 ) .
however , an independent comparison of the most common single - target pcr assays for b pertussis has not been reported to date .
therefore , the results of the present study could be useful for guiding clinical microbiologists in the selection of an appropriate real - time pcr assay for b pertussis .
the new por gene - based pcr assay described may serve as a valuable new tool for diagnostic laboratories seeking to improve the specificity of b pertussis detection , with sensitivity comparable with that of is481 pcr assays . | background : bordetella pertussis infections continue to be a major public health challenge in canada .
polymerase chain reaction ( pcr ) assays to detect b pertussis are typically based on the multicopy insertion sequence is481 , which offers high sensitivity but lacks species specificity.methods:a novel b pertussis real - time pcr assay based on the porin gene was tested in parallel with several previously published assays that target genes such as is481 , ptx - promoter , pertactin and a putative thialase .
the assays were evaluated using a reference panel of common respiratory bacteria including different bordetella species and 107 clinical nasopharyngeal specimens .
discrepant results were confirmed by sequencing the pcr products.results:analytical sensitivity was highest for the assay targeting the is481 element ; however , the assay lacked specificity for b pertussis in the reference panel and in the clinical samples .
false - positive results were also observed with assays targeting the ptx - promoter and pertactin genes . a pcr assay based on the thialase gene was highly specific but failed to detect all reference strains of b pertussis .
however , a novel assay targeting the porin gene demonstrated high specificity for b pertussis both in the reference panel and in clinical samples and , based on sequence - confirmed results , correctly predicted all b pertussis - positive cases in clinical samples . according to probit regression analysis ,
the 95% detection limit of the new assay was 4 colony forming units / reaction.conclusion : a novel porin assay for b pertussis demonstrated superior performance and may be useful for improved molecular detection of b pertussis in clinical specimens . |
cells respond to environmental
signals and stresses using a variety
of mechanisms that link the external stimuli to changes in cell phenotype
by myriad biochemical reactions that ultimately lead to changes in
gene expression and protein activity .
well - defined pathways of signal
transduction affect transcription , mrna stability , protein levels ,
and protein secondary modification , with the altered protein function
and metabolite levels defining a new cell state . among the mechanisms
of cell response ,
the link between translation and environmental changes
is the least understood . here , we describe the emerging evidence for
a new system by which cells respond and adapt to environmental stresses
by reprogramming dozens of modified ribonucleosides in trna , which
leads to selective translation of codon - biased mrnas .
this system
exploits features of both genetics , in the form of a code of codon
use in families of genes , and postsynthetic dna and protein modifications ,
in the idea that editable modifications affect gene expression , with
signal transduction pathways linking the environmental stress to controlled
changes in gene expression at the level of translational elongation .
as a parallel to the system
of rna modifications in control of
translation , epigenetics is classically defined as heritable changes
in gene expression without changing the dna sequence .
the prime example
involves formation of mc by dna methyltransferases ( dnmt s )
as a well - established regulator of gene expression , with methylation patterns in promoter
regions dramatically altered in response to environmental stimuli
or in different cancers .
although dna methylation
patterns are heritable and the patterns previously presumed to be
stable in a specific cell type , global reprogramming of mc patterns in the genome has now been observed in response to exposure
to drugs and toxicants , which illustrates
a dynamic role for epigenetic signals in cellular response and adaptation .
methylation of histone tails by protein methyltransferases ( pmt s )
functions in a similar manner to dna methylation as a well - recognized
regulator of gene expression . as part
of an integrated system with dna methylation ,
histone methylation
is theorized to be part of a complicated histone code that is composed
of a variety of other posttranslational modifications and that is
altered by environmental signals and disease pathologies to control
gene expression .
for example , lysine n - methylation ( h3k4 , h3k36 ) in histone h3 and subsequent demethylation
are considered dueling signals that regulate transcription . at their simplest ,
both promoter and histone
methylation affect gene expression by regulating how much of a transcript
is made , with these signals altered in cancer pathogenesis and reprogrammed
after some environmental exposures ( figure 1a ) .
however , the complexity of epigenetic dna marks has become more
complicated by the emergence of 5-hydroxylmethylcytosine , 5-formylcytosine ,
and 5-carboxycytosine modifications of dna and by the diversity of histone modifications , including acetylation
and phosphorylation . in this context of
transcriptional control by dna and histone modification , we introduce
the concept of a system of dozens of rna modifications , including
rna methylation , that reprogram in response to environmental changes
and control gene expression at the level of translation .
( a ) dna and histone methylation
marks regulate transcript levels to affect gene expression .
dynamic
rna methylation signals have recently been demonstrated to regulate
how well a transcript is translated to affect gene expression .
( b )
structures of 5-methylcytosine ( mc ) in dna ( x = h ) and
rna ( x = oh ) and the trna wobble modification mcmu .
( c )
structures of four of > 120 modified ribonucleosides in prokaryotes
and eukaryotes . the rna modification database can be accessed to view
more modification structures ( http://mods.rna.albany.edu/ ) .
the nucleosome image was prepared
by david s. goodsell and the rcsb pdb ( http://www.rcsb.org/pdb/101/motm.do?momid=7 ) and is used with permission .
similar to dnmt s and pmt s , rna methytransferases
( rnmt s ) have also been implicated in the pathophysiology of
human disease , but a clear understanding of
their mechanism of action in human cells has been elusive until recently .
for example , the modification of mrna with n - methyladenosine ( ma ) by the methyltransferase
mettl3 and removal of ma by the fto demethylase are emerging
as determinants of mrna stability and translational efficiency . here , we focus on trna , with recent work with
the model eukaryote saccharomyces cerevisiae demonstrating that rna - methylation enzymes specific to trna ( trna )
are vital to cell viability after exposure to agents that generate
reactive oxygen species ( ros ) and dna damage . specifically , defects
in the mc trna methyltransferase 4 ( trm4 , also called
ncl1 ) and mcmu trna methyltransferase 9 ( trm9 ) lead to
damage - induced growth and cell cycle phenotypes .
this highlights an important connection between trna and stress
response : modified ribonucleosides in trna .
trna molecules are initially
transcribed with u , a , c , and g bases , but the nucleobases and ribose
sugars in a trna molecule are subject to chemical modification by
a large system of enzymes .
there are > 100 chemical trna modifications
throughout phylogeny , with 2530 in all trna species
in a cell , including s. cerevisiae and
humans.s .
cerevisiae have an average of 11 modifications spread throughout the 70
bases in trna , whereas the average mammalian trna contains 13 modifications
( figure 1b , c ) . in general
, trna methyltransferases transfer the methyl group from s - adenosyl methionine ( sam ) to the 2-oh of the ribose
sugar , to the heterocyclic or exocyclic nitrogen atoms of the nucleobase ,
or to nucleophilic sites in modification intermediates .
there are
18 known trm enzymes in s. cerevisiae , with genomic analyses predicting 36 human trms . in many cases , and for both trm4 and trm9 , there are two
or more human homologues for each yeast trna methyltransferases , which
suggests diversification or specialization of trm activity to new
modifications in humans , modification of different trnas , or functions
other than trna modification .
regardless of enzyme identify or regulation ,
modified ribonucleosides can promote trna structural stability and
folding , translational fidelity , frame - shift prevention , and translation
efficiency , with evidence for roles in trna quality control , cellular
stress responses , and cell growth .
the modifications are located in conserved positions throughout
the four loops and termini of the trna molecule , with a large diversity
of chemical structures occurring in the anticodon loop and the wobble
position of the anticodon in particular .
this is not surprising in light of the role of the wobble ribonucleotide
in determining anticodon codon interactions between trna and
mrna during translational elongation .
the diversity of both
the chemical structures and locations of
trna modifications suggests a role for modified ribonucleosides in
controlling translation as part of a regulatory system .
codon
interactions , which gives them great regulatory potential as a result
of their ability to control the rate of translational elongation .
some wobble base modifications are only found on a subset of trnas
for specific amino acids that interact with select codons , which supports
the idea that regulation by trna modification can be very specific
to a particular codon .
if trna modifications are part of a such a
regulatory system , then they must satisfy at least two criteria : ( 1 )
that they increase or decrease in response to specific changes in
cell state and ( 2 ) that changes in the levels of the modifications
alter the codon - reading properties of the associated trna and , in
some cases , the selection of redundant codons .
these behaviors transcend
the chemical structure or location of individual ribonucleoside modifications
and require a coordinated system with rules beyond the primary genetic
code .
only recently have analytical and informatic technologies provided
a means to define these transcendent properties of trna modifications .
in the field of systems biology , the development
of convergent
technologies to quantify the thousands of individual components of
the transcriptome , proteome , and metabolome has led to the discovery
of regulatory networks and interactions that would not have been observed
in single - molecule or -pathway analyses .
the power of liquid chromatography - coupled
mass spectrometry ( lc ms ) for identifying and quantifying modified
ribonucleosides has recently been recognized by several groups . to explore the regulatory potential for trna modifications in cellular
stress responses
ms platform
to measure changes in the relative quantities of all trna modifications
in an organism ( figure 2 ) .
the platform involves artifact - free rna isolation , purification
of individual noncoding rna species by hplc , hydrolysis and hplc resolution of individual ribonucleosides , and
mass spectrometric identification and quantification of stress - induced
changes in all modified ribonucleosides by quadrupole time - of - flight
and tandem quadrupole mass spectrometry , respectively .
the data set
is subjected to bioinformatic and statistical analysis to define patterns
of change and then to define pathways linked to altered ribonucleosides .
as shown in figures 2 and 3 , our lc
ms method is capable of quantifying 23 of
the 25 known ribonucleoside modifications in cytoplasmic trna
in s. cerevisiae , with limited detection of two modifications ( ar(p ) and ncmum ) in positive ion mode . of critical importance here is the sensitivity
of detection , because low - level modifications are those most likely
to be found at wobble positions of specific trna species , as opposed
to more abundant modifications found in many trna species .
ms
analysis reveals that modifications occur roughly at high ( d , mc , mg , m2 g , ma , and y ) , medium ( acc , ta , mu , cm , gm , mg , mg , ia , and am ) , and low levels ( ncmum , mcmsu , ncmu , mcmu , um , mi , i , and mc ) , which generally
reflects their presence in all or specific trna species as well as
their presence at multiple or single positions in trna . these features
make the sensitivity , precision , and accuracy of the analytical method
particularly important in first - pass studies of stress - induced changes
in trna modifications .
for example , trm4 catalyzes the formation of
mc in over 34 species of trna , yet trna is the only trna
with mc at the anticodon wobble position 34 in addition
to position 48 between the variable and tc loops .
the observation of stress - dependent changes
in mc levels may thus depend on the ability to detect
small changes in the total quantity of mc in the trna
population .
similarly , trm9 catalyzes two modifications , mcmsu and mcmu , at wobble positions in five
trna species ( trna , trna , trna , trna , and
trna ) such that changes could
occur in any or all of the trna species . ultimately , individual trna
species must be isolated and analyzed for changes in trna - modification
levels in an analysis of the regulatory properties of modified ribonucleosides ,
and this is accomplished by quantitative localization of modifications
using combinations of rnase cleavage and oligonucleotide - based affinity
purification along with lc
ms analysis . platform for trna modification analytics and computational analysis
of codon usage , which allows definition of the link between trna modifications
and selective translation of motts in the cell stress response .
trna
modifications are identified and quantified by hplc - coupled mass spectrometry
techniques to identify highly up- and downregulated ribonucleosides .
critical modifications are then mapped to wobble positions in specific
trna species , the anticodon of which specifies a codon that is subjected
to genomic analysis .
biased use of this codon in gene families specifies
potential motts . in parallel ,
proteomic analysis of stress - altered
protein levels reveals codon - biased translation of motts .
ultimately ,
the stress - altered trna reprogramming is linked to selective translation
of codon - biased mrnas , with patterns of gene expression unique to
each stress .
hierarchical clustering
of exposure- and genetic - induced changes
in rna modification levels .
rna modification data from wild - type cells
exposed to different agents and mock - treated cells were identified
and quantitated by mass spectrometry .
log - based fold - change values
were determined relative to untreated , wild - type cells , and these
data where hierarchically clustered .
as noted earlier , trna modifications fulfill
a regulatory function
in cell response if they increase or decrease following specific stresses .
application of the trna - modification analysis platform to yeast exposed
to different chemical stresses revealed that this is indeed the case
in yeast .
cells were exposed to three equitoxic doses
of hydrogen peroxide ( h2o2 ) , methyl methanesulfonate
( mms ) , sodium arsenite ( naaso2 ) , and sodium hypochlorite
( naocl ) , and changes in the levels of 23 modified ribonucleosides
in total trna were quantified by lc
application
of multivariate statistical analysis to the fold - change data ( e.g. ,
hierarchical clustering ) revealed that specific groups of trna modifications
were uniquely up- or downregulated for each agent and for individual
doses of each agent , as shown in figure 3 .
the highly reproducible changes in trna - modification spectra demonstrate
that the exposures promote reprogramming of the system of rna modifications , which has been referred to as the ribonucleome .
more recently , we quantitatively compared changes in the
complete set of trna modifications in yeast exposed to four different
oxidizing agents and five different alkylating agents .
multivariate
statistical analysis revealed class - specific features that distinguished
oxidizing agents from alkylating agents , with 14 modifications forming
the basis for a data - driven model that predicted the chemical class
of toxicant exposure with greater than 80% sensitivity and specificity
( chan et al . , submitted ) .
furthermore , signature changes in trna modification
spectra distinguished sn1 from sn2 alkylating
agents ( chan et al . ,
these systems - level changes in trna
modifications are analogous to the stress - specific patterns of changes
in mrna levels in transcriptional profiling or to proteomic and metabolomic
signatures of cell state , which suggests a role for rna modifications
in regulating gene expression after ros stress and dna damage .
recent
evidence for codon biases across the genome has provided a basis for
linking trna - modification reprogramming to selective translation of
codon - biased transcripts as the regulatory mechanism in question .
the second criterion for a translational regulatory role for trna
modifications involves their ability to recognize information in mrnas
that is separate from the simple amino acid - specifying codon .
more
specifically , understanding how changes in the levels of specific
trna modifications can affect gene expression requires insight into
codon anticodon interactions and the patterns of usage of so - called
redundant codons in the genome .
the general dogma in thinking about
codons is that the rate by which they are translated by the ribosome
is tightly linked to the concentration of the decoding trna , with
reported correlations between genome - wide codon bias , trna copy number ,
and gene - expression levels in many model organisms .
simply put , current models correlate the most highly translated
transcripts with possession of the most frequently used codons , which
are specified and decoded by corresponding trna species whose genes
have the highest number of copies in the genome and the highest number
of trna copies in the pool .
although
this model holds true for the expression of many genes , it suffers
from being a static model : it can not account for stress - induced regulation
of translation .
there are also many exceptions to the model , as revealed
by transcripts showing clustering of low - frequency codons , distinct
mrna secondary structure , and internal ribosome binding sites .
so there is a need to better understand the information content
of biased codon usage in genes and to identify a mechanistic link
between codon - usage patterns and specific trna modifications . developing
rules , though , is a challenge because of the fact that there are 20
standard amino acids , 64 codons , 76 unique trna species , 300 trna
genes , and > 23 trna modifications in s. cerevisiae as a model organism
. the complexity can be simplified by concentrating
on wobble base modifications in specific trnas and then analyzing
patterns of codon usage in specific transcripts , but an appreciation
of the degeneracy of the genetic code is required to move the model
to the next level .
there are 64 standard codons possible from the
four canonical bases found in mrna , with several ( i.e. , synonymous )
codons translated into the same amino acid .
both amino acids have the maximum number
of six degenerate codons in whole- ( four ) and split- ( two ) codon boxes
( figure 4a ) . in split - codon boxes ,
the wobble
base trna modifications mc and mcmu can influence
codon anticodon affinity by dramatically enhancing interactions
with one codon ( i.e. , ttg for leu and aga for arg ) .
transcripts in
which one codon from the split box is over - represented therefore have
great potential for their translational efficiency to be tied to the
levels of specific wobble base modifications .
we can extend this idea
further by proposing that specific transcripts may have over - representation
of many specific codons from split boxes for multiple amino acids ,
which could lead to translational regulation by multiple trna modifications .
we term the mrnas from these codon - biased genes as modification tunable
transcripts ( motts ) , and we have identified 425 of them in s. cerevisiae using a recently developed codon - analysis
algorithm ( figure 4 ) .
these 425 genes contain
statistically significant deviations in the usage of 29 codons when
compared to all transcripts in the s. cerevisiae genome .
several recent studies have validated the
use of the term mott by establishing a link between the dynamics of
stress - induced tuning of trna wobble modifications and the selective
translation of codon - biased mrnas that represent critical stress response
genes .
( a ) fold - difference
in the average codon frequency of the 425 identified yeast motts when
compared to genome averages is noted .
those codons overused in the
motts are colored yellow ( p < 10 ) , whereas those under - represented are colored purple ( p < 10 ) , with the sum frequency that all degenerate
codon are used for each amino acid = 1 .
( b ) run of 25 codons used
at the c - terminal end of the yef3 transcript is highly
enriched ( n = 24 ) for those over - represented in motts .
notably , there are two ( aag)4 codon runs and one ( aag)5 codon run represented in this sequence , with 21 codons specific
to trm9 ( aag , gaa , and aga ) and trm4 ( uug ) .
the evidence for stress - induced reprogramming
of trna modifications
and for a link between specific trna modifications and biased codon
used in motts suggests that the system of trna modifications composes
a mechanism for regulating cellular responses to environmental changes
at the level of translation ( figure 5 ) .
several
recent studies confirm this model , with regulation of codon - biased
transcripts demonstrated for mott s linked to mc and mcmu modifications .
specifically , trm4-catalyzed
modification of c to mc at the wobble position of anticodon
of trna has been shown to
increase in response h2o2 exposure , with this
increase driving increased translation of mrnas ( motts ) derived from
the 38 genes in yeast in which 90% or more of the leucines are encoded
by uug . among these uug - enriched motts
is the fact that rpl22a is one of two alternative
proteins for rpl22 , with the gene for its paralogue , rpl22b , lacking
significant enrichment of uug .
h2o2 exposure
did not increase the rate of translation of rpl22b , and only loss
of the gene for rpl22a rendered the cells sensitive to killing by
oxidative stress .
these results provide
a direct link between stress - induced increases in a specific wobble
trna modification and selective translation of codon - biased mrnas
for critical stress response genes .
rna modifications and biased codon use
form a system that controls
cellular stress response at the level of translation . emerging evidence
supports a model in which stress induces a reprogramming of trna modifications
that leads to selective translation of codon - biased mrnas ( i.e. , motts )
representing critical stress response proteins .
similar to trm4 , trm9-specific mcmu and mcmsu modifications have been demonstrated to be
regulated
in response to dna damage and are required to increase the translation
of codon - biased motts . in s. cerevisiae , the mrnas for yeast translation elongation factor 3 ( yef3 ) and ribonucleotide reductases 1 ( rnr1 ) and 3 ( rnr3 ) are over - represented with aga , gaa , and aag codons ,
and the basal translation of these proteins is dramatically decreased
in trm9 cells lacking mcmu and
mcmsu .
proteins corresponding to transcripts with average
codon usage ( i.e. , non - motts ) were found to occur at similar levels
in wild - type and trm9 cells .
notably , mrna levels for rpl22a , yef3 , rnr1 , and rnr3 were
identical in wild - type , trm4 ( for rpl22a ) ,
and trm9 ( for yef3 , rnr1 , and rnr3 ) cells , which further demonstrates
that trna - modification - dependent gene expression operates at the level
of translational regulation . computational analysis of the motts rpl22a , yef3 , rnr1 , and rnr3 indicates
that each of these transcripts is significantly over - represented in
specific groups of codons , with protein analysis technologies clearly
demonstrating that their protein levels can be regulated by specific
trna modifications .
the computed codon signature is an indicator of
a mott and is limited at this point from the perspective of developing
regulatory rules , as it is a trend for the whole gene .
notably , it
does not provide any location - specific information detailing where
these over - represented codons fall in the gene , and there will most
likely be transcript regions that are more severely biased in certain
codons and thus represent key regulatory motifs . as an example , the
3/c - terminal region of yef3 is shown in figure 4b . in a span of 25 codons ,
it contains 21 codons
whose regulation can be linked to trm4 ( ugu ) and trm9 ( aga , gaa , and
aag ) , thus representing a local transcript region that is predicted
to be highly dependent on specific trna modifications for translation .
furthermore , development and testing of computational rules governing
the precise mechanism of translational regulation of motts is needed
to identify the most significant regulatory regions where modifications
regulate the translation of specific transcripts .
motts are
a new regulatory term , but they have been described before .
there is an important precedent in the form of selenocysteine - containing
proteins that also illustrates the concept of motts , rna modifications ,
and stress response proteins .
selenocysteine ( sec ) is commonly called
the 21st amino acid , and it is found in cellular detoxification and
stress response proteins that include members of the glutathione peroxidase
( gpx ) and thioredoxin reductase ( trxr ) families .
importantly , these sec - containing proteins can detoxify h2o2 ( gpx1 and gpx3 ) and lipid peroxidation products ( gpx6 )
and contribute to the regulation of ribonucleotide reductase enzymes
( trxr1 and trxr2 ) .
sec lacks its own dedicated codon , and it is incorporated
into proteins using a novel mechanism termed stop - codon recoding ,
which requires a number of key signals .
the uga codon ( i.e. , the stop
codon ) is normally used to signal the end of translation , but in stop - codon
recoding , an internal uga codon is used in conjunction with other
factors to signal for the insertion of sec .
importantly , the wobble
base trna modification mcmu , which is catalyzed by human
and mouse alkbh8 , is required for efficient stop - codon recoding .
as a side note , it has been proposed that the oxidative demethylation
activity of alkbh8 could serve as an off switch by reversing wobble
methylation modifications , akin to dna and histone demethylation ,
but no such activity has been demonstrated .
transcripts that encode sec - containing proteins can be thought of
as extreme motts because they are over - represented with stop codons
and need mcmu for efficient translation .
transcripts for
sec - containing proteins also fit into the theme of motts because they
correspond to important stress response proteins , with specific gpx
and trxr activities well established as cellular detoxification enzymes . in conclusion
, the connection between rna modifications , biased
codon use , and translational regulation of stress response protein
highlights a complicated set of mechanisms to regulate gene expression .
this parallels other methylation - based signals , as understanding regulation
of transcription by mc and histone methylation is also
complicated , required new tools at their outset , and can have species - specific
rules .
it is important to note that the dna , protein , and rna modification
activities and modifications specified by dnmt s , pmt s ,
and rnmt s share a common theme of regulating gene expression
by enzyme - catalyzed methylation , with all of them reprogrammable by
environmental conditions and during some disease pathologies .
there
are significant challenges for better defining the roles and mechanisms
of motts and rna modifications because codon usage and modification
patterns change when studying different organisms and there are numerous ,
varied , and specialized instrumentation required for the study of
rna modifications .
the study of motts and rna modifications is therefore
required in multiple model systems and settings to define further
and to develop general and then species - specific rules .
we note that
one possible path to make the study of rna modifications simpler and
more accessible is to follow the example set by researchers studying
dna and histone methylation signals : to develop antibodies for each
modification . | cells
respond to environmental stressors and xenobiotic exposures
using regulatory networks to control gene expression , and there is
an emerging appreciation for the role of numerous postsynthetic chemical
modifications of dna , rna , and proteins in controlling transcription
and translation of the stress response . in this perspective
, we present
a model for a new network that regulates the cellular response to
xenobiotic exposures and other stresses in which stress - induced reprogramming
of a system of dozens of post - transcriptional modifications on trna
( trna ) promotes selective translation of codon - biased mrnas for critical
response proteins . as a product of novel genomic and bioanalytical
technologies ,
this model has strong parallels with the regulatory
networks of dna methylation in epigenetics and the variety of protein
secondary modifications comprising signaling pathways and the histone
code .
when present at the trna wobble position , the modified ribonucleosides
enhance the translation of mrnas in which the cognate codons of the
trnas are highly over - represented and that represent critical stress
response proteins .
a parallel system may also downregulate the translation
of families of proteins .
notably , dysregulation of the trna methyltransferase
enzymes in humans has also been implicated in cancer etiology , with
demonstrated oncogenic and tumor - suppressive effects . |
improvement of science and technology considered as the essential requirement for social development all over the world . in each country , the knowledge production outputs reflect the interesting topics of researches .
knowledge creation process provide the fundamental integral part of knowledge life cycle for creating , sharing , translating , and applying the generated knowledge for health promotion .
research - based knowledge could be followed by the trends of scientometrics indicators that quantify documented research outputs in the scholarly communication framework .
these indicators for different fields of sciences show the scientific outputs , as a part of research activity of each country or even scientific unit .
the special aim of this indicator system is characterizing and comparing the contribution of research units in the world 's scientific research activities and clarifying points that need to be strengthened .
comparisons between different countries results of these metrics determine their prospects and performance of research fields . for each country ; the quantity and quality of indexed articles in international databases is an important indicator of its contribution to specific field knowledge production . in recent decades , noncommunicable diseases ( ncds )
have been intended as the most significant health problem with increasing trend , thus knowledge production in this field has been important .
one of the leading cause of death due to ncds is diabetes . in the middle east and north africa ( mena ) ; disabilityadjusted life year ( daly ) rank 's percentage change of diabetes was 87% from 1990 to 2010 . in this region ,
diabetes was the fifth leading cause of disability while in other regions this rank varies from six to ten . ascending trend of diabetes prevalence in these countries leads to more premature heart disease and stroke that should be more emphasized through preventive health policies .
authorship as indicator of research activity and citation as indicator of reception of research are deterministic indicators that measured through international databases . in addition , collaboration measurement is an indicator of research systems structure at the macro level .
as we know , the scientometric study was not performed in the diabetes field in mena .
this paper aims to assess the trends of published articles and citations in the diabetes field as the proxy of these countries research activities during the past 23-year period and review the collaborative researches between regions country .
in addition , we specifically focused on iran and assessed its contribution in diabetes researches by details of scientometrics outputs of iranian articles .
the present study is scientometric analysis of diabetes research articles among middle east countries from 1990 to 2012 .
we searched three international indexing databases with the most coverage in health and biomedicine discipline .
these are isi web of science ( isi / wos ) , pubmed / medline , and scopus . in this study
, we considered 23-year , and all databases searched for published articles related to diabetes by middle east countries .
the middle east countries consist of bahrain , cyprus , egypt , iraq , iran , israel , jordan , kuwait , lebanon , oman , palestine , qatar , saudi arabia , syria , turkey , united arab emirates ( uae ) , and yemen ( in alphabetical order ) .
as , these databases covered same journals in some cases , all results stored in endnote x5 software , thomson reuters , usa and duplicate cases was excluded .
based on the controlled vocabulary of medical subject heading , and emtree , the term of diabetes used as a major quarry for indexing and extracting in mentioned database . through that , we retrieved all records indexed under our predefined search strategy .
limitations were the period of papers ( 19902012 ) , the countries that papers were submitted from ( based on authors affiliation or address ) .
search strategy for each database present in box 1 box 1 : used search strategies in three international databases box 1 : search strategies
search strategy in isi web of sciencetime span = 1990 - 2012 .
databases = sci - expanded , ssci , cpci - s , cpci - ssh.topic= ( diabetes ) and address= ( country 's name)search strategy in pubmed / medline((diabetes [ title / abstract ] or diabetes [ transliterated title ] ) or ( diabetes mellitus[mesh terms ] ) ) and country 's name [ affiliation ] and ( 1990/01/01[pdat ] :
2012/12/31[pdat])search strategy in scopus(title - abs - key ( diabetes ) and affil ( country 's name ) ) and pubyear > 1989 and pubyear < 2013
search strategy in isi web of science time span = 1990 - 2012 .
databases = sci - expanded , ssci , cpci - s , cpci - ssh .
topic= ( diabetes ) and address= ( country 's name ) search strategy in pubmed / medline ( ( diabetes [ title / abstract ] or diabetes [ transliterated title ] ) or ( diabetes mellitus[mesh terms ] ) ) and country 's name [ affiliation ] and ( 1990/01/01[pdat ] :
2012/12/31[pdat ] ) search strategy in scopus ( title - abs - key ( diabetes ) and affil ( country 's name ) ) and pubyear > 1989 and pubyear < 2013 in this way , systematic search was performed in each databases and the number of published articles related to diabetes by each country in mena determined . among these databases , pubmed provides the opportunity of article type analysis .
thus , the article type was investigated among those papers that were retrieved from pubmed .
citation analysis was performed based on scopus database . as citation to published articles by middle east countries regarding diabetes
also , collaboration of middle east countries in diabetes knowledge production was studied based on scopus results . for estimating of p trends , the number of articles and citations imported to stata software , version 11 , statacorp lp , usa and by pearson chi - square , p trends were estimated .
mannkendall ( mk ) test is a statistical test commonly used for trend analysis in time series data .
the null hypothesis of this test assumes that there is no trend that is , the data is independent and randomly ordered .
we designed figures using r software 3.1 ( the free software foundation 's gnu general public license ) .
in the later part of this article , we specifically focus on diabetes research in iran .
the information about research budget and academic members of iran retrieved from ministry of health website .
iranian published articles were categorized according to subject area , document type , affiliation of the authors and source title . to demonstrate the indexing pattern of diabetes articles terms in scopus databases , we used vosviewer mapping software of centre for science and technology studies , leiden university , the netherlands .
we considered scopus database because the number of diabetes articles of iran in this database was more than the others . using the vosviewer and thresholds of minimally 10 fractionally counted papers for each term ,
each term meet the threshold , a relevance score will be calculated and based on the score , the most relevant terms were selected . for mapping scopus diabetes articles terms , maps created based on title and abstract field .
through a systematic search for diabetes publication by middle east countries , we found 14,022 article in isi / wos , 11366 articles in pubmed / medline , and 20,707 articles in scopus .
the details presented in appendix a. after deletion of duplications , the number of articles reaches 31,143 . the time trend of these articles has been showed in figure 1 .
the trend of publications on diabetes in middle east countries from 1990 to 2012 pearson chi - square test showed that time trends of articles in various countries are different significantly ( p = 0.000 ) .
the mk test provides remarkable insight about annual publication on diabetes for turkey , iran and israel .
the mk correlation ( rho ) indicates that there is an increasing publication trend for these countries ( p = 0.968 , 0.936 , 0.919 respectively ) .
the correlation , however , is not strong for countries like syria , cyprus and yemen ( p = 0.587 , 0.691 , 0.701 respectively ) compared to other countries . as shown in figure 1 , more than 80%
of articles had been published after 2000 . according to figure 1 , turkey and iran from 2001 to 2003
have the steepest slope . from 1990 to 2012 , about 70% of knowledge production on diabetes was done by three counterparts ; turkey ( 30.2% ) , israel ( 27.4% ) , and iran ( 12.7% ) .
however , since 2011 , iran took the second place with 22.1% knowledge production in this region .
as shown in table 1 , syria ( 1.5% ) , yemen ( 2.5% ) and palestine ( 2.8% ) had the lowest contribution in diabetes knowledge production among middle east 's countries .
distribution of publications on diabetes in middle east countries from 1990 to 2012 based on pubmed database , 11366 articles on diabetes in middle east during 1990 - 2012 were divided to original article ( 72.44% ; except clinical trials ) , review article ( 9.28% ) , clinical trial ( 8.71% ) , case report ( 8.03% ) , systematic review ( 1.17% ) , meta - analysis ( 0.26% ) , letter ( 0.07% ) , and editorial ( 0.04% ) . along the time , type of article was varied within each country .
the pattern of diabetes article types in 2000 and 2012 based on pubmed database based on scopus results from 1990 to 2012 , the most collaborative country with middle east countries in diabetes articles is usa . as shown in table 2 , in some region 's country such as lebanon , joint articles with usa
international collaboration of middle east countries in knowledge production on diabetes based on scopus database citations based on scopus database among israel , turkey and iran have ascending trend . from 1990 to 2012
, syria has the less citation ( 349 ) and israel has the most citation ( 191,740 ) .
this index in other middle east countries are as follow ; lebanon ( 23 ) , uae ( 12 ) , cyprus ( 11 ) , kuwait and syria ( 10 ) , jordan , oman , palestine and turkey ( 9 ) , egypt , qatar , yemen saudi arabia ( 8) , and iran , iraq , and bahrain ( 6 ) .
as citation needs time , we present trend of citation to diabetes publications of middle east countries from 2000 .
the trend of citation to diabetes articles in middle east countries from 1990 to 2012 iran has published 1697 article related to diabetes indexed in isi web of science , 1470 article in pubmed and 3013 article in scopus from 1990 to 2012 . by excluding duplicates ,
3958 article was remained . according to isi web of science database , clinical biochemistry ( 3.6% ) , diabetes research and clinical practice ( 3.4% ) , journal of research in medical sciences ( 3.0% ) , transplantations proceedings ( 1.9% ) , pakistan journal of medical sciences ( 1.8% ) , archives of iranian medicine ( 1.6% ) , and iranian journal of public health ( 1.5% )
were most frequent sources title in iran . about 85.3% of iranian articles about diabetes published in foreign journals . based on scopus database ,
the five top rank journals for diabetes publications are iranian journal of diabetes and lipid disorders ( 4.8% ) , iranian journal of endocrinology and metabolism ( 3.5% ) , journal of research in medical sciences ( 2.3% ) , journal of medicinal plants ( 2.1% ) , and acta medica iranica ( 1.9% ) respectively . from medical sciences universities in iran , tehran university of medical sciences had most publication on diabetes ( 38.1% in isi / wos , 30.6% in scopus ) and then shahid beheshti university of medical sciences ( 14.2% in isi / wos , 11.5% in scopus ) and isfahan university of medical sciences ( 10.2% in isi / wos , 11% in scopus ) have second and third position . assess the proportion of diabetes publications to research budget and academic members among mentioned universities in iran show academic members , as professional resource is compatible with diabetes publication and trend of research budget is different from diabetes publications trend .
figure 4 shows the trend of three top rank universities of iran in diabetes publication adjusted to research budget and academic member in recent years .
the trend of three top rank universities of iran in diabetes publication adjusted to research budget ( a ) and academic member ( b ) in mapping the scopus terms frequency network , from the 9922 terms , 412 terms meet the threshold . then
map of frequent terms in iranian diabetes articles indexed in scopus figure 5 demonstrates the network of frequently used terms in diabetes - related iranian articles .
some common terms between the borders of the major region refer to common research fields .
based on the map the terms of woman and effect had most frequency .
it is noticeable that less frequent terms , for example coronary artery , have inconspicuous view .
through a systematic search for diabetes publication by middle east countries , we found 14,022 article in isi / wos , 11366 articles in pubmed / medline , and 20,707 articles in scopus .
the details presented in appendix a. after deletion of duplications , the number of articles reaches 31,143 . the time trend of these articles has been showed in figure 1 .
the trend of publications on diabetes in middle east countries from 1990 to 2012 pearson chi - square test showed that time trends of articles in various countries are different significantly ( p = 0.000 ) .
the mk test provides remarkable insight about annual publication on diabetes for turkey , iran and israel .
the mk correlation ( rho ) indicates that there is an increasing publication trend for these countries ( p = 0.968 , 0.936 , 0.919 respectively ) .
the correlation , however , is not strong for countries like syria , cyprus and yemen ( p = 0.587 , 0.691 , 0.701 respectively ) compared to other countries . as shown in figure 1 , more than 80%
of articles had been published after 2000 . according to figure 1 , turkey and iran from 2001 to 2003
have the steepest slope . from 1990 to 2012 , about 70% of knowledge production on diabetes was done by three counterparts ; turkey ( 30.2% ) , israel ( 27.4% ) , and iran ( 12.7% ) .
however , since 2011 , iran took the second place with 22.1% knowledge production in this region .
as shown in table 1 , syria ( 1.5% ) , yemen ( 2.5% ) and palestine ( 2.8% ) had the lowest contribution in diabetes knowledge production among middle east 's countries .
based on pubmed database , 11366 articles on diabetes in middle east during 1990 - 2012 were divided to original article ( 72.44% ; except clinical trials ) , review article ( 9.28% ) , clinical trial ( 8.71% ) , case report ( 8.03% ) , systematic review ( 1.17% ) , meta - analysis ( 0.26% ) , letter ( 0.07% ) , and editorial ( 0.04% ) . along the time , type of article was varied within each country .
figure 2 shows this difference between 2000 and 2012 . the pattern of diabetes article types in 2000 and 2012 based on pubmed database
based on scopus results from 1990 to 2012 , the most collaborative country with middle east countries in diabetes articles is usa . as shown in table 2 , in some region 's country such as lebanon , joint articles with usa
the most collaborative country is saudi arabia . also , uae collaborates with four countries in this regard .
citations based on scopus database among israel , turkey and iran have ascending trend . from 1990 to 2012
, syria has the less citation ( 349 ) and israel has the most citation ( 191,740 ) .
this index in other middle east countries are as follow ; lebanon ( 23 ) , uae ( 12 ) , cyprus ( 11 ) , kuwait and syria ( 10 ) , jordan , oman , palestine and turkey ( 9 ) , egypt , qatar , yemen saudi arabia ( 8) , and iran , iraq , and bahrain ( 6 ) .
as citation needs time , we present trend of citation to diabetes publications of middle east countries from 2000 .
iran has published 1697 article related to diabetes indexed in isi web of science , 1470 article in pubmed and 3013 article in scopus from 1990 to 2012 . by excluding duplicates ,
3958 article was remained . according to isi web of science database , clinical biochemistry ( 3.6% ) , diabetes research and clinical practice ( 3.4% ) , journal of research in medical sciences ( 3.0% ) , transplantations proceedings ( 1.9% ) , pakistan journal of medical sciences ( 1.8% ) , archives of iranian medicine ( 1.6% ) , and iranian journal of public health ( 1.5% )
were most frequent sources title in iran . about 85.3% of iranian articles about diabetes published in foreign journals . based on scopus database ,
the five top rank journals for diabetes publications are iranian journal of diabetes and lipid disorders ( 4.8% ) , iranian journal of endocrinology and metabolism ( 3.5% ) , journal of research in medical sciences ( 2.3% ) , journal of medicinal plants ( 2.1% ) , and acta medica iranica ( 1.9% ) respectively . from medical sciences universities in iran ,
tehran university of medical sciences had most publication on diabetes ( 38.1% in isi / wos , 30.6% in scopus ) and then shahid beheshti university of medical sciences ( 14.2% in isi / wos , 11.5% in scopus ) and isfahan university of medical sciences ( 10.2% in isi / wos , 11% in scopus ) have second and third position . assess the proportion of diabetes publications to research budget and academic members among mentioned universities in iran show academic members , as professional resource is compatible with diabetes publication and trend of research budget is different from diabetes publications trend .
figure 4 shows the trend of three top rank universities of iran in diabetes publication adjusted to research budget and academic member in recent years .
the trend of three top rank universities of iran in diabetes publication adjusted to research budget ( a ) and academic member ( b ) in mapping the scopus terms frequency network , from the 9922 terms , 412 terms meet the threshold . then
map of frequent terms in iranian diabetes articles indexed in scopus figure 5 demonstrates the network of frequently used terms in diabetes - related iranian articles .
. some common terms between the borders of the major region refer to common research fields .
based on the map the terms of woman and effect had most frequency .
it is noticeable that less frequent terms , for example coronary artery , have inconspicuous view .
many countries attend to diabetes researches because , priority based researches could produce scientific evidence for addressing health threatening problems such as diabetes . in middle east , turkey , israel , and iran have considerable ascending trends of diabetes publication . in turkey and iran , size of the population and consequently the size of professional researchers , and developing specialized research centers are effective factors in this progress .
israel has lower population size but strongly collaborate with international researchers so that about half of their publications on diabetes are in collaborating with usa . as shown in table 3 , despite appropriate human and financial resources , some countries have been shown less interest in diabetes publications . in some cases such as bahrain and
similarly , this mismatch between the burden of disease and knowledge production is seem in india and china .
health relies on valid , reliable , and accessible knowledge thus attention of policy makers on proper resource allocation could provide the appropriate context for research , knowledge production and consequently healthy situation .
comparison resources , diabetes daly , and publications on diabetes among middle east countries in this regards , maintain ascending trend of publication needs more attention . a bibliometric study on diabetes literature from 1995 to 2005 revealed doubling time of diabetes publication increase every year . as shown in table 1 ,
pattern of article types in 2000 and 2012 shows attention to clinical trials that have been increased during the time of the study . also review articles , which have the essential role in citation increase ,
joint project , as the essential factor of this more effective scientific collaboration should be more attention and effort .
similarities such as regional conditions , lifestyle , and common health problems could intend middle east countries to emerging a regional network to benefit from shared potential and investments .
citation could be one of the representative indexes of paper 's quality . in some country such as iran , trend of citation
diabetes care , diabetes , diabetic medicine , diabetologia , and diabetes research and clinical practice are five top ranks of journals in diabetes research .
only 3.4% of isi indexed articles on diabetes in iran published in diabetes research and clinical practice journal .
in iran , presence endocrinology and metabolism diseases research centers in tehran , shahid beheshti and isfahan medical science universities and the role of their researchers are some of the main potentials in iran .
assess the frequent terms used in iranian articles about diabetes revealed that the terms of effect ,
is used in biomedical articles , but frequently used of woman may be signed of researcher attention to more prevalence of diabetes in iranian women .
it is noticeable ; heart disease despite its importance in this area was not considered .
first , we focused on one subject of endocrinology domain to clarify the situation of knowledge production about diabetes as growing and threatening factor .
third , we assess collaboration between middle east countries in this area based on the international database and fourth , we explain the situation of iran as a knowledge productive country in the region in details .
of course , in our study , we faced with some limitation such as ; presence multidisciplinary subject category and overlap between databases categories that considered in scientometric analysis .
this study provides a scientometrics analysis about diabetes research in middle east region and collaboration analysis among this region 's countries . ascending trend of diabetes research outputs in this region
is appreciated but encouraging to strategic planning for maintaining this trend , and more collaboration between researchers is needed to regional health promotion .
np , shdj , ak , ff and bl contributed in the conception of the work , conducting the study , revising the draft , approval of the final version of the manuscript , and agreed for all aspects of the work . | background : diabetes burden is a serious warning for urgent action plan across the world .
knowledge production in this context could provide evidences for more efficient interventions . aimed to that
, we quantify the trend of diabetes research outputs of middle east countries focusing on the scientific publication numbers , citations , and international collaboration.materials and methods : this scientometrics study was performed based on the systematic analysis through three international databases ; isi , pubmed , and scopus from 1990 to 2012 .
international collaboration of middle east countries and citations was analyzed based on scopus .
diabetes publications in iran specifically were assessed , and frequent used terms were mapped by vosviewer software.results:over 23-year period , the number of diabetes publications and related citations in middle east countries had increasing trend .
the number of articles on diabetes in isi , pubmed , and scopus were respectively ; 13,994 , 11,336 , and 20,707 .
turkey , israel , iran , saudi arabia , and egypt have devoted the five top competition positions . in addition
, israel , turkey , and iran were leading countries in citation analysis .
the most collaborative country with middle east countries was usa and within the region , the most collaborative country was saudi arabia .
iran in all databases stands on third position and produced 12.7% of diabetes publications within region .
regarding diabetes researches , the frequent used terms in iranian articles were effect , woman , and metabolic syndrome.conclusion:ascending trend of diabetes research outputs in middle east countries is appreciated but encouraging to strategic planning for maintaining this trend , and more collaboration between researchers is needed to regional health promotion . |
human papillomavirus ( hpv ) is the most common sexually transmitted virus , infecting 75% of the sexually active canadian population [ 1 , 2 ] .
it can cause cancer of the cervix , vulva , oropharynx , penis , and anus , as well as genital warts [ 35 ] . until recently
, there were no medical interventions against hpv infections except monitoring the virus 's progress through the papanicolaou test ( or pap test ) and treating the ailments that developed [ 6 , 7 ] .
one ( cervarix ) protects against types 16 and 18 while the other ( gardasil ) also protects against hpv types 6 and 11 .
these vaccines offer protection for at least eight years . in 2007 , the canadian federal government provided the provinces and territories with $ 300 million to spend over 3 years for hpv immunization programs . by 2009 , every province and territory in canada had an hpv vaccination program in place .
the creation and implementation of the programs are the responsibility of the individual provinces and territories .
this results in eight distinct strategies throughout canada , which can be seen in table 1 .
these strategies vary in the number of doses of the quadrivalent vaccine given ( only two doses in british columbia and quebec , with three doses administered elsewhere ) , the grade of the girls who are given the vaccine ( ranging from 4 to 10 ) , and the coverage rate ( 4986% within the first two years of the program ) .
although the implementation of these programs differs between provinces , they all share a common goal of reducing the impact of hpv on the canadian population .
this is measured through the morbidity and mortality rates of hpv . since the vaccine 's introduction
, there have been a number of mathematical models assessing its impact on several populations . in terms of the cost - effectiveness of the vaccine ,
brown and white used an optimal control model for vaccinating adolescent females and males in the united kingdom .
brisson et al . used a cohort model to estimate how many girls need to be vaccinated to prevent cervical cancer and genital warts in canada . in terms of the disease impact on a population , barnabas et al . developed a transmission model to measure the impact of vaccinating against hpv-16 in finland for both women and men .
although it does consider the effects caused by hpv later in life , this model neglects to consider herd immunity .
there are current clinical trials in british columbia looking at the effect of taking two or three doses of the quadrivalent vaccine
. this will provide valuable information about the levels of protection provided by two or three doses .
llamazares and smith ? developed an epidemiological model that includes a widespread childhood vaccination program with voluntary adult vaccination .
the model is used as a preliminary investigation to determine whether provincial healthcare programs in canada should fund adult hpv vaccination .
the authors determined a threshold of eradication for the targeted types of hpv and critical efficacy and immunogenicity levels such that eradication is not possible at any coverage rate . here
we develop a mathematical model to explore the current vaccination strategies across canada as well as potential alternative strategies .
these strategies are defined by the number of doses given , the grade of the girls the vaccine is given to , and the coverage rate achieved .
our model will provide similar insight into the effectiveness of two and three doses of the quadrivalent vaccine .
this model also provides insight into what the programs should look like in order to achieve a desired outcome . this model provides a unique perspective in its evaluation of the provincial programs based on the grade of vaccination , number of doses given , and the necessary coverage rate .
we address the following research questions : ( 1 ) does the grade at which the girls are vaccinated significantly affect the outcome of the program ?
( 2 ) what coverage rate must the provinces reach in order to reduce the impact of hpv on the canadian population ?
( 3 ) what are the implications of vaccinating with two versus three doses ? this paper is organized as follows .
section 4 gives the expressions for critical thresholds of efficacy and probability of protection .
section 5 explores how the parameters were estimated , the results on the infection when varying the grade , dosage and coverage rate , and the sensitivity analysis of the model .
our model is composed of 19 equations , 13 that describe the childhood vaccination strategy and six that describe the disease propagation through adults .
the female children are broken up into seven grades ( 4 through 10 ) . within each appropriate grade class , there are unvaccinated ( c
iu , where 4 i 10 ) and vaccinated female children ( c
iv , where 4 i 10 ) . in the adult portion of the model , women are broken up into unvaccinated uninfected susceptible women ( a
u ) , vaccinated uninfected susceptible women ( a
v ) , unvaccinated and infected women ( i
u ) , or vaccinated infected women ( i
v ) .
girls in grade 4 ( approximately 9 years old ) are described as ( 2)dc4dt=w1+cc4.for girls in grade i , where 5 i 10 , we have ( 3)dc(i+1)udt=1piciu1+cci+1u , dc(i+1)vdt=piciu+civ1+cc(i+1)v.uninfected adult women are described as ( 4)daudt=(1u)c10u+uiuf(wpw)au waunaau+av , davdt=(1v)c10v+viv+f(wpw)au (1)wavnaavav.infected adult women are described as ( 5)diudt=uc10u+waunuiuaiu+iv , divdt=vc10v+(1)wavnvivaiviv.uninfected men are described as ( 6)dmdt=mmiummivm+mnam.infected men are described as ( 7)dndt=mium+mivmmnan .
a girl enters the model unvaccinated in grade 4 at a constant rate
w. at some grade between 4 and 10 , a proportion of the girls become vaccinated at rate p
i ( where p
i is the proportion of girls given the vaccine in grade i and represents the vaccine efficacy for girls ) . when the girls enter grade 11 , a proportion of unvaccinated girls ( 1
u ) grow up to become uninfected unvaccinated women ( a
u ) , while the remaining unvaccinated girls (
u ) are categorized as infected unvaccinated women ( i
u ) to account for early childhood sexual activity .
a proportion of vaccinated girls ( 1
v ) grow up to become uninfected vaccinated women ( a
v ) , while the rest of the vaccinated girls (
v ) are categorized as infected vaccinated women ( i
v ) to account for a proportion of girls who have contracted at least one of the targeted types before receiving the vaccine . once in the adult stage ( grade 11 to around the age of 26 )
unvaccinated adult women ( a
u ) can become vaccinated at rate f(
w
p
w ) , where
w is the efficacy of the vaccine for adult women and p
w is the proportion of women who get the vaccine .
the vaccine wanes at rate . unvaccinated adult women become infected ( i
u ) at rate
w when coming into contact with an infected man ( n ) .
vaccinated adult women ( a
v ) can also become infected ( i
v ) at rate ( 1 )
w , where represents the ability of the vaccine to protect against all targeted types .
men enter the model as susceptible ( m ) through a constant rate
m and stay in the model for approximately 10 years . unvaccinated susceptible men ( m )
can become infected after sexual activity with an infected woman ( i
u or i
v ) with transmission rate
m. infection clears at rate
u for unvaccinated women , at rate
v for vaccinated women , and
m for men .
each compartment also includes a natural leaving rate ,
c or
a , depending on child or adult status .
parameter descriptions , ranges , and sample values can be found in table 2 . for this model , it is assumed that hpv is only heterosexually transmitted .
female children are in grades 4 through 10 , which are the years childhood vaccination can take place .
it is assumed that vaccination only occurs in one year , that male vaccination is negligible , and that the proportion of female children vaccinated during other years is negligible .
the rate at which the hpv infection is cleared is independent of previous infection status .
the transmission from women to men is higher than the transmission from men to women .
both women and men are active in the adult model for approximately 10 years because the vaccine is not recommended for women over the age of 26 .
we keep men in the model for the same length of time as women , on the grounds that , while there may be an age difference between young women and older men , such men are aging out with their sexual cohorts .
that is , they may choose new partners within this cohort ( e.g. , friends of existing partners ) , but we assume they do not revert to an even younger cohort after such a cohort ages out .
( however , we have explored this assumption in more detail elsewhere and shown that relaxing it has a negligible effect on the epidemic , so it is only included here for analytical convenience . )
the assumptions made about the vaccine are that the vaccine does not wane in children , that it may not protect 100% ( this is based on the probability of protection and the efficacy ) , and that the vaccine does not protect someone who is already infected with the virus [ 32 , 33 ] . the difference between a two- and three - dose schedule is the efficacy of the vaccine .
lastly , we do not consider disease - induced death since it does not play a role in removing sexually active individuals from the pool that we are considering ( complications due to hpv , such as cervical cancer , are generally seen much later in life ) [ 7 , 34 , 35 ] .
the disease - free equilibrium is(8)c4,c5u,c5v,c6u,c6v,c7u,c7v,c8u,c8v,c9u,c9v , c10u,c10v,au,av,iu,iv,m,n,where ( 9)c4=w1+c , and we define c4u=c4 , c4v=0.then , for 5 i 10 , we have ( 10)ciu=1pi1c(i1)u1+c , civ=p(i1)c(i1)u+c(i1)v1+c , au=1uc10ufw pw+a , av=fw pwau+1vc10va , iu=0 , iv=0,m=ma , n=0 .
the jacobian matrix for this model evaluated at the disease - free equilibrium is j
dfe = [ j
dfe
| j
dfe
| j
dfe
] , where(11)the characteristic polynomial of the jacobian is ( 12)detji=1c13adethdetl , where ( 13)h=fafa,l=uawau0va(1)wavmmmmma.
solving det(h ) = 0 , we get(14)2+f+2a++af+a+=0,which has only eigenvalues with negative real part . solving det(l ) = 0 , we get ( 15)3+2++=0,where ( noting that m= ) ( 16)=3a+u+v+m+,=3a2+uv++um+v+m + 2au+v+m+1wm av wmau,=a3+a2u+v+m+ + auv++um+v+m + u(v+)m 1wm ava+u+ wmaua+v+.
in order to determine the stability of the disease - free equilibrium in the linearized system , we use the routh hurwitz stability criterion . for our cubic characteristic polynomial ( 15 ) , we have four routh hurwitz conditions that must all be satisfied in order for the disease - free equilibrium to be locally stable:(1 )
> 0;(2 )
>
0;(3 )
> 0;(4 )
> .
the first condition is satisfied , since all of the parameters are positive .
the second condition is our threshold . if < 0 , then we are guaranteed to have at least one positive real root of the characteristic polynomial .
if = 0 , then we have a nonhyperbolic fixed point and must use stability theory to determine the stability of the disease - free equilibrium .
we determine stability for the case when = 0 = > 0 ( where is a small positive perturbation ) .
we will show that > 0 and > 0 in order to satisfy the routh hurwitz criterion .
we find ( 18)(1)wmav = 1a+u+a3+a2+a = 1a+u+wmaua+v+ = 1a+u++ uv+ma3 .
substituting this into , we have ( 19)=3a2 + 2a+ 1a+u+ a3+a2+ awmaua+v+ + u(v+)mwmauwmau.
to require > 0 , we need ( 20)(a+u+)3a2 + 2a+ + wmaua+v++ >
a3+a2+ a+wmaua+u+ + uv+m,3a3 + 2a2+ a+3a2u+2au+ u+3a2 + 2a+ +(a+v+)wmau+ > a3+a2+ a+(a+u+)wmau + uv+m,2a3+a2(+3u+3)+2au+ uu(v+)m + 2a+ +(vu)wmau+>0.note that ( 21)uuv+m = uuv++um+v+m uvmum>0.it follows that > 0 if ( 22)vu , that is , if the recovery rate from infection is faster for vaccinated individuals ( or at least not worse ) , which we expect to be the case . when = 0 , the routh
hurwitz conditions are satisfied , all roots have a negative real part , and our system is stable at the disease - free equilibrium . since > 0 and > 0 when = 0 , then = 0 is our threshold of stability .
we used the product =
w
m to numerically examine the routh
and . figure 2(a ) shows the scenario before vaccination while figure 2(b ) shows the scenario with 100% children and 100% adult vaccination .
the region of stability does not significantly change depending on the efficacies (
c and
w ) or the probability of protection ( ) . in figure 2 ,
is represented by the solid red line while is represented by the dashed blue line . when > 0 , > 0 .
the disease - free equilibrium is stable only if > 0 and > . the value of
indicated on the inset graph of figure 2 shows the threshold of transmission between a stable and unstable disease - free equilibrium .
if <
, then > 0 and > . this shows that we can use as a ( local ) threshold of stability
the basic reproductive number r
0 is a threshold that represents the average number of secondary infections caused by one infectious person in a completely susceptible population . from the analysis in section 3.1 , the r
0 threshold is ( 23)r0=wm((1)(a+u+)av+(a+v+)au)[a3+a2+ a+u(v+)],where au and av are the values at the disease - free equilibrium . knowing this threshold is significant , since it will tell us which parameters are involved in shifting the disease from persistence to eradication .
this threshold will be used to determine which parameters have the greatest influence on r
0 , which in turn will give us insight into the most effective intervention strategies ( section 5.5 ) .
there are critical vaccine efficacies for both children (
) and women (
w
) and there is a critical probability of protection ( ) that serves as a threshold where , even with 100% vaccination , the targeted types of hpv can not be eradicated in the population .
these values are determined by first setting r
0 = 1 and rearranging for the desired parameter . in order to simplify the expression for r
0 , we rewrite the equilibrium values for our population in a general form . note here that k
represents the grade of childhood vaccination . for 4 k 10 ,
we find ( 24)cku=w1+ck3 for kk,cku=w1pk11+ck3 for k > k,ckv=0 for kk,ckv=w1+ck3 for k > k,au=wfpww+a1c7,av=wffpww+a1c7 .
to determine the expression for
, we look at only childhood vaccination ( i.e. , no adult vaccination ) , which we get by setting p
w = 0 . since childhood vaccination only occurs during one year , then(25)pk=1,when k = k,0,when kk.
rearranging the expression r
0 = 1 and solving for
, we find(26)=1+c7a2wm(a+u+)(1u)wa
noticing that 0 ( i.e. , is not constant ) , we construct the following inequality from the definition of . we know that(27)wamc,w=wcwa=g , where w is the size of the female population when there are only women and g is the size of the female population when there are only girls .
<
( w ) , then 100% coverage rate of girls will not be sufficient for eradication , since r
0 > 1 .
if >
( g ) , then with a sufficient coverage rate
it is possible for female - only vaccination to eradicate the targeted types of hpv .
if is between the bounds , we can not accurately predict the outcome of the vaccine intervention .
the critical adult efficacy occurs when there is no childhood vaccination but there is 100% adult vaccination . using a similar approach to the critical childhood efficacy , we find(29)w=(1+)wmw(a+u+)(1u)a ad = ld(ca)wmw(a+u+)=lllll1u1ca1,where d = ( 1 +
c)
a(
a
+
a
+
a
+
u(
v + ) ) .
w
is bounded as in ( 30)www()wg.the interpretation of
w
is similar to that of
.
if the transmission in canada is below
= 1.02 , then the disease would be eradicated without any intervention .
since hpv is endemic throughout the world and has not become eradicated , we conclude that the actual transmission parameters must be greater than 1.02 ( assuming all other parameters used in the simulation are correct ) .
figure 2(b ) shows the region of stability for 100% childhood and 100% adult vaccination . as long as the transmission is lower than
= 2.95
however , if the transmission is above 2.95 , then even 100% vaccination can not eradicate hpv .
these values allow us to estimate a range of likely transmission values , which are currently unknown .
the probability of protection , , was estimated by including the transmission of the targeted hpv types and an estimate for the proportion of girls who become sexually active before grade 11 ( 30% ) . assuming all of the sexually active girls came into contact with someone able to transmit one of the targeted hpv types , the infection for this grade class would spread at a rate of 30% , giving a range of 0 0.3 .
the recovery rate was found by determining the average infectious period for both high- and low - risk types of hpv ( 1/ ) and solving for [ 26 , 27 ] .
we looked at the significance of vaccinating girls at different ages by constructing a box plot as seen in figure 3 . for a specific grade
, the coverage rate was varied between 0 and 100% . in figures 3(a ) and 3(b ) , the vaccine efficacy in adults was used to represent two ( 5096% ) and three ( 7188% ) doses , respectively .
latin hypercube sampling was used to sample parameter values from the given ranges in table 2 using a uniform distribution .
latin hypercube sampling is a statistical sampling method in which parameters are assigned a range of values , and a distribution of plausible collections of these parameter values are created .
note that only one grade ( or adult ) is considered during each run ; for example , a box plot representing grade 4 only takes into account vaccinating girls in grade 4 and does not include vaccinating any other grade or adults .
the thick red horizontal lines represent the median value of r
0 , while the box indicates the location of the upper and lower quartiles . comparing the median values in figures 3(a ) and 3(b )
, we notice that when girls are between grades 4 and 10 , the values are all around 1 , whereas the median r
0 is always above one for adult vaccination regardless of the number of doses given . the upper quartile values and lower quartile values also follow this trend , where the childhood vaccination values all have a similar range that is noticeably smaller than when adult vaccination occurs .
figure 4 shows the mean r
0 values , rather than the median . while there is more variation between grades , vaccinating with 3 doses
is always superior to vaccinating with 2 doses . due to transmission rates ( see figure 5 ) , neither form of vaccination will lead to eradication .
however , childhood vaccination is always superior to adult vaccination . comparing figures 3(a ) and 3(b )
, we notice that the r
0 values are generally smaller in the case of three doses , which is expected since the vaccine is less effective on a two - dose schedule .
this can also be validated by figure 5 , which shows a low correlation between or
w and r
0 .
figure 4 shows the difference in the mean r
0 when using a two - dose versus a three - dose regime . once again , having three doses is clearly superior to two , but the difference is not significant . since the true value of each parameter may fluctuate , we explore the sensitivity of r
0 to the parameter values in table 2 using latin hypercube sampling .
this in turn allows us to use partial rank correlation coefficients to rank the parameters in terms of their influence on r
0 , be it a positive or negative influence .
here r
0 is most sensitive to the transmission of hpv from men to women ,
w , and from women to men ,
m. the next influential parameter is the probability of protection , . notice that the coverage rates ( p
i , where 4 i 10 , and p
w ) and vaccine efficacy ( and
w ) do not significantly affect the value of r
0 . figure 6 shows the output of the latin hypercube sampling for the three most influential parameters as well as some other parameters of interest : the recovery rates and the waning rate . note the extremely weak correlation between the latter parameters and r
0 .
the coverage rate required to eliminate the targeted high - risk hpv types can be seen in figure 7 . above the curves
is the region where r
0 < 1 and the targeted types are theoretically eradicated , whereas below the curves is the region where the disease persists in the population .
the curves indicate when r
0 = 1 for either two doses ( lower red curves ) or three doses ( upper black curves ) for different values of the waning rate . as expected , the curve for two doses is higher than three , since the efficacy for two doses is slightly lower .
the targeted types can be eradicated if childhood vaccination is supplemented with significant adult vaccination .
however , as the waning rate of the vaccine increases , the window for eradication shrinks , requiring a significant amount of both childhood and adult vaccination .
a childhood - only vaccination program can theoretically eradicate the targeted hpv types with either two or three doses , as long as the appropriate coverage rate is achieved ( figure 7 ) .
for example , if 80% of children are vaccinated with a 3-dose vaccine that does not wane ( the lowest of the curves ) , then at least 40% of adults must be vaccinated to achieve eradication of targeted types .
these requirements become harsher as the number of doses decreases or as the vaccine wanes .
based on the results of the model , we conclude that , with sufficient childhood and adult vaccination , it is theoretically possible to eradicate targeted hpv types .
we also determine that the grade of vaccination before sexual debut does not significantly affect the prevalence of the targeted hpv types for the canadian population . comparing figures 3(a ) and 3(b ) , we observe that the median value of r
0 is always close to one when children are vaccinated , while the median value of r
0 is above one when only adult women are vaccinated , independent of the number of doses given .
this suggests that the targeted types are candidates for eradication if significant childhood vaccination can be achieved , whereas eradication is less likely from adult - only vaccination . looking only at childhood - only vaccination ,
there does not seem to be a large difference between the grades . from a mathematical standpoint
, it is easy to understand why the grade of vaccination does not matter in the model , since there is no impact in terms of the disease dynamics , whether girls are vaccinated in grade 4 or 10 .
the large jump is due to the change in vaccine effectiveness for those previously exposed to the considered hpv types , as well as the possibility of overvaccinating women , since they remain within the vaccination cohort for 10 years , whereas children are only vaccinated within a single year .
this is important to determine because it shows there are likely no underlying grade - dependent trends .
however , from an epidemiological standpoint , we know that the vaccine has little to no impact on those individuals who have a previous infection with a targeted type [ 32 , 33 ]
. therefore public vaccination strategies should choose the grade of vaccination based on epidemiological and vaccination program limitations .
assuming that the number of doses only changes the efficacy of the vaccine , the evidence from this model suggests that the number of doses does not significantly change the outcome of the vaccine strategy .
figure 7 shows that the possibility of eradication of targeted hpv types exists with either two or three doses , independent of the grade the childhood vaccine is given in .
this evidence follows the findings by several clinical studies that suggest that two doses of the bivalent vaccine may protect just as well as three doses [ 5 , 15 , 22 ] .
the higher the coverage rate , the higher the success of the vaccine program ( figure 7 ) . based on this evidence
, we suggest each province chooses the number of doses based on cost or ease of program implementation , as long as an appropriate coverage rate is matched to achieve the desired level of infection in the community .
the value of r
0 is mainly affected by the ability of the targeted hpv types to transmit and the ability of the vaccine to prevent the infection .
thus the most effective way to decrease r
0 is to decrease the transmission of the targeted types of hpv .
this could be done through increasing condom use , reducing the number of sexual partners , or increasing the heterogeneity of the sexual contact network of the population [ 16 , 38 ] .
since hpv has been infecting humans for millions of years and has not been eradicated , the real transmission rate in the canadian population must be greater than 1.02 . by studying the critical transmission value for 100% childhood and adult vaccination , we know that if the actual transmission rate is greater than 2.95 , then , even with 100% vaccine coverage , the targeted hpv types could not be eradicated . looking at the stability of the disease - free equilibrium based on the transmission is reasonable since the transmission parameters are the only two parameters that significantly affect the value of the basic reproductive number , as shown by the sensitivity analysis .
we chose the estimates for the efficacies ( and
w ) based on clinical evidence of both the bivalent and the quadrivalent vaccine .
obviously , a single vaccine program will only use either the bivalent or the quadrivalent vaccine . since
this model does not differentiate between different hpv types and the efficacies for either vaccine do not differ greatly , the numerical results hold for either vaccine . through sensitivity analysis , using larger ranges of the efficacy than observed for either vaccine , we see in figure 5 that it is not important to narrow down the range of efficacies since neither nor
w affects the value of r
0 significantly . in order to estimate the efficacy of a two - dose schedule of the quadrivalent vaccine , we used the efficacy of the bivalent vaccine from the costa rica clinical trial .
this model should be updated once better data is available for the efficacy and effectiveness of a two - dose quadrivalent hpv vaccine .
it is important to note that our model has several limitations . in terms of vaccination programs ,
the model does not include catch - up programs unless included in the initial population conditions .
each person receiving the vaccine with either two or three doses must complete the regimen within one year .
the possibility of male vaccination is not included , although it is now approved by health canada [ 1 , 5 , 11 ] .
the model does not differentiate in hpv 6 , 11 , 16 , or 18 or include any other hpv types .
we have also not ruled out the possibility of a backward bifurcation , meaning the disease may persist even for r
0 < 1 , complicating eradication efforts . from the base adult model , we assume that men who have sexual relations with women in the sexually active cohort ( i.e. , women who are eligible for adult vaccination ) do not continue to find new partners in this age group as time goes on . the sexually active cohorts of men and women are linked only for the time ( approximately 10 years ) that adult women are sexually active and eligible for vaccination .
note , however , that we have previously shown that this cohort assumption is negligible . since the provincial vaccination programs are already in place and each province has chosen their dosing schedule ( although it is possible they may change ) , we recommend that all of the provinces focus on increasing their coverage rate for both the public vaccination of girls and private vaccination of women to at least 80% .
this number is realistic for many provinces , since several have already reached this goal ( quebec , nova scotia , newfoundland , and pei ) . for the other provinces ,
increasing their coverage rate may be accomplished by creating public - education campaigns or may involve making protocols similar to vaccines necessary to attend school such as those for hepatitis b [ 17 , 39 ] .
our model suggests that the grade of vaccination does not affect the outcome of the vaccination program .
therefore we suggest provinces vaccinate girls as early as possible to avoid vaccine failure due to previous infection .
the main focus should be on obtaining large enough coverage rates for children and/or adults in order to achieve the desired outcome : using the vaccine to reduce the prevalence of hpv types 6 , 11 , 16 , and 18 in the population . | human papillomavirus ( hpv ) infection is the most common sexually transmitted infection , which is linked to several cancers and genital warts . depending on the canadian province ,
the quadrivalent vaccine is given to girls in grades 4 through 10 with either a two- or three - dose schedule .
we use a mathematical model to address the following research questions : ( 1 ) does the grade at which the girls are vaccinated significantly affect the outcome of the program ?
( 2 ) what coverage rate must the provinces reach in order to reduce the impact of hpv on the canadian population ?
( 3 ) what are the implications of vaccinating with two versus three doses ?
the model suggests the grade of vaccination and the number of doses do not make a significant difference to the outcome of the public vaccination program .
the most significant factor is the coverage rate of children and adults .
we recommend that provinces vaccinate as early as possible to avoid vaccine failure due to previous infection .
we also recommend that the main focus of the program should be on obtaining a large enough coverage rate for children and/or adults in order to achieve the desired outcome with either two or three doses of the vaccine . |
pulmonary gas exchange and respiratory mechanics are usually impaired during general anesthesia and muscle paralysis .
functional residual capacity ( frc ) decreases approximately 20 % during induction of anesthesia .
reduced frc can be restored somewhat through ventilation using positive end - expiratory pressure ( peep ) .
nevertheless , few reports in the literature describe studies of the influence of peep level upon frc in ventilated patients during administration of general anesthesia .
frc was reduced by induction of anesthesia , but no report in the literature has described a comparison of frc values with those of stepwise increase of peep during upper abdominal surgery .
we measured frc for mechanically ventilated general anesthesia with different peep levels during upper abdominal surgery and subsequently compared them with awake levels measured for the subject in the supine position .
we hypothesized that frc , compliance , and arterial oxygenation show positive effects of increased peep , and that 5 cmh2o peep or 10 cmh2o peep increased frc nearly to the awake level .
the wash - in or wash - out of a tracer gas in a multiple breath maneuver is apparently best applied at bedside .
promising techniques for nitrogen or oxygen wash - in / wash - out with reasonable accuracy and repeatability have been presented .
this technique , which estimates frc with good precision using a change of fio2 of only 0.1 , has been evaluated in volunteers and in patients with acute respiratory distress syndrome .
we studied nine patients with american society of anesthesiologists physical status classification system grade 1 or grade 2 who had been scheduled for elective upper abdominal surgery .
the arterial line , inserted under local anesthesia , was used for the study protocol .
monitoring included invasive arterial blood pressure , a continuous electrocardiogram , and peripheral oxygen saturation ( spo2 ) .
after induction of anesthesia and tracheal intubation , end - tidal carbon dioxide concentration ( etco2 ) , airway pressure , tidal volume , and respiratory rate were monitored and recorded .
before induction of anesthesia ( awake ) , a tight - seal facemask was used for spontaneous ventilation ( engstrm carestation ; ge healthcare , madison , wi , usa ) .
the facemask was applied with 2 cmh2o pressure support ventilation using an inspired oxygen fraction ( fio2 ) of 1.0 during frc measurement ( frc inview ) .
the frc was designated as the facemask frc value minus facemask dead space ( 100 ml ) .
arterial blood gas samples were collected from subjects who were in a supine position ( fig . 1 ) .
anesthesia was induced as target - controlled infusion of propofol 45 g / ml with a calculated target plasma , fentanyl 12 g / kg i.v . , and rocuronium bromide 0.6 mg / kg i.v.fig .
ps pressure support ventilation , frc functional residual capacity , abg arterial blood gases , tv tidal volume , peep positive end - expiratory pressure . predicted body weight : men , 49.9 + 0.91 [ height ( cm ) , 152.4 ] ; women , 45.4 + 0.91 [ height ( cm ) , 152.4 ] schematic protocol used for this study .
ps pressure support ventilation , frc functional residual capacity , abg arterial blood gases , tv tidal volume , peep positive end - expiratory pressure . predicted body weight : men , 49.9 + 0.91 [ height ( cm ) , 152.4 ] ; women , 45.4 + 0.91 [ height ( cm ) , 152.4 ] predicted body weight was calculated as 45.4 + 0.91 [ height ( cm ) 152.4 ] for women or 49.9 + 0.91 [ height ( cm ) 152.4 ] for men .
after tracheal intubation , anesthesia was maintained with a propofol target plasma drug concentration of 2.53 g / ml and remifentanil 0.20.4 g / kg / min with thoracic epidural analgesia .
lungs were ventilated in bilevel airway pressure with volume guarantee ( pressure - regulated volume control ) .
the tidal volume of 7 ml / kg was estimated according to the predicted body weight .
frc measurements and arterial blood gas samples were taken from 30 min after intubation ( pre - ope ) . from 1 h after intubation ,
frc measurements and arterial blood gas samples were obtained during upper abdominal surgery ( opep0 ) .
after 1 and 2 h peep 5 cmh2o , frc measurements and arterial blood gas samples were collected ( opep5.1 and opep5.2 ) .
frc measurements and arterial blood gas samples were also collected at 1 and 2 h after the stepwise increase of peep 10 cmh2o ( opep10.1 and opep10.2 ) .
the quasistatic compliance of the respiratory system was calculated as the tidal volume divided by the inspiratory plateau pressure minus the end - expiratory pressure .
comparison among groups was performed using two - way analysis of variance for repeated one - way measurements .
correlation between peep and frc or the pao2/fio2 ratio was analyzed using pearson s correlation .
no premedication was administered . before induction of anesthesia ( awake ) , a tight - seal facemask was used for spontaneous ventilation ( engstrm carestation ; ge healthcare , madison , wi , usa ) .
the facemask was applied with 2 cmh2o pressure support ventilation using an inspired oxygen fraction ( fio2 ) of 1.0 during frc measurement ( frc inview ) .
the frc was designated as the facemask frc value minus facemask dead space ( 100 ml ) .
arterial blood gas samples were collected from subjects who were in a supine position ( fig . 1 ) .
anesthesia was induced as target - controlled infusion of propofol 45 g / ml with a calculated target plasma , fentanyl 12 g / kg i.v . , and rocuronium bromide 0.6 mg / kg i.v.fig .
ps pressure support ventilation , frc functional residual capacity , abg arterial blood gases , tv tidal volume , peep positive end - expiratory pressure . predicted body weight : men , 49.9 + 0.91 [ height ( cm ) , 152.4 ] ; women , 45.4 + 0.91 [ height ( cm ) , 152.4 ] schematic protocol used for this study .
ps pressure support ventilation , frc functional residual capacity , abg arterial blood gases , tv tidal volume , peep positive end - expiratory pressure . predicted body weight : men , 49.9 + 0.91 [ height ( cm ) , 152.4 ] ; women , 45.4 + 0.91 [ height ( cm ) , 152.4 ] predicted body weight was calculated as 45.4 + 0.91 [ height ( cm ) 152.4 ] for women or 49.9 + 0.91 [ height ( cm ) 152.4 ] for men .
after tracheal intubation , anesthesia was maintained with a propofol target plasma drug concentration of 2.53 g / ml and remifentanil 0.20.4 g / kg / min with thoracic epidural analgesia .
lungs were ventilated in bilevel airway pressure with volume guarantee ( pressure - regulated volume control ) .
the tidal volume of 7 ml / kg was estimated according to the predicted body weight .
frc measurements and arterial blood gas samples were taken from 30 min after intubation ( pre - ope ) . from 1 h after intubation ,
frc measurements and arterial blood gas samples were obtained during upper abdominal surgery ( opep0 ) .
after 1 and 2 h peep 5 cmh2o , frc measurements and arterial blood gas samples were collected ( opep5.1 and opep5.2 ) .
frc measurements and arterial blood gas samples were also collected at 1 and 2 h after the stepwise increase of peep 10 cmh2o ( opep10.1 and opep10.2 ) .
the quasistatic compliance of the respiratory system was calculated as the tidal volume divided by the inspiratory plateau pressure minus the end - expiratory pressure .
comparison among groups was performed using two - way analysis of variance for repeated one - way measurements .
correlation between peep and frc or the pao2/fio2 ratio was analyzed using pearson s correlation .
the value of frc awake was significantly higher than that for pre - ope or opep0 ( p < 0.01 ) .
the frc ope10.1 and ope10.2 values were significantly higher than that for opep5.1 ( p < 0.01 ) .
the frc for opep10.2 was significantly higher than that for ope5.2 ( p < 0.01).fig .
2functional residual capacity for peep levels studied . * * p < 0.01 versus preoperative ( pre - op ) and opep0 , p < 0.01 versus opep5.1 , p < 0.01 versus opep5.2 pre - induction ( pre - ind ) .
p postoperative , frc measured pre - ind frc mask dead space ( 100 ml ) functional residual capacity for peep levels studied . * * p < 0.01 versus preoperative ( pre - op ) and opep0 , p < 0.01 versus opep5.1 , p < 0.01 versus opep5.2 pre - induction ( pre - ind ) .
p postoperative , frc measured pre - ind frc mask dead space ( 100 ml ) figure 3 presents oxygenation data .
the pao2/fio2 awake was significantly higher than that for pre - ope , opep0 , or ope5.1
( p < 0.01 ) . peep 5 cmh2o and peep 10 cmh2o were significantly higher than either pre - ope or opep0 ( p < 0.01 ) ( table 1).fig . 3pao2/fio2 for peep levels studied . * * p < 0.01 versus pre - op and opep0 , p < 0.01 versus opep5.1table 1patient characteristics ( n = 9)gender ( male : female)5:4age ( years)69 7weight ( kg)64 8height ( cm)158 6predicted body weight ( kg)53 6body mass index ( bmi)26 43pancreaticoduodectomy4pancreatectomy2hepatic lobectomy2reconstructive operation of biliary tract1values are mean sd pao2/fio2 for peep levels studied . * * p < 0.01 versus pre - op and opep0 , p < 0.01 versus opep5.1 patient characteristics ( n = 9 ) peep levels at opep0 , opep5.2 , and opep10.2 were correlated with frc ( r = 0.671 , p < 0.01 ) and pao2/fio2 ( r = 0.642 , p < 0.01 ) ( fig
4peep levels at opep0 , opep5.2 , and opep10.2 were correlated with frc ( r = 0.671 , p < 0.01 ) and pao2/fio2 ( r = 0.642 , p < 0.01 ) peep levels at opep0 , opep5.2 , and opep10.2 were correlated with frc ( r = 0.671 , p < 0.01 ) and pao2/fio2 ( r = 0.642 , p <
0.01 ) compliance was significantly higher for opep10.2 than for opep5.1 ( p < 0.01).table 2respiratory mechanics and hemodynamic datapre - opeopep0opep5.1opep5.2opep10.1opep10.2pip ( cmh2o)12 213 217 2 * 16 2 * 21 2 * 20 2*paco2 ( mmhg)44 544 643
642 643 643 6compliance(ml / cmh2o)36 1035 935
937 938 941 10map ( mmhg)98 12114 9 * 117 22 * 109 14110 19113 14heart rate ( bpm)63 1072 1675 16 * 76 15 * 82 17 * 81 17*data are expressed as mean sdquasistatic compliance = tidal volume/(inspiratory plateau pressure peep)ope operation , p postoperative day , pip peak inspiratory pressure , map maximum arterial pressure *
p < 0.01 versus pre - operative ( pre - ope ) , p < 0.01 versus opep0 , p < 0.01 versus opep5.1 , p < 0.01 versus opep5.2 respiratory mechanics and hemodynamic data data are expressed as mean sd quasistatic compliance = tidal volume/(inspiratory plateau pressure peep ) ope operation , p postoperative day , pip peak inspiratory pressure , map maximum arterial pressure * p < 0.01 versus pre - operative ( pre - ope ) , p < 0.01 versus opep0 , p < 0.01 versus opep5.1 , p < 0.01 versus opep5.2 maximum arterial pressure and heart rate were lowest at pre - ope . no significant difference was found in hemodynamics among different peep groups .
peep levels at opep0 , opep5.2 , and opep10.2 were correlated with frc ( r = 0.671 , p < 0.01 ) and pao2/fio2 ( r = 0.642 , p < 0.01 ) ( fig .
opep10.2 were correlated with frc ( r = 0.671 , p < 0.01 ) and pao2/fio2 ( r = 0.642 , p < 0.01 ) peep levels at opep0 , opep5.2 , and opep10.2 were correlated with frc ( r = 0.671 , p < 0.01 ) and pao2/fio2 ( r = 0.642 , p < 0.01 )
compliance was significantly higher for opep10.2 than for opep5.1 ( p < 0.01).table 2respiratory mechanics and hemodynamic datapre - opeopep0opep5.1opep5.2opep10.1opep10.2pip ( cmh2o)12 213 217 2 * 16 2 * 21 2 * 20 2*paco2 ( mmhg)44 544 643
642 643 643 6compliance(ml / cmh2o)36 1035 935 937 938 941 10map ( mmhg)98
12114 9 * 117 22 * 109 14110 19113 14heart rate ( bpm)63 1072 1675 16 * 76 15 * 82 17 * 81 17*data are expressed as mean sdquasistatic compliance = tidal volume/(inspiratory plateau pressure peep)ope operation , p postoperative day , pip peak inspiratory pressure , map maximum arterial pressure * p < 0.01 versus pre - operative ( pre - ope ) , p < 0.01 versus opep0 , p < 0.01 versus opep5.1 , p < 0.01 versus opep5.2 respiratory mechanics and hemodynamic data data are expressed as mean sd quasistatic compliance = tidal volume/(inspiratory plateau pressure peep ) ope operation , p postoperative day , pip peak inspiratory pressure , map maximum arterial pressure * p < 0.01 versus pre - operative ( pre - ope ) , p < 0.01 versus opep0 , p < 0.01 versus opep5.1 , p < 0.01 versus opep5.2
maximum arterial pressure and heart rate were lowest at pre - ope . no significant difference was found in hemodynamics among different peep groups .
the first main finding of our study was that peep showed positive effects on frc , compliance , and pao2/fio2 ratio , indicating that patients undergoing upper abdominal surgery might benefit from peep 10 cmh2o .
the frc examinations were performed using an engstrm carestation ( ge healthcare ) equipped with the frc inview monitoring feature .
the frc is determined using the change of lung nitrogen volume after a step change in the inspired oxygen fraction . in the engstrm carestation , when ventilated with fio2 1.0 , where the o2 consumption was calculated from the co2 production with a default rq of 0.85 , very high measurement precision was attained .
results show that frc was reduced by 37 % from the awake level after the induction of anesthesia .
as previously reported , frc is reduced by approximately 15 % during anesthesia induced with thiopentone and maintained with halothane at fio2 0.35 .
our protocol was fio2 1.0 at induction of anesthesia , which engendered absorption atelectasis because of the high concentration of oxygen .
the mechanisms underlying reduction of frc include muscle paralysis , decreased chest wall recoil , increased abdominal pressure , atelectasis formation , and gas entrapment behind a closed airway .
our results showed no significant change in frc between that after induction of anesthesia and that during operation at zero end - expiratory pressure ( zeep ) . during abdominal operation ,
however , during the surgery procedure , frc decreased and returned to post - induction levels at the end of the procedure . indeed ,
pulmonary gas exchange , which is impaired following induction of anesthesia , deteriorates during laparotomy but not during peripheral surgery .
the difference is probably explained by the effect of surgical influences ( packs , retractors , etc . ) on frc . at opep0 ,
results of this study demonstrated that peep 10 cmh2o reached nearly the same frc level as that of awake frc .
no data exist to elucidate waking frc for different peep levels , showing intraoperative frc changes .
presented measured mean frc data for postoperative patients at different peep levels ( 0 , 5 , 10 cmh2o ) .
their study revealed that peep increased frc at levels of peep 5 and 10 cmh2o ; peep decreased frc after reversion to peep of 0 cmh2o .
pelosi et al . reported that peep 10 cmh2o did not improve respiratory function in paralyzed or anesthetized postoperative patients .
their mean pao2/fio2 ratio was 436 mmhg , and their tidal volume was 812 ml / kg ideal body weight .
our mean pao2/fio2 ratio was 253 mmhg , and tidal volume was 7 ml / kg ideal body weight at peep 0 cmh2o intraoperatively .
lower tidal volume might engender atelectasis , especially if peep is low or not used at all .
described a randomized controlled nonblinded preventive trial comparing mechanical ventilation with tidal volume of 10 versus 6 ml / kg in critically ill patients without acute lung injury ( ali ) at the onset of mechanical ventilation .
mechanical presentation with 10 ml / kg is associated with sustained cytokine production in plasma .
those results suggest that mechanical ventilation with conventional tidal volumes contributed to the development of lung injury in patients without ali at the onset of mechanical ventilation .
administration of peep alone increased the normally aerated lung fraction , which combined with a reduction of poorly aerated lung tissue while atelectasis remained unchanged .
an earlier study of postoperative mechanically ventilated obese patients ( mean body mass index , 51 ) after abdominal surgery showed that peep 10 cmh2o increased pao2 , respiratory compliance , and frc .
reportedly , peep higher than 10 cmh2o is associated with marked derangement of hemodynamics .
reported that static compliance and the amount of atelectasis estimated using computed tomography ( ct ) did not change in parallel .
described that compliance indicates an optimal level of peep after recruitment in anesthetized patients , reporting that optimal peep was 10 cmh2o because , at that pressure level , the highest compliance value in conjunction with the lowest dead space fraction revealed a maximum amount of effectively expanded alveoli .
our results demonstrated that the quasistatic compliance was significantly higher for opep 10.2 than for opep 5.1 .
peep 10 cmh2o did not produce the pre - operation level of pao2/fio2 , probably because of a ventilation and circulation mismatch by mechanical ventilation or because recruitment maneuvers were not used .
the increased intrapleural pressure caused by peep might also increase the risk of barotrauma and cause changes to cardiovascular dynamics .
two trials reported postoperative barotrauma in both peep and zeep [ 14 , 15 ] .
their comparison revealed rr of 0.3 for the peep group , which was not statistically significant .
it remains unclear whether the increases in frc and p / f ratio are attributable to the increase in peep level , or to a time - dependent effect of peep , or both . during a time study of 5 h operation , pao2 showed constant values at much lower tidal volumes of 6 ml / kg and peep 10 cmh2o ( 21 patients were enrolled ; 13 cases were upper abdominal surgeries with abdominal opening ) .
following coronary artery bypass grafting , significant reduction of p ( a a ) o2 during positive pressure ventilation at 10 cmh2o peep was compared with 0 cmh2o peep and 5 cmh2o peep during 6 h . these data show that pulmonary oxygenation might maintain constant values at equal peep levels . as one limitation of our study , peep was applied in a stepwise increasing fashion .
we have no data for p / f ratio and frc on time - dependent effect of peep .
however , the same peep level was maintained for 2 h. two hours after changing peep , the frc data were slightly higher than at 1 h after changing peep , but the values were not significantly different .
the peep levels at opep0 , opep5.2 , and opep10.2 were correlated with frc and with pao2/fio2 .
the peep levels might be applied in a random sequence to mitigate this potential bias in the results .
data reported herein suggest that if low tidal volume ventilation is used during upper abdominal surgery , then peep 10 cmh2o is necessary to maintain lung function . | purposeseveral reports in the literature have described the effects of positive end - expiratory pressure ( peep ) level upon functional residual capacity ( frc ) in ventilated patients during general anesthesia .
this study compares frc in mechanically low tidal volume ventilation with different peep levels during upper abdominal surgery.methodsbefore induction of anesthesia ( awake ) for nine patients with upper abdominal surgery , a tight - seal facemask was applied with 2 cmh2o pressure support ventilation and 100 % o2 during frc measurements conducted on patients in a supine position .
after tracheal intubation , lungs were ventilated with bilevel airway pressure with a volume guarantee ( 7 ml / kg predicted body weight ) and with an inspired oxygen fraction ( fio2 ) of 0.4 .
peep levels of 0 , 5 , and 10 cmh2o were used .
each level of 5 and 10 cmh2o peep was maintained for 2 h. frc was measured at each peep level.resultsfrc awake was significantly higher than that at peep 0 cmh2o ( p < 0.01 ) .
frc at peep 0 cmh2o was significantly lower than that at 10 cmh2o ( p < 0.01 ) .
pao2/fio2 awake was significantly higher than that for peep 0 cmh2o ( p < 0.01 ) .
pao2/fio2 at peep 0 cmh2o was significantly lower than that for peep 5 cmh2o or peep 10 cmh2o ( p < 0.01 ) .
furthermore , peep 0 cmh2o , peep 5 cmh2o after 2 h , and peep 10 cmh2o after 2 h were correlated with frc ( r = 0.671 , p < 0.01 ) and pao2/fio2 ( r = 0.642 , p < 0.01).conclusionsresults suggest that peep at 10 cmh2o is necessary to maintain lung function if low tidal volume ventilation is used during upper abdominal surgery . |
within a cohort of insulin - treated type 2 diabetic patients , those with incident cancer during a longitudinal follow - up were identified as case subjects and compared for treatments received with matched control subjects from the same cohort . of a consecutive series of 1,533 diabetic outpatients , referred to the diabetes clinics of the university of florence , italy , and starting insulin therapy between 1 january 1998 and 31 december 2007 ,
those free of previous malignancies ( n = 1,340 ) were enrolled in the study , provided that they met the following criteria :
they were living in the city of florence at the date of enrolment;they had a clinical diagnosis of type 2 diabetes;they had no reported insulin therapy during the previous 5 years ; andthey had no reported previous malignancy or hospital admission for malignancy after 1 january 1998 and before initiation of insulin therapy .
the participating hospital - based clinics provided outpatient care for diabetes ; a large majority of patients initiating insulin therapy are referred to specialist clinics , which also provided subsequent follow - ups , at least yearly .
all patients were treated by specialists who did not receive incentives for the use of less expensive drugs .
they were living in the city of florence at the date of enrolment ; they had a clinical diagnosis of type 2 diabetes ; they had no reported insulin therapy during the previous 5 years ; and they had no reported previous malignancy or hospital admission for malignancy after 1 january 1998 and before initiation of insulin therapy . demographic and clinical information was obtained from clinical records , including a detailed medical history , self - reported smoking habits and alcohol intake , prescribed medications , a1c ( measured every 34 months with high - perfomance liquid chromatography [ menarini diagnostics , florence , italy ] , upper normal limit 5.9% ) , lipid profile , and serum creatinine , and assessed as a part of routine follow - up .
weight , height , and blood pressure were routinely measured at each visit following world health organization recommendations ( 8) . the nearest - available measure within a 3-month interval from enrollment
comorbidity was assessed with the charlson comorbidity score ( ccs ) , which includes diabetes and its complications , malignancies , arthritis / arthrosis , hiv - infections , chronic cardiovascular disease , skin ulcers , renal insufficiency , liver diseases , and obstructive pulmonary disease ( 9 ) .
patients were observed until 31 december 2008 or to the date in which they moved to another city .
cancer incident case subjects were identified as first hospital admission or death with icd-9 codes 140209 .
information on hospital admission was obtained through the regional hospital discharge system , an administrative database containing icd codes of current diagnoses , which collects all reimbursed admissions ( in public or private hospitals ) , with no possibility of loss at follow - up .
case finding was therefore performed on 1,340 patients ( 746 women and 594 men ) , aged ( means sd ) 63.1 14.9 years , with a median duration of diabetes of 7.5 years ( interquartile range 0.519.2 ) , a1c 8.7 1.9% , and bmi 27.9 5.4 kg / m .
a nested case - control study dataset was generated from the cohort study dataset by sampling control subjects from the risk sets .
for each case subject , the control subjects ( up to five ) were chosen randomly from those members of the cohort who are at risk for the same follow - up time of the case subject .
five - year age classes , sex , and bmi classes ( < 18.5 , 18.524.9 , 2529.9 , and 30 kg / m ) were considered as additional categorical variables for matching control subjects to case subjects .
exposure to hypoglycemic drugs was assessed from enrollment to incident cancer in case subjects and during the corresponding time from initiation of insulin therapy in matched control subjects . to compare case and control subjects for differences in exposure to each insulin type ,
the mean daily dose ( mdd ) was calculated , dividing total insulin units prescribed for duration of observation ( days ) and for weight ( kg ) , thus accounting for both time of treatment and daily dose during treatment .
mdd was calculated separately for basal and prandial formulations of each insulin . for premixed insulin ,
the fraction of the dose attributed to basal or prandial therapy was calculated and used in subsequent analyses . for oral hypoglycemic drugs ,
the length of exposure during the 10 years preceding the end of observation was considered .
drug exposure was obtained from clinical records containing a self - reported history of hypoglycemic treatment before the first contact and all drug prescriptions afterward .
information retrieval for each case and control subject was performed independently by two investigators , and conflicts were resolved by a third investigator ( e.m . ) .
if the last available visit had occurred > 3 months before the event ( or the matching index date ) , a telephone contact was attempted to collect further information on subsequent drug use ; if unsuccessful , the patient was assumed to have continued the last reported therapy .
three cases with a last visit occurring > 2 years from the end of follow - up were excluded from analysis .
the main predefined analysis was the comparison of case and control subject for proportion of patients exposed and mdd ( units / kg / day ) for each insulin .
unpaired student t test and mann - whitney test were used to compare continuous variables whenever appropriate .
the statistical analysis was done by conditional logistic regression , which takes into account the matching structure .
this was especially important because for some case subjects , less than five control subjects were available in the risk set , so that an unequal number of control subjects were sampled by case subject . in all models , total insulin mdd , ccs , and metformin exposure
alternative models in which mdd for basal and prandial insulins were entered separately were applied .
furthermore , an analysis with ccs , metformin exposure , total insulin mdd , glargine exposure time , and mean glargine dose during exposure was performed .
all analyses were carried out with spss 15.0 statistical package and stata 9.0 , and a p < 0.05 was considered statistically significant .
exposure to hypoglycemic drugs was assessed from enrollment to incident cancer in case subjects and during the corresponding time from initiation of insulin therapy in matched control subjects . to compare case and control subjects for differences in exposure to each insulin type , the mean daily dose ( mdd )
was calculated , dividing total insulin units prescribed for duration of observation ( days ) and for weight ( kg ) , thus accounting for both time of treatment and daily dose during treatment .
mdd was calculated separately for basal and prandial formulations of each insulin . for premixed insulin ,
the fraction of the dose attributed to basal or prandial therapy was calculated and used in subsequent analyses . for oral hypoglycemic drugs ,
the length of exposure during the 10 years preceding the end of observation was considered .
drug exposure was obtained from clinical records containing a self - reported history of hypoglycemic treatment before the first contact and all drug prescriptions afterward .
information retrieval for each case and control subject was performed independently by two investigators , and conflicts were resolved by a third investigator ( e.m . ) .
if the last available visit had occurred > 3 months before the event ( or the matching index date ) , a telephone contact was attempted to collect further information on subsequent drug use ; if unsuccessful , the patient was assumed to have continued the last reported therapy .
three cases with a last visit occurring > 2 years from the end of follow - up were excluded from analysis .
the main predefined analysis was the comparison of case and control subject for proportion of patients exposed and mdd ( units / kg / day ) for each insulin .
unpaired student t test and mann - whitney test were used to compare continuous variables whenever appropriate .
the statistical analysis was done by conditional logistic regression , which takes into account the matching structure .
this was especially important because for some case subjects , less than five control subjects were available in the risk set , so that an unequal number of control subjects were sampled by case subject . in all models , total insulin mdd , ccs , and metformin exposure
alternative models in which mdd for basal and prandial insulins were entered separately were applied .
furthermore , an analysis with ccs , metformin exposure , total insulin mdd , glargine exposure time , and mean glargine dose during exposure was performed .
all analyses were carried out with spss 15.0 statistical package and stata 9.0 , and a p < 0.05 was considered statistically significant .
during a median follow - up of 75.9 months ( interquartile range 27.4133.7 ) , 112 patients had new diagnoses of cancer ( incidence 1.9/100 person - years ) ; in 1 case subject , 2 different cancers were observed , for a total number of 113 registered cancers ( 11 colorectal , 18 other gastrointestinal / hepatic , 14 pancreatic , 16 lung , 10 leukemia / lymphoma , 7 breast , 5 female urogenital , 9 male urinary , 4 prostate , and 20 other cancers ) .
the demographic and clinical characteristics of case and control subjects are summarized in table 1 .
case subjects had a greater comorbidity and a nonsignificant trend toward less exposure to metformin in the previous 10 years , whereas the mean daily metformin dose ( median [ interquartile range ] ) was not significantly different between case and control subjects ( 16.0 mg / kg / day [ 11.821.4 ] vs. 18.2 mg / kg / day [ 10.020.9 ] , p = 0.63 ) .
after adjusting for ccs and mean daily total insulin dose , time of exposure to metformin ( hazard ratio [ hr ] 0.993 [ 95% ci 0.9860.999 ] , p = 0.046 , for each month ) and metformin doses ( 0.943 [ 0.9150.971 ] , p < 0.01 , for each mg / kg / day ) were both inversely associated with incident cancer .
the proportion of patients who discontinued insulin treatment during follow - up was similar in case and control subjects ( 34.3 vs. 41.2% , p = 0.54 ) .
main demographic and clinical characteristics of enrolled sample data are expressed as means sd , median ( interquartile range ) , or n ( % ) . * in exposed patients .
no significant difference was observed between case and control subjects in the proportion of patients exposed to each insulin . among those exposed ,
a significantly higher mdd ( iu / kg / day ) was observed in case subjects than in control subjects for glargine only . in those receiving glargine ,
median duration of glargine treatment was 20.0 months ( 9.028.0 ) and 14.5 months ( 4.726.0 ) in case and control subjects , respectively ( p = 0.16 ) ; corresponding figures for human basal insulin were 19.4 months ( 5.748.2 ) and 21.0 months ( 7.045.7 ) ( p = 0.86 ) .
the proportion of patients receiving 0.3 iu / kg / day of glargine was higher in case subjects ( 13/112 [ 11.6% ] vs. 14/370 [ 3.8% ] , p = 0.002 ) , whereas no such difference was observed for human insulin ( 27/112 [ 24.1% ] vs. 107/370 [ 28.8% ] , p = 0.33 ) , lispro ( 19/112 [ 17.0% ] vs. 92/370 [ 24.9% ] , p = 0.08 ) , or aspart ( 2/112 [ 1.8% ] vs. 2/370 [ 4.2% ] , p = 0.24 ) .
median doses of basal insulin did not differ between case and control subjects ( 0.16 [ 0.080.31 ] vs. 0.16 [ 0.07027 ] , p = 0.59 ) .
mean total insulin dose was not significantly different between case and control subjects , even after adjusting for metformin exposure and ccs ( odds ratio 1.024 [ 95% ci 0.9461.108 ] , p = 0.56 , for each 0.1 iu / kg / day increment in mdd ) .
after adjusting for the same confounders , exposure to a mean daily total insulin dose 0.3
iu / kg / day was not significantly associated with incident cancer ( hr 0.75 [ 95% ci 0.501.17 ] , p = 0.21 ) .
a majority of patients were treated with combinations of basal and prandial insulin at some time during follow - up , in both case and control subjects . in particular , 82.9 and 86.2% of patients receiving glargine
were also exposed to prandial insulin among case and control subjects , respectively ; the corresponding figures for human basal insulin were 58.9 and 55.1% .
of those treated with glargine , 23.8 and 37.9% in control and case subjects , respectively , also received nph at some time during follow - up ; a large majority ( 94.5% ) of those were treated initially with nph and with glargine afterward .
iu / kg / day insulin doses , adjusted for ccs , metformin exposure , and mean daily total insulin dose , is reported in fig . 1 for each preparation , with the exception of detemir and basal lispro , which could not be analyzed for insufficient case numbers . at total of 0.3
iu / kg / day basal insulin was associated with incident cancer ; this phenomenon was due to the association of cancer with high - dose glargine , which was not observed with basal ( nph ) human insulin .
in fact , 0.3 iu / kg / day glargine was associated with increased risk of cancer , while no effect was observed for human insulin or aspart ; this association was confirmed after exclusion from analysis of cases with cancer occurring within the first 12 months of observation and of their corresponding matched control subjects ( fig .
lispro was associated with a marginally lower risk of cancer , which was not confirmed after exclusion of cases occurring within the first 12 months of observation .
an alternative model , adjusting for metformin exposure , ccs , exposure to prandial insulin , and doses of other basal insulins , provided similar results ( odds ratio 4.76 [ 95% ci 1.9911.40 ] , p < 0.001 , and 0.73 [ 0.341.53 ] , p = 0.40 , for glargine and nph , respectively ) . in a further model , adjusting for the same confounders plus any exposure to other basal insulins during follow - up , use of 0.3
iu / kg / day glargine was still associated with cancer ( 5.83 [ 2.3414.30 ] , p < 0.001 ) .
iu / kg / day , adjusted for comorbidity , exposure to metformin , and doses of other types of insulin .
, all case subjects ; , after exclusion of case subjects with cancer occurring within the first 12 months of observation and of their matched control subjects .
iu / kg / day glargine with incident cancer was evident in younger , but not in older , subjects , while it was similar in men and women and in leaner and overweight / obese patients ( fig .
a-1 in the online appendix , available at http://care.diabetesjournals.org/cgi/content/full/dc10-0476/dc1 ) . considering different cancer types ,
the odds ratio for breast cancer ( n = 7 ) was 5.46 ( 95% ci 0.4566.1 ) ( p = 0.18 ) ; corresponding figures for gastrointestinal / hepatic ( n = 29 ) , pulmonary ( n = 16 ) , pancreatic ( n = 14 ) , and leukemia / lymphoma ( n = 10 ) were 0.58 [ 0.065.87 ] , 2.89 [ 0.3722.23 ] , 4.00 [ 0.2563.95 ] , and 2.83 [ 0.1747.15 ] , respectively .
when insulin doses ( iu / kg / day ) were considered as a continuous variable , no significant unadjusted association between mdds and risk of incident cancer was found for any insulin preparation , although a nonsignificant trend could be observed for glargine . when adjusting for total insulin dose , comorbidity , and exposure to metformin ,
glargine ( adjusted odds ratio 1.33 [ 95% ci 1.071.65 ] , p = 0.011 , for each 0.1 iu / kg / day increment ) , but not basal human insulin ( 1.06 [ 0.911.24 ] , p = 0.11 ) , was associated with a greater risk of incident cancer ; similar results were obtained when adding current smoking as a covariate ( 1.35 [ 1.041.76 ] , p = 0.020 , and 1.04 [ 0.871.29 ] , p = 0.18 , for each 0.1 iu / kg / day increment of glargine and human basal insulin , respectively ) .
this association was maintained after exclusion of case subjects occurring in the first 12 months of observation and of their matching control subejects ( 1.34 [ 1.041.73 ] , p = 0.024 , for each 0.1 iu / kg / day increment ) .
to better discriminate the effect of duration of treatment with glargine and daily dose during treatment , an alternative analysis was performed entering those two variables together with ccs , metformin exposure , and total insulin dose ; incident cancer was associated with higher mean glargine dose during treatment ( 1.21[1.011.47 ] , p = 0.047 , for each 0.1 iu / kg / day ) but not with duration of glargine exposure ( 0.99 [ 0.971.01 ] , p = 0.41 , for each month ) .
the proportion of case subjects exposed to each insulin was not different from that of control subjects , whereas higher mdds of glargine , but not of other types of insulin , were associated with cancer after adjusting for confounders .
several hypoglycemic agents have been reported to modify the risk of malignancies in type 2 diabetic patients ( 1014 ) .
the link between hypoglycemic treatment and cancer has been suggested only by observational studies ; however , randomized clinical trials , designed to assess the effects of drugs on metabolic and/or cardiovascular outcomes , can not have an adequate sample size and length of follow - up to provide relevant information on incident malignancies .
in observational studies , different agents are prescribed to patients with different characteristics , and those diversities can not be entirely eliminated by statistical adjustments ( 6,7 ) .
previous studies suggesting an association between glargine and cancer ( 24 ) suffered from further limitations , including limited information on comorbidities ( 24 ) , short duration of observation , the inclusion of probably pre - existing cases of cancer diagnosed shortly after the initiation of insulin ( 2,3,5 ) , and the failure to discriminate between basal and prandial human insulin used as comparator ( 24 ) . in one study ( 3 )
, the inclusion of time - dependent covariates within a traditional cox model ( 7 ) could have interfered with results ( 2 ) .
on the other hand , because of the frequent change of insulin regimens in clinical practice , the use of baseline therapy for analysis ( 2,4,5 ) is a limitation in studies with longer follow - up .
the prolonged follow - up allowed to identify relevant numbers of incident case subjects whose onset reasonably occurred after the initiation of insulin therapy .
the persistent association of high - dose glargine with cancer after exclusion of cases occurring within the first 12 months of observation suggests that differences can not be attributed to pre - existing , but still subclinical , malignancies .
the adjustment for a comorbidity index confirms that treatment - related differences in cancer incidence can not be considered the effect of comorbid conditions .
moreover , the association of high - dose glargine with incident cancer can not be regarded as a class effect of any formulation of basal insulin or as a consequence of the characteristics of patients prescribed any type of basal insulin , since it was not observed for basal ( nph ) human insulin .
several observational studies have shown that oral hypoglycemic treatments can be associated either with increased or reduced incidence of cancer ( 1014 ) . in particular , a dose - dependent inverse association of metformin with incident cancer has been reported ( 11,12 ) and confirmed in our sample .
notably , the association of high - dose glargine with cancer was confirmed even after adjusting for metformin exposure
. a recent meta - analysis of randomized clinical trials failed to show any association between glargine and cancer ( 15 ) , but the limited number of cases produced wide cis in risk estimates .
the short duration of most randomized trials is a further limitation to that meta - analysis .
these considerations also apply to another meta - analysis showing a lower incidence of cancer with detemir than with human nph insulin ( 16 ) .
the two meta - analyses enrolled both type 1 and type 2 diabetic patients , limiting their interpretation for each of those two individual conditions .
short - acting insulin analogues , lispro and aspart , did not appear to be associated with a higher risk than prandial ( regular ) human insulin .
the limited proportion of patients treated with aspart suggests a special caution in the interpretation of results on this analog , while data on detemir were too limited to provide any valuable information .
two previous studies ( 2,4 ) reported an association of glargine with breast cancer but not with other types of malignancy .
the present sample does not have an adequate size to provide detailed information on specific types of tumors ; although it is possible that glargine treatment has a greater association with breast cancer , the significant association between high - dose glargine and cancer in men suggests that malignancies other than breast cancer could be involved .
discrepancies between the present results and some previous data ( 2,4,5 ) could have been determined , at least in part , by differences in duration of follow - up .
it should be recognized that even the longer follow - up of this study could be insufficient to detect differences with respect to cancer incidence .
furthermore , we observed a dose - dependent association of glargine with incident malignancies , consistent with one previous observation ( 3 ) .
the mechanisms that could account for differences in cancer risk in type 2 diabetic patients treated with different types of insulin are uncertain .
the mitogenic properties of glargine observed in some preclinical models ( 1719 ) , but not consistently confirmed in others ( 20,21 ) , have been reported to be either specific for glargine ( 18,19 ) or shared with other insulin analogues ( 19 ) .
compared with human insulin , glargine has a higher affinity for the igf-1 receptor , which could translate into a greater stimulation of cell growth and transformation , leading to increased cancer risk ( 17 ) ; however , the impact of the igf-1 axis on malignancies is still controversial ( 22 ) .
furthermore , insulin , particularly when at high doses , has a mitogenic and transforming effect independent of its interaction with the igf-1 receptor ( 23 ) . in the present study ,
higher doses of insulin other than glargine were not associated with incident cancer , but this result could be due to an insufficient sample size
no detailed information on reproductive history in women , potentially affecting the incidence of breast cancer , was available .
information on treatments was based on drug prescription , rather than output from pharmacies , as in previous studies ( 25 ) ; in the case of reduced compliance , this should lead to overestimating actual insulin consumption .
however , assessment of insulin delivered from pharmacies could also overestimate actual exposure , because a fraction of the insulin bought in pharmacies is wasted for several reasons .
it is possible that patients receiving higher basal insulin doses were at greater risk and that they had a greater chance of receiving glargine instead of nph , although median basal insulin doses did not differ between case and control subjects .
it should also be considered that cases of incident cancer were identified through hospital admissions and death certificates so that few cases of nonfatal malignancies not receiving treatment in public hospitals could have been missed .
furthermore , the choice of 0.3 iu / kg / day as a threshold for high - dose treatment is somewhat arbitrary ; a larger sample size is needed for exploring more thoroughly the dose - response relationship , identifying a more reliable cutoff .
another relevant limitation is represented by the observational nature of our study , which prevents the entire elimination of prescription biases , although the multiple adjustments performed attenuate the problem .
notably , local guidelines ( www.siditalia.it ) recommend either nph or analogues as basal insulins , recognizing that the former is associated with higher hypoglycemic risk . some potentially relevant confounders affecting treatment choices , such as insulin resistance , are not available .
further , glargine insulin could have been used as a rescue therapy in cases not adequately controlled with other , less expensive , insulin preparations , such as nph , making glargine a marker of severity of disease .
this is a relevant issue , considering that diabetes itself is associated with increased risk of cancer .
it should also be considered that available evidence shows that glargine reduces the risk of hypoglycemia without improving a1c ( 1 ) , as stated also in italian national guidelines ( www.siditalia.it ) ; therefore , the shift from nph to glargine is likely to be motivated by hypoglycemic episodes , rather than to inadequate control of fasting glucose .
furthermore , the association between use of higher glargine doses and incident cancer was confirmed after adjusting for previous therapy with nph insulin .
we should all be aware that the epidemiological approach can not provide definitive conclusions on the effects of any pharmacological treatment .
in fact , not all the motivations underlying a therapeutic decision can be formally explored and adjusted for in multivariate analyses .
for example , patients treated with insulin analogues ( either basal or prandial ) could be different for some clinical or sociocultural characteristics not reported in clinical records ; however , only high - dose glargine , and not other ( rapid - acting ) analogues , was associated with incident cancer . the only fully reliable instrument to assess the effects of glargine on the incidence of cancer
would be an adequately sized , long - term , randomized trial with other basal insulins as active comparators and the onset of malignancies as the predefined , primary end point .
unfortunately , such a trial is not likely to be performed , so that information on safety of glargine , and of other insulin analogues , will inevitably derive from observational studies , such as the present one .
clinical decisions should be based on a careful evaluation of risk / benefit ( and cost / benefit ) ratios .
glargine has some advantages over nph human insulin , such as a lower risk of nocturnal hypoglycemia in head - to - head comparisons ( 1 ) .
the only other available long - acting insulin analog , detemir , is absorbed more rapidly after subcutaneous injection and it has therefore a shorter duration of action ( 24 ) ; as a consequence , many patients treated with detemir need a twice - daily administration , while glargine usually is effective with once - a - day administration ( 25 ) .
conversely , long - acting insulin analogues have a higher cost than nph insulin . the potentially greater risk for cancer with glargine should be weighed against those advantages .
further , larger - scale observational studies on the incidence of malignancies in patients treated with glargine should be performed taking into account insulin doses , in order to quantify the actual impact of different insulin analogues on the onset of new cases of cancer and identify patients at higher risk .
| objectiverecent epidemiological studies suggested that some insulin analogues could be associated with increased risk of cancer .
the present study is aimed at assessing the long - term association of different insulin analogues with cancer incidence.research design and methodsa nested case - control study dataset was generated from the cohort study dataset ( n = 1,340 insulin - treated diabetic outpatients ) by sampling control subjects from the risk sets . for each case
subject , the control subjects ( up to five ) were chosen randomly from those members of the cohort who are at risk for the same follow - up time of the case subject .
five - year age classes , sex , and bmi classes ( < 18.5 , 18.524.9 , 2529.9 , and 30 kg / m2 ) were considered as additional categorical matching variables.resultsduring a median follow - up of 75.9 months ( interquartile range 27.4133.7 ) , 112 case subjects of incident cancer were compared with 370 matched control subjects . a significantly higher mean daily dose of glargine was observed in case subjects than in control subjects ( 0.24 iu / kg / day [ 0.100.39 ] versus 0.16 iu / kg / day [ 0.120.24 ] , p = 0.036 ) .
incident cancer was associated with a dose of glargine 0.3
iu / kg / day even after adjusting for charlson comorbidity score , other types of insulin administration , and metformin exposure ( odds ratio 5.43 [ 95% ci 2.1813.53 ] , p < 0.001 ) .
no association between incident cancer and insulin doses was found for human insulin or other analogues.conclusionsthe possibility of association between cancer and higher glargine doses suggests that dosages should always be considered when assessing the possible association of insulin and its analogues with cancer . |
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